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Nuclear Shuttling Proteins of
Paramyxoviruses: Identification of
Previously Unknown Function of
Respiratory Syncytial Virus Matrix
Protein in Viral Proliferation and Potential
Localisation Signals in Human
Metapneumovirus Nucleocapsid Protein
A thesis submitted for the
Degree of Master in Applied Science (Research)
October, 2012
Wahyu Nawang Wulan
Biomedical Sciences
Faculty of Applied Science
University of Canberra
Canberra, Australia
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Abstract
Respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) are the major
causes of viral lower respiratory tract infections (LRTIs). There are currently no licensed
vaccines or antivirals for RSV and hMPV; and so research is directed towards the
development of live-attenuated vaccines. One pathway to attenuation is to disrupt the
nucleocytoplasmic transport of structural proteins of a virus. The nuclear localisation of a
structural protein is a strategy used by many viruses to inhibit host antiviral responses.
RSV matrix (M) protein shuttles between the cytoplasm and the nucleus, with nuclear
localisation occurring early in infection (around 18 – 24 hours post infection) and
cytoplasmic localisation later. The disruption of RSV M nuclear import decreases the titre of
infectious particles while the disruption of nuclear export ceases the production of infectious
particles. The nucleocytoplasmic transport of RSV M is regulated by CK2 phosphorylation
although the detailed mechanism is not well understood. RSV M(205) (T), which is adjacent
to the nuclear export signal, has been predicted as a putative regulatory site. The
nucleocytoplasmic transport of hMPV nucleocapsid (N) protein, however, has never been
described. The hMPV N has been shown to localise in the nucleus late in the replication cycle
(day 5 post infection), but further research is required to fully understand the mechanism.
Published literature (RSV M) and unpublished results (hMPV N) from the Ghildyal group led
to a hypothesis that disruption of the nucleocytoplasmic transport of a viral structural protein
can lead to attenuation. In this study, two hypotheses were tested: (1) mutation in RSV M205
reduces the production of infectious progeny and the induction of pro-inflammatory
responses; and (2) the hMPV N has inherent nucleocytoplasmic transport ability. The
hypotheses were tested in two aims. Firstly, to investigate the role of the RSV M(205) (T) in
virus growth and induction of pro-inflammatory response in Vero E6 and A549 cells; and
secondly, to investigate the nucleocytoplasmic transport ability of hMPV N in transfected
Cos-7 cells.
To understand the role of RSV M(205) (T), cells were infected with recombinant RSV strain
A2 having threonine (T) substitution to alanine (A) at M205 (rRSV A2 M(T205A)). Viral
titres and expression of IL-8 and RANTES were determined using plaque assay and ELISA,
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following single cycle and multiple cycle replication assays. To define hMPV N
nucleocytoplasmic transport, full-length and truncated hMPV N constructs fused to green
fluorescent protein were expressed in transfected Cos-7 cells.
rRSV A2 M(T205A) was found to be capable of infecting Vero E6 and A549 cells, but was
unable to transmit the infection between cells. The inability to spread leads to a reduced
induction of IL-8 and RANTES expression, which was significantly suppressed in the
interferon (IFN)-α/β-producing A549 cells, compared to the wild type rRSV A2 M205 (T).
hMPV N was found to have inherent ability of nucleocytoplasmic transport, with a potential
nuclear export signal (NES) identified at amino acid 1 – 15 and a region containing a nuclear
localisation signal (NLS) identified at amino acid 192 – 250.
This study shows for the first time that the RSV M(205) (T) is an important site for the
success of transmission of infection and RSV M has a role in the suppression of IFN-α/β.
This study was also the first to show that hMPV N is capable of nucleocytoplasmic transport,
with potential nuclear transport motifs have been identified. By describing the
nucleocytoplasmic transport function of the RSV M and hMPV N proteins, this study
contributes to attempts to develop live-attenuated vaccines for RSV and hMPV.
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Table of Contents
Abstract………………………………………………………………………………………………………..iii
Table of Contents………………………………………………………………………………..…………..v
List of Tables and Figures………………………………………………………………….……………..ix
Form B – Certificate of Authorship of Thesis…………………………………………………...……xi
Acknowledgement………………………………………………………………….………………………xiii
List of Abbreviation………………………………………………...……………………………..………xv
1 Introduction .........................................................................................................................1
1.1 Viral respiratory tract infections ....................................................................................3 1.2 Respiratory syncytial virus ............................................................................................3
1.2.1 Epidemiology................................................................................................... 4 1.2.2 Clinical disease ................................................................................................ 4
1.2.3 Taxonomy ........................................................................................................ 5 1.2.4 The structure of an RSV particle (the virion) .................................................... 6
1.2.5 The life cycle of RSV....................................................................................... 8 1.3 The nucleocytoplasmic transport of viral proteins .........................................................9
1.3.1 Overview ......................................................................................................... 9 1.3.2 The components of nucleocytoplasmic transport ............................................ 10
1.3.3 The mechanism of nuclear import .................................................................. 13 1.3.4 The mechanism of nuclear export ................................................................... 14
1.3.5 Regulation of nucleocytoplasmic transport by phosphorylation ...................... 14 1.3.6 Regulated nucleocytoplasmic transport of viral proteins ................................. 17
1.4 The nucleocytoplasmic transport of the matrix (M) protein of RSV ............................. 19 1.4.1 Overview of the M protein of RSV ................................................................ 19
1.4.2 The nucleocytoplasmic transport of the M protein in infected cells................. 20 1.5 Regulation of cellular innate immunity against RSV infection ..................................... 22
1.5.1 Overview ....................................................................................................... 22 1.5.2 Innate immune responses against RSV infection ............................................ 23
1.5.3 Activation of NF-κB transcription factor ........................................................ 24 1.5.4 Activation of IRF3 transcription factor ........................................................... 25
1.6 RSV vaccine ............................................................................................................... 26 1.7 Human metapneumovirus (hMPV) .............................................................................. 28
1.7.1 Epidemiology................................................................................................. 28 1.7.2 hMPV infection ............................................................................................. 29
1.7.3 Taxonomy ...................................................................................................... 30 1.7.4 The structure of an hMPV particle (the virion) ............................................... 30
1.7.5 The life cycle of hMPV .................................................................................. 32 1.7.6 The role of N protein in virus replication ........................................................ 32
1.7.7 The nucleocytoplasmic transport of nucleocapsid proteins ............................. 34 1.8 Hypotheses and aims ................................................................................................... 36
2 Materials and Methods ....................................................................................................... 39 2.1 Materials ..................................................................................................................... 41
2.1.1 Cell lines ........................................................................................................ 41 2.1.2 Viruses........................................................................................................... 41
2.1.3 Plasmids and bacterial cultures....................................................................... 41 2.1.4 Primers .......................................................................................................... 42
2.1.5 Enzymes/buffers for molecular biology assays ............................................... 43 2.1.6 Antibodies ..................................................................................................... 43
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2.1.7 Kits ................................................................................................................ 43 2.1.8 Buffers, media, and solutions ......................................................................... 44
2.2 Methods ...................................................................................................................... 46 2.2.1 Cell culture .................................................................................................... 46
2.2.2 Virus propagation .......................................................................................... 48 2.2.3 Replication kinetics assay .............................................................................. 51
2.2.4 Enzyme-linked immunofluorescence assay (ELISA) ...................................... 52 2.2.5 hMPV N constructs ........................................................................................ 53
2.2.6 Cloning of hMPV N gene using GatewayTM
Technology ............................... 55 2.2.7 Confirmation of the hMPV N gene inserted into vectors using PCR and DNA
sequencing method .............................................................................................................. 57 2.2.8 Measurement of nucleic acid concentration .................................................... 59
2.2.9 Agarose gel electrophoresis ............................................................................ 59 2.2.10 Plasmid storage in glycerol solution ............................................................... 59
2.2.11 hMPV N protein expression ........................................................................... 60 2.2.12 Bacterial culture ............................................................................................. 61
3 Replication Efficiency of RSV in Cell Culture ................................................................... 63 3.1 Introduction ................................................................................................................ 65
3.2 Methods ...................................................................................................................... 66 3.2.1 Cells and virus ............................................................................................... 66
3.2.2 Single cycle growth curve .............................................................................. 67 3.2.3 Multiple cycle growth curve ........................................................................... 67
3.2.4 Data analysis .................................................................................................. 67 3.2.5 Confirmation of the stability of rRSV M constructs ........................................ 68
3.3 Results ........................................................................................................................ 68 3.3.1 Single cycle replication kinetics ..................................................................... 68
3.3.2 Multiple cycle replication kinetics .................................................................. 72 3.3.3 Confirmation of sequence of rRSV A2 and rRSV A2 M(T205A) ................... 74
3.4 Discussion................................................................................................................... 76 3.5 Summary .................................................................................................................... 78
4 Pro-Inflammatory Response in RSV Infection .................................................................... 79 4.1 Introduction ................................................................................................................ 81
4.2 Methods ...................................................................................................................... 82 4.2.1 Cells and virus ............................................................................................... 82
4.2.2 Single cycle replication assay ......................................................................... 82 4.2.3 Multiple cycle replication assay ..................................................................... 83
4.2.4 Enzyme-linked immunosorbent assay (ELISA) .............................................. 83 4.2.5 Data analysis .................................................................................................. 84
4.3 Results ........................................................................................................................ 84 4.3.1 Single cycle replication assay ......................................................................... 84
4.3.2 Multiple cycle replication assay ..................................................................... 88 4.4 Discussion................................................................................................................... 92
4.5 Summary .................................................................................................................... 94 5 The Subcellular Localisation of hMPV N Protein in Transfected Cells ............................... 95
5.1 Introduction ................................................................................................................ 97 5.2 Methods ...................................................................................................................... 99
5.2.1 Generation of constructs and recombination cloning ...................................... 99 5.2.2 Transient transfection into Cos-7 cells.......................................................... 101
5.2.3 Confocal laser scanning microscopy (CLSM) .............................................. 103
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5.3 Results ...................................................................................................................... 104 5.3.1 The generation of hMPV N constructs ......................................................... 104
5.4 The GatewayTM
recombination cloning (RC) technology system ............................... 107 5.4.1 BP recombination reaction ........................................................................... 107
5.4.2 LR recombination reaction ........................................................................... 109 5.5 Expression of hMPV N constructs in transfection cells .............................................. 111
5.5.1 Expression of full-length hMPV N (1-394) observed in live Cos-7 cells ....... 111 5.5.2 Expression of hMPV N deleted constructs observed in Cos-7 cells............... 113
5.6 Discussion................................................................................................................. 115 6 General Discussion .......................................................................................................... 119
6.1 Respiratory syncytial virus ........................................................................................ 121 6.2 Human metapneumovirus.......................................................................................... 124
6.3 Conclusion ................................................................................................................ 125 7 Reference List .................................................................................................................. 127
8 Appendix ......................................................................................................................... 147
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List of Tables and Figures
Figure 1-1 Syncytia. ............................................................................................................. 5
Figure 1-2 The structure of respiratory syncytial virus .......................................................... 7 Figure 1-3 The life cycle of respiratory syncytial virus.. ....................................................... 9
Figure 1-4 Overview of a nuclear pore complex (NPC).. .................................................... 10 Table 2-1 Cell lines used in this study ................................................................................. 41
Table 2-2 Viruses used in this study .................................................................................... 41 Table 2-3 Plasmid/bacteria used in this study ...................................................................... 41
Table 2-4 Primers used in this study .................................................................................... 42 Table 2-5 Enzymes used in this study .................................................................................. 43
Table 2-6 Antibodies used in this study ............................................................................... 43 Table 2-7 Kits used in this study ......................................................................................... 43
Table 2-8 Culture media used in this study .......................................................................... 44 Table 2-9 List of commercial chemicals used in this study .................................................. 44
Table 2-10 List of in-house prepared buffers and solution ................................................... 45 Table 2-11 Components of PCR for Mycoplasma sp. detection ........................................... 47
Table 2-12 The reaction mix I to generate cDNA from hMPV RNA.................................... 53 Table 2-13 The reaction mix II to generate cDNA from hMPV RNA .................................. 54
Table 2-14 The PCR components to generate hMPV N constructs....................................... 54 Table 2-15 List of primer pairs used to generate hMPV N construct .................................... 54
Table 2-16 The components of BP recombination reaction .................................................. 55 Table 2-17 The components of LR recombination reaction .................................................. 56
Table 2-18 PCR components to confirm the presence of hMPV N insert in cloning vectors. 58 Table 2-19 Components of DNA sequencing reaction, with PCR product as template ......... 58
Table 2-20 Components of DNA sequencing reaction, with ds plasmid as template ............. 58 Figure 3-1 The growth curve of single cycle replication kinetics of rRSV A2 and rRSV A2
M(T205A) in Vero E6 and A549 cells. ........................................................................ 70 Figure 3-2 The growth curve of multiple-step replication kinetics of rRSV A2 and rRSV A2
M(T205A) in Vero E6 and A549 cells.. ....................................................................... 73 Figure 3-3 Chromatogram of DNA sequencing of the matrix gene of rRSV A2 and rRSV A2
M(T205A) showing the difference between the threonine and alanine codons at amino
acid 205. ...................................................................................................................... 75
Figure 4-1 Expression of interleukin 8 (IL-8) in cells infected with rRSV A2 or rRSV A2
M(T205A) in single cycle replication assay.. ............................................................... 85
Figure 4-2 Expression of regulated upon activation, normal T-cell expressed, and secreted
(RANTES) in cells infected with respiratory syncytial virus (RSV) in single cycle
replication assay. ......................................................................................................... 87 Figure 4-3 Expression of interleukin 8 (IL-8) in cells infected with respiratory syncytial virus
(RSV) in multiple cycle replication assay.. .................................................................. 89 Figure 4-4 Expression of regulated upon activation, normal T-cell expressed, and secreted
(RANTES) in cells infected with respiratory syncytial virus (RSV) in multiple cycle
replication assay.. ........................................................................................................ 91
Figure 5-1 Schematic representation of hMPV N constructs generated in this study.......... 105 Figure 5-2 Human metapneumovirus N (hMPV N) gene constructs and vectors used to
investigate the subcellular localisation of the constructs.. ........................................... 106 Figure 5-3 Screening for E. coli colonies containing the pDONR
®207 – hMPV N constructs
using PCR. ................................................................................................................ 108
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Figure 5-4 Screening for E. coli colonies containing the pEPI-DESTC – hMPV N constructs
using PCR. ................................................................................................................ 110
Figure 5-5 Subcellular localisation of GFP-tagged full-length hMPV N (1-394) protein
observed in Cos-7 cells.. ............................................................................................ 112
Figure 5-6 Subcellular localisation of GFP-tagged hMPV N constructs in live Cos-7 cell. 114 Appendix: Confirmation of hMPV N constructs ligated in the entry and expression clones by
DNA sequencing ....................................................................................................... 149
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Form B
Certificate of Authorship of Thesis
Except where clearly acknowledged in footnotes, quotations and the bibliography, I certify
that I am the sole author of the thesis submitted today entitled –
Nucleocytoplasmic Transport of Respiratory Syncytial Virus Matrix Protein and Human
Metapneumovirus Nucleocapsid Protein in Infection
I further certify that to the best of my knowledge the thesis contains no material previously
published or written by another person except where due reference is made in the text of the
thesis.
The material in the thesis has not been the basis of an award of any other degree or diploma
except where due reference is made in the text of the thesis.
The thesis complies with University requirements for a thesis as set out in Gold Book
Part 7: Examination of Higher Degree by Research Theses Policy, Schedule Two (S2).
Refer to http://www.canberra.edu.au/research-students/goldbook
31 October 2012
Signature of Candidate
Wahyu Nawang Wulan Signature of chair of the supervisory panel
Date: ……………………………..
xiii
Acknowledgment
I would like to thank my supervisors, Dr Reena Ghildyal and Dr Michelle Gahan for great
supervision, guidance, patience, and encouragement. Their vast knowledge tells that they are
world class scientists, and it is truly a great honour for me to be under their supervision.
Other than my supervisors, I would like to thank all the people who have helped me
accomplishing my study, in particular Dr Erin Walker and Dijana Townsend for huge
research assistance. A huge thank you to Parisa, Raj, Robert, James, Hamid, and Kylia from
the Ghildyal lab, for providing technical assistance and making the lab a great place to work.
I would also like to thank the BMS students for sharing the experience of research works and
the friendship, particularly the members of room 3D29: Guifang Shang, Khloud, Bilquis,
Sandy, Matt, Jade, Jessica, Rebecca, Fan, Sherry, and Andrew. Also, a big thank you to Ke
Jun, Marwa, Rafat, and other BMS staffs/ post-docs; the 3D level is a supportive place to
work because of them.
My master’s education could not have been carried out without the Australian Development
Scholarship, for which I am greatly indebted. I am particularly grateful to Rozana Muir, the
AUSAid Liaison Officer, for her invaluable assistance during my study.
Thank you to my family, relatives, and friends, for the support and encouragement. I am
particularly indebted to the lengthy support and encouragement given by my mother and my
aunt Djarwani, who have inspired me to pursue my dreams and kept on cheering me through
the ups and downs.
Finally, I would like to thank God for giving me the strength in accomplishing this study.
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List of Abbreviation
A Alanine
BDV Borna disease virus
BRAP2 BRCA1 binding protein 2
BRCA1 breast cancer antigen 1
CARD caspase recruitment domain
Cdk cyclin-dependent kinase
cDNA complementary deoxyribonucleic acid
CDV canine distemper virus
CK2 casein kinase 2
CLSM confocal laser scanning microscopy
cp cold passaged
CPE cytopathic effect
Crm chromosomal region maintenance
CTD carboxy terminal domain
D aspartic acid
d.p.i day post infection
DEN dengue virus
dsRNA double-stranded ribonucleic acid
EAV equine arteritis virus
ELISA enzyme-linked immunosorbent assay
F phenylalanine; fusion protein
FG phenylalanine glycine
FI formaline-inactivated
G glycine; attachment glycoprotein
GAP GTPase-activating protein
GDP guanosine diphosphate
GE gene end
GEF guanine–nucleotide exchange factor
GFP green fluorescent protein
GS gene start
GTP guanosine triphosphate
h.p.i hour post infection
HCMV human cytomegalovirus
hMPV human metapneumovirus
I isoleucine
IFN interferon
IKK inhibitor of κB kinase
IL interleukin
ILI influenza-like illness
xvi
Imp importin
IPS1 interferon-β promoter stimulator 1
IRAK IL-1 receptor-associated kinases
IRF interferon regulatory factor
ISG interferon-stimulated gene
ISGF IFN-stimulated growth factor
IκB inhibitor of κB
JAK Janus kinase
K lycine
kDa kilo dalton
L leucine
LRTIs lower respiratory tract infections
M methionine; matrix
MAVS mitochondrial antiviral signalling protein
MOI multiplicity of infection
MV measles virus
N nucleocapsid; asparagine
NES nuclear export signal
NF-κB nuclear factor kappa B
NLS nuclear localisation signal
NODSH+
nucleolar detention signal regulated by H+
NoLS nucleolar localisation signal
NPC nuclear pore complex
NS non-structural protein
NTD amino terminal domain
NTF nuclear transport factor
Nups nucleoporins
ORF open reading frame
PAMP pathogen-associated molecular pattern
PKC protein kinase C
ppp triphosphate
PRR pathogen recognition receptor
Q glutamine
R arginine
RABV rabies virus
RANTES regulated upon activation, normal T-cell expressed, and secreted
RC recombinational cloning
RCC regulator of chromosome condensation
RIG-1 retinoic-acid inducible gene
RPV rinderpest virus
rRNA ribosomal RNA
rRSV recombinant respiratory syncytial virus
xvii
RSV respiratory syncytial virus
RTIs respiratory tract infections
S serine
SEM standard error of the means
SH small hydrophobic
ssRNA single-stranded ribonucleic acid
T threonine
TAB TAK1-binding protein
TAK tumour growth factor-β-activated kinase
TLR Toll-like receptor
TNF tumour necrosis factor
TRAF tumour necrosis factor receptor-associated factor
ts temperature sensitive
Tyk tyrosine kinase
URTIs upper respiratory tract infections
UV ultraviolet
V valine
VISA virus-induced signalling adaptor
VSV vesicular stomatitis virus
ZFD zinc finger domain
1
CHAPTER 1
INTRODUCTION
1 Introduction
2
3
1.1 Viral respiratory tract infections
Viral respiratory tract infections (RTIs) are the most common infections in humans
(Lieberman et al., 2002), and morbidity and mortality contribute to a significant loss of
productivity that puts a strong economic burden on society. RTIs are highly infectious due to
the ease of the transmission of the respiratory viruses and therefore there is a need to prevent
the feasibility of RTIs distribution in the population. The disease is mostly self-limiting with
symptoms that include ‘runny nose’ (rhinorrhea), nasal congestion, throat inflammation
(pharyngitis), and cough, which are most commonly known as influenza-like illness (ILI)
(Kesson, 2007). However, disease severity varies according to the infecting virus, the age of
the infected person, as well as the host inflammatory responses. In general, upper respiratory
tract infections (URTIs), which general symptoms include runny/stuffy nose and sneezing,
are usually benign, transitory, and self-limiting; while lower respiratory tract infections
(LRTIs), which mostly progress to bronchitis/bronchiolitis and pneumonia, can be fatal
(Dasaraju and Liu, 1996). The viruses that are associated with serious LRTIs are the closely-
related negative single-stranded ribonucleic acid ((-)ssRNA) viruses of the Paramyxoviridae
family: respiratory syncytial virus (RSV) and human metapneumovirus (hMPV) (Simoes,
1999, Wat, 2004, Kesson, 2007), to which there are no licensed vaccines available for the
general public.
1.2 Respiratory syncytial virus
Respiratory syncytial virus (RSV) was first isolated in 1956 from a laboratory chimpanzee
showing symptoms of ILI (Collins and Crowe Jr., 2007). It is the primary cause of severe
LRTIs among infants and very young children (less than 2 years old), among the elderly,
particularly those who have underlying cardiopulmonary diseases, as well as among
immunocompromised people of any age (Englund et al., 1988, Falsey and Walsh, 2005,
C.D.C, 2011). RSV infection in immunocompetent adults is seldom asymptomatic, generally
only develops into mild ILIs, and is not treated as threatening illness; yet poses a potential
risk of transmission to the susceptible age groups because immunity against subsequent
infection is short-lived (< 2 months) and reinfections are common (Anderson and Heilman,
1995, Hashem and Hall, 2003, Falsey et al., 2006). The virus is transmitted by large droplets
and fomites (Hall, 1982). The development of an RSV vaccine has been hindered by
immaturity of the immune system and immunosuppressive effects of maternally-derived
4
antibodies in the young, as well as difficulties in conducting research due to poor virus
growth in vitro and physical instabilities of virus particles (Dudas and Karron, 1998).
1.2.1 Epidemiology
RSV infection is highly associated with bronchiolitis and pneumonia in infants/ very young
children and the elderly (Ruuskanen et al., 2011). The global burden of RSV disease is
estimated at 64 million cases and 160,000 deaths annually (W.H.O, 2011). In regions where
RSV infection is well documented, such as in the United States of America, the mortality rate
of RSV infection is recorded as the highest in people aged less than 1 year and more than 65
years old, reaching 5.4 and 29.6 cases per 100,000 person-years, respectively (Thompson et
al., 2003). Epidemics occur throughout the year worldwide and are characterised by a strong
pattern of seasonality, particularly in temperate regions. The epidemics mostly occur during
the winter/spring months, with a peak of incidence in January – March in the northern
hemisphere and July – September in the southern hemisphere (Al-Toum et al., 2006, Checon
et al., 2002, Roche et al., 2003, Tatochenko et al., 2010). In the tropical regions, the epidemic
period is less clear-cut, but most cases occur during the rainy season (Weber et al., 1998).
1.2.2 Clinical disease
RSV-associated illness starts as an URTI, as a result of the replication of the virus in the
nasopharyngeal epithelial cells; followed by the migration of the virus to the lower part of the
respiratory tract, where the virus targets the ciliated cells of the small bronchioles and the
type-1 pneumocytes to develop LRTI (Brooks et al., 2007, Collins and Graham, 2008). The
spread of virus particles from cell to cell is facilitated by the beating of the epithelial cilia
(Stokes-Peebles-Jr. and Graham, 2005). The incubation period between exposure and onset of
illness is 3 – 5 days; however, once symptoms have developed, progress to death may occur
rapidly (Brooks et al., 2007). Virus spread induces the fusion between adjacent plasma
membranes, leading to the development of the characteristic cytopathic effect (CPE) of
multinucleated giant cells known as syncytia (sing.: a syncytium), which can contain 20 – 30
nuclei (Figure 1-1).
5
Figure 1-1 Syncytia (circled, and shown by arrow head) induced by respiratory syncytial
virus (RSV) infection in Vero E6 cells, 48 hours after infection.
RSV infection leads to the production of various inflammatory mediators that signal and
recruit immune cells to the infection sites, such as RANTES (eosinophil chemoattractant) and
interleukin 8 (neutrophil chemoattractant). However, overexpression of the mediators that
leads to the over recruitment of the immune cells may exaggerate the inflammatory responses
that subsequently promote airway damage and enhanced disease pathogenesis, which
manifests primarily as bronchiolitis and/or pneumonia (Domachowske and Rosenberg, 1999,
Openshaw and Tregoning, 2005, Oshansky et al., 2009). Bronchiolitis is a condition where
sloughed necrotic infected cells and intense inflammatory mediators infiltrate and obstruct
the bronchial lumen, while pneumonia refers to inflammation in the alveoli or when the lungs
are filled with fluid (Brooks et al., 2007, Williams and Crowe Jr., 2007). RSV-associated
bronchiolitis and pneumonia often contribute to respiratory failure and fatalities (Dasaraju
and Liu, 1996).
1.2.3 Taxonomy
RSV is classified in the order Mononegavirales, family Paramyxoviridae, subfamily
Pneumovirinae, and genus Pneumovirus (Collins, 1991, Lamb and Parks, 2006, Collins and
Graham, 2008). Other viruses of significant importance for human health in the family
Paramyxoviridae include measles virus (genus Morbillivirus), mumps virus (genus
6
Rubulavirus), human metapneumovirus (genus Metapneumovirus), and the deadly emerging
virus, Nipah virus (genus Henipaviruses). RSV is differentiated into antigenic subgroups A
and B based on the neutralisation with monoclonal antibody against the G protein (Collins,
2011).
1.2.4 The structure of an RSV particle (the virion)
RSV particles are pleomorphic, having various shapes that include round, kidney, and
filamentous, with the latter the most observed (Norrby et al., 1970, Bachi and Howe, 1973).
The size of RSV is approximately 80-350 nm in diameter as a spherical unit and
approximately 60-100 nm in diameter and up to 10 µm in length as a filamentous unit
(Collins, 1991, Ogra, 2004). The main architecture of RSV is a nucleocapsid core that is
enclosed by a lipid bilayer envelope structure, with a matrix layer that bridges between the
two structures, as seen in Figure 1-2 (Collins, 1991, Lamb and Parks, 2006). The viral
genome is a negative sense single-stranded ribonucleic acid ((-)ssRNA) sized 15.2 kilobase
(kb) and contains 10 genes that are arranged in the order of 3’-NS1-NS2-N-P-M-SH-G-F-
M2-L-5’, which encode for 11 proteins, in sequential order, the non-structural 1 and 2 (NS1,
NS2), the nucleocapsid (N), the phosphoprotein (P), the matrix (M), the small hydrophobic
(SH), the attachment glycoprotein (G), the fusion (F), the transcription elongation factor
(M2-1) and a replication-regulator factor (M2-2), and the large polymerase subunit (L)
(Collins and Murphy, 2005).
The internal virion is built from the helical nucleocapsid complex that consists of the
(-)ssRNA genome, N protein, the L and P polymerase complex (García-Barreno et al., 1996,
Collins, 2011). Viral envelope is derived from host plasma membrane and contains the SH
protein and the two RSV major neutralising antigens: the attachment G protein and the fusion
F protein. The G and F proteins mediate the binding of virion to cellular receptors to establish
efficient infection (Collins, 2011). The homotrimeric-arranged F protein specifically
functions to fuse between viral and host membranes to initiate infection and between the
plasma membranes of infected cells to develop the characteristic syncytial CPE (Morton et
al., 2003). RSV F is capable of attachment function in the absence of the G protein (Lamb
and Parks, 2006, Collins, 2011). The G protein is less neutralising and more diverged than the
F protein (Collins, 1991). However, together with the SH protein, the G protein is not
required for growth in cell culture (Karron et al., 1997a, Teng and Collins, 1998). The RSV
7
M protein underlies the viral envelope and is associated with the nucleocapsid complex; its
positioning is the result of a hydrophobic-electrostatic interaction with the plasma membrane
and the binding to the cytoplasmic tail of the G glycoprotein and to the M2-1 protein of the
nucleocapsid core (Lenard, 1996, Gaudier et al., 2002, Ghildyal et al., 2002, Latiff et al.,
2004, Ghildyal et al., 2005b, Li et al., 2008).
The proteins that comprise the virion are called the structural proteins, while the non-
structural proteins, which comprise M2-1, M2-2, NS1, and NS2, are either underrepresented
or absent in the virion but synthesised in infected cells to function in regulation of viral
proliferation (Collins, 1991, Lamb and Parks, 2006, Collins, 2011). For example, the NS1
and NS2 proteins facilitate successful replication by counteracting the antiviral action of the
host innate immune response, the M2-1 protein is the transcription elongation factor, and the
M2-2 protein is the regulatory switch between transcription and replication of viral RNA
(Hardy and Wertz, 1998, Bermingham and Collins, 1999, Spann et al., 2004, Spann et al.,
2005).
Figure 1-2 The structure of respiratory syncytial virus. The general structure of an RSV
virion is a nucleocapsid core that is sheathed by an envelope structure, which is the host-
derived lipid bilayer plasma membrane. The nucleocapsid core consists of the negative-sense
(-) single-strand (ss) RNA genome bound by the nucleocapsid (N) protein and is associated
with the phosphoprotein (P) and large (L) polymerase subunit. The nucleocapsid core
interacts with the envelope structure by the mediation of the matrix (M) protein that layers
the inner surface of the envelope. The surface glycoproteins consisting of the fusion (F),
attachment glycoprotein (G), and the small hydrophobic (SH) proteins are inserted into the
envelope
8
1.2.5 The life cycle of RSV
The main stages in the life cycle of RSV are adsorption/entry, RNA synthesis, and
assembly/budding (Figure 1-3). Adsorption/entry involves the binding between the viral and
cellular receptors, followed by fusion of the F protein with the plasma membrane, and the
release of the nucleocapsid core into the cytoplasm, which is known as uncoating (Collins,
1991, Lamb and Parks, 2006, Collins and Crowe Jr., 2007). RNA synthesis consists of
transcription of monocistronic mRNAs from (-)ssRNA genome and genome replication. The
initiation and termination of transcription is signalled by specific internal sequences, namely
gene start (GS) and gene end (GE), that flank each gene; and transcription is attenuated at
succeeding genes such that the relative abundance of each mRNA decreases in the 3’ – 5’
direction of the (-)ssRNA genome (Lamb and Parks, 2006). Genome replication involves the
production of the complementary (+)ssRNA antigenome that acts as an intermediate template
for the synthesis of new (-)ssRNA (Collins, 1991, Lamb and Parks, 2006). Virion assembly is
initiated by the formation of the nucleocapsid core in the cytoplasm and the arrangement of
the envelope glycoproteins in the plasma membrane (Collins, 1991, Lamb and Parks, 2006).
New infectious particles are formed by the joining together between the nucleocapsid and
envelope structures by the M protein, followed by maturation via budding through plasma
membrane, during which the cellular membrane is acquired as virion envelope (Norrby et al.,
1970, Bachi and Howe, 1973, Roberts et al., 1995, Garoff et al., 1998).
The single cycle growth of RSV in cell culture takes place between 20 – 40 hours post
infection (h.p.i) and the viral spread from cell to cell induces syncytia formation that has been
shown to peak between 72 and 96 h.p.i (Bachi and Howe, 1973, Lamb and Parks, 2006).
However, it is arguable whether the production of infectious particles is the highest at this
point since syncytia formation can be induced by RSV F protein independent of other viral
components (Morton et al., 2003).
9
1.3 The nucleocytoplasmic transport of viral proteins
1.3.1 Overview
The nucleocytoplasmic transport of molecules inside a eukaryotic cell plays an important role
in the integration of DNA replication/RNA biogenesis and protein synthesis, since the first
occurs in the nucleus and the latter in the cytoplasm. The two compartments are separated by
the nuclear envelope, which is a double membrane system perforated by nuclear pores
(Alberts et al., 2008). Molecules are transported through the pore, which itself is a complex
protein structure built from subunits called nucleoporins (Nups). The nuclear pore complex
(NPC) consists of three domains: the cytoplasmic filaments, the central core, and the nuclear
basket (Sorokin et al., 2007) (Figure 1-4). The spatial segregation provides the cells with a
Figure 1-3 The life cycle of respiratory syncytial virus. All stages of replication occur in the
cytoplasm of infected cells. (1) The RSV virion attaches to the cellular receptor via G
glycoprotein, followed by (2) the fusion of viral membrane with the cellular plasma membrane
mediated by F glycoprotein. The consequence of which is the release of the helical nucleocapsid
containing the (-)ssRNA genome into the cytoplasm. The (-)ssRNA is transcribed into mRNAs
(3) and replicated into progeny genome (4). Viral proteins are translated (5) from the mRNAs.
The nucleocapsid complex is built in the cytoplasm (6) from free N subunits, the (-)ssRNA
genome, the P(hospho) protein, L polymerase subunits, and the M2-1 protein. Meanwhile, the
glycoproteins are transported via the exocytic pathway to the plasma membrane where they are
arranged at the assembly sites (7). The matrix (M) protein interacts with the cytoplasmic tail of G
glycoprotein of the envelope structure and with the M2-1 protein of the nucleocapsid complex in
the cytoplasm; then brings the two structures together to form new infectious particles, which
mature by budding out of the plasma membrane (8).
10
means of regulation of gene expression. In terms of virus infection, nucleocytoplasmic
transport of viral genomes or proteins is crucial for the successful replication of many
viruses, one of the reasons is as a strategy to evade the host immune responses (Fulcher and
Jans, 2011). The transport of viral proteins between the cytoplasm and the nucleus is most
commonly facilitated by a mechanism known as Ran-dependent nucleocytoplasmic transport
of proteins. Nucleocytoplasmic transport is bidirectional, consisting of nuclear import and
nuclear export, and is regulated at the level of individual cargo molecule, nuclear transport
receptors, and nucleoporins; with the efficiency of nuclear transport is determined by the
affinity between nuclear transport factors and nuclear transport signals (Nigg, 1997,
Henderson and Eleftheriou, 2000, Lam et al., 2001, Sorokin et al., 2007, Terry et al., 2007,
Chumakov and Prasolov, 2010).
1.3.2 The components of nucleocytoplasmic transport
The main components in nuclear transport are transport factors, nuclear transport signals of a
cargo molecule, phenylalanine glycine nucleoporins (FG-Nups) of the NPC, and GTPase Ran
with its main regulators (RanGAP1 and RanGEF) (Gorlich and Mattaj, 1996, Nigg, 1997,
Terry et al., 2007, Sorokin et al., 2007, Alberts et al., 2008, Chumakov and Prasolov, 2010).
Figure 1-4 Overview of a nuclear pore complex (NPC). The cytoplasmic filaments, outer
and inner ring, the NPC core, nuclear basket and distal ring are shown with their constituent
nucleoporins (Nups).
11
1.3.2.1 Transport factors
Transport factors are proteins from the large evolutionarily-conserved karyopherin β family,
which carry the cargo molecule across the NPC and can be differentiated into importins and
exportins (Sorokin et al., 2007). Importins transport the cargo molecule from the cytoplasm to
the nucleus, while exportins travel in the opposite direction. Importin α (Imp-α) and importin
β (Imp-β) are members of the importin family that are known to play a major role in nuclear
import; while the most widely characterized exportin is the Crm1 (chromosomal region
maintenance 1), which is a homologue of Imp-β (Alberts et al., 2008, Chumakov and
Prasolov, 2010). The nucleocytoplasmic transport of the M protein of RSV has been shown to
be facilitated by Imp-β1 and Crm1 (Ghildyal et al., 2005a).
1.3.2.2 Nuclear transport signals
The nuclear localisation signal (NLS) specifies translocation across the NPC; it provides a
high affinity interaction between a cargo and an importin (Imp) and is typically present as a
repeat (monopartite) of positively charged basic residues (lysine (K) or arginine (R)), or two
clusters of the repeat (bipartite) separated by a spacer (Corbett and Silver, 1997). The most
well-studied monopartite NLS is of the SV40 T antigen, PKKKRKV132
, while the
nucleoplasmin’s KRPAATKKAGQAKKKK, is the best-studied bipartite NLS (Gorlich and
Mattaj, 1996, Nigg, 1997, Sorokin et al., 2007). Bold characters indicate the key residues of
an NLS motif. The interaction of the positively-charged basic NLS with the acidic residues in
the inner surface of the importin results in the strong affinity of the cargo-importin complex
(Jamali et al., 2011). This binding affinity governs the formation of a functional nuclear
import complex that eventually determines the nuclear import rate (Timney et al., 2006).
It has been suggested that proteins having NLS rich in arginines can be transported directly
by Imp-β; while proteins with NLS rich in lysines require the mediation of Imp-α for binding
to Imp-β (Palmeri and Malim, 1999). The differentiation between direct binding to Imp-β1
and binding with mediation of Imp-α is specific, since the NLS of a protein that directly binds
to Imp-β1 tends to bind to an area of Imp-β1 that recognises Imp-α; therefore there is a
competition to use the Imp-β1 binding region between Imp-α and cargo, making the presence
of Imp-α inhibits Imp-β1-cargo binding (Henderson and Percipalle, 1997).
12
The nucleolar localisation signal (NoLS) ensures accumulation of a cargo protein in the
nucleolus. There is not much known about the consensus sequence of a NoLS; however it is
believed that the motif is also rich in basic residues like NLS and tends to be located at both
ends of a protein (Scott et al., 2011). Commonly, cytoplasmic proteins that localise in the
nucleolus also possess NLS to facilitate transport across the NPC (Scott et al., 2010). A
protein can also localise in the nucleolus as a result of retention in the nucleolus following a
high affinity binding to the nucleolar components such as ribosomal DNA or RNA (rDNA or
rRNA), where this high binding affinity is also facilitated by a specific sequence known a s
nucleolar detention signal regulated by H+ (NoDS
H+) (Carmo-Fonseca et al., 2000, Mekhail et
al., 2007). In this case, it was proposed that the transport signal in these proteins is most
likely NLS, but then the proteins have the capability of binding to nucleolar components, and
this binding facilitates the nucleolar localisation (Jans and Hübner, 1996).
The nuclear export signal (NES) is a strong cytoplasmic localisation signal that is recognised
by Crm1 and consists of a short hydrophobic sequence most commonly conforms to the motif
LXXXLXXLXL, where L can be the hydrophobic residues of leucine (L), methionine (M),
phenylalanine (F), valine (V), or isoleucine (I), and X is any amino acid, as exemplified by
the HIV Rev NES LQLPPLERLTL83
(Gorlich and Mattaj, 1996, Nigg, 1997, Hutten and
Kehlenbach, 2007, Sorokin et al., 2007, Jamali et al., 2011). The transport signal is usually
positioned in an exposed region or in a flexible region that unfolds easily (la-Cour et al.,
2004).
1.3.2.3 FG-Nups
FG-Nups fill the central core of a NPC and impose a selective transport following a specific
interaction with transport factors (Grunwald et al., 2011). FG-Nups that are critical for
nucleocytoplasmic transport are characterised by the GLFG, FxFG, or FG repeats motif,
separated from each other by spacers rich in glutamine (Q), serine (S), threonine (T), or
asparagine (N). The F and L residues of this motif produce weak hydrophobic interactions
with karyopherins, facilitating translocation of transport complex across a nuclear pore by
acting as docking sites for the translocation of transport factor (Sorokin et al., 2007, Jamali et
al., 2011).
13
1.3.2.4 Ran and its regulators
The formation and dissociation of the transport complex are controlled by GTPase Ran
through hydrolysis of GTP into GDP. The hydrolysing activity of Ran is induced by
RanGTPase activating protein 1 (RanGAP1) that is compartmentalized in the cytoplasm
(Nigg, 1997, Sorokin et al., 2007, Chumakov and Prasolov, 2010, Jamali et al., 2011). Ran-
bound GDP is converted into a RanGTP by the nuclearly-compartmentalised guanine–
nucleotide exchange factor (RanGEF), which in mammals is known as the regulator of
chromosome condensation (RCC1) (Gorlich and Mattaj, 1996, Nigg, 1997, Sorokin et al.,
2007). Another important factor is the nuclear transport factor 2 (NTF2), which does not play
a direct role in translocating a cargo protein but is involved in governing the directionality of
nucleocytoplasmic transport/karyopherin recycling by maintaining a high concentration of
nuclear Ran-GTP and cytoplasmic Ran-GDP (Chumakov and Prasolov, 2010, Jamali et al.,
2011, Grunwald et al., 2011).
1.3.3 The mechanism of nuclear import
In terms of Imp-β1-facilitated nuclear import, the transport of proteins from the cytoplasm to
the nucleus occurs through three major steps: firstly, the association of cargo-Imp-β1 and
subsequent docking to the NPC, secondly, the translocation of nuclear import complex
through the NPC, and thirdly, the dissociation of nuclear import complex. In the cytoplasm,
the Imp-β1 binds to a cargo protein at its NLS to form an import complex, which
subsequently docks at FxFG repeats of the Nup358 that is positioned in the cytoplasmic
filaments of the NPC (Sorokin et al., 2007, Chumakov and Prasolov, 2010). From Nup358,
the complex translocates to the nuclear side by multiple associations-dissociations between
the import complex and FxFG repeats, particularly those contained in Nup62 of the central
core of the NPC (Gorlich and Mattaj, 1996, Nigg, 1997, Sorokin et al., 2007). Recent
findings suggest that translocation is energy-independent, taking place only through repeated
low-affinity hydrophobic interactions between Imp-β1 and the phenylalanine residues of
FxFG repeats (Sorokin et al., 2007, Terry et al., 2007, Chumakov and Prasolov, 2010, Jamali
et al., 2011). Reaching the nuclear side of the NPC, the Imp-β1 docks the import complex to
Nup153 of the nuclear filaments of NPC (Sorokin et al., 2007). RanGTP, which is highly
concentrated in the nucleus, breaks this connection by binding to Imp-β1, thus releasing
cargo protein into the nucleoplasm. RanGTP has a very strong affinity for karyopherins, and
14
its association to Imp-β1 disassembles the import complex: possibly due to the competition
with the binding domain in Imp-β1 for NLS or because RanGTP interaction with Imp-β1
drives conformational change that detaches cargo (Henderson and Percipalle, 1997, Lam et
al., 2001, Pemberton and Paschal, 2005, Chumakov and Prasolov, 2010, Jamali et al., 2011).
The RanGTP–Imp-β1 complex is recycled back into the cytoplasm, where RanGTP is
hydrolysed into RanGDP by RanGAP.
1.3.4 The mechanism of nuclear export
In contrast to nuclear import, translocation of export complex from nucleus to cytoplasm is
an energy-dependent process. A functional nuclear export complex commonly comprises
Crm1, a cargo protein, RanGTP, and RanBP3, which is a cofactor that enhances the binding
of Crm1 to RanGTP as well as increases the affinity of Crm1 to NES (Hutten and
Kehlenbach, 2007, Sorokin et al., 2007, Chumakov and Prasolov, 2010). The complex
translocates through the NPC in a mechanism that has not been well understood but is
believed to involve the FG-Nups (Macara, 2001). The translocation ends at the terminal
docking sites, which, although still in debate, are thought to involve the cytoplasmic Nup358
(Hutten and Kehlenbach, 2007, Sorokin et al., 2007, Chumakov and Prasolov, 2010).
In the terminal docking sites, the export complex dissociates through the hydrolysis of
RanGTP, which causes a conformational change that breaks the strong bond of RanGTP –
Crm1. The hydrolysis is facilitated by RanGAP1, which itself is targeted to the Nup358 by
the Sumo E3 ligase (Jamali et al., 2011). RanGTP – karyopherin association is very stable
and requires RanBP1 to first penetrate the bond (Chumakov and Prasolov, 2010, Jamali et al.,
2011). The Crm1 affinity to NES weakens as RanGTP is hydrolysed into RanGDP, thus
allowing the release of cargo protein into the cytoplasm. Meanwhile, RanGDP is transported
back to the nucleus by NTF2, where it is converted into RanGTP by RanGEF (Chumakov
and Prasolov, 2010, Jamali et al., 2011).
1.3.5 Regulation of nucleocytoplasmic transport by phosphorylation
Phosphorylation is a common means of regulating the subcellular localisation of various
proteins in eukaryotic cells. It is a reversible posttranslational modification of the proteins
that consists of phosphorylation and dephosphorylation. Phosphorylation is the transfer of a
phosphate group (PO32-
) from the kinase-facilitated hydrolysis of adenosine tri-phosphate
15
(ATP) into adenosine di-phosphate (ADP), mainly to the amino acid residues serine (S),
threonine (T), or tyrosine (Y) (Alberts et al., 2008, Nelson and Cox, 2008).
Dephosphorylation is phosphatase-catalysed removal of the phosphate group from a
phosphorylated amino acid residue (Alberts et al., 2008). To phosphorylate or
dephosphorylate, kinases or phosphatases, specifically recognise common structural motifs.
Kinases that phosphorylate serine and threonine cluster in one subfamily, the most prominent
of which are casein kinase 2 (CK2) and cyclin-dependent kinase (cdk) cdc2 that recognise the
motifs –X-(S/T)-X-X-(E/D)-X- and –X-(S/T)-P-X-(K/R)-, respectively (Alberts et al., 2008,
Nelson and Cox, 2008). Whereas the tyrosine kinase subfamily clusters in another
evolutionarily-related group, the most prominent enzyme of which is Src (Alberts et al.,
2008).
Phosphorylation, particularly at an individual amino acid residue that plays a critical role for
the tertiary or quaternary structure of polypeptides, can dramatically change the overall
structure of a protein, hence altering its interaction with other proteins (Jans and Hübner,
1996, Harreman et al., 2004). As a result, phosphorylation is an important mechanism that
regulates the nucleocytoplasmic transport of viral proteins, as described in the following
models.
1.3.5.1 Phosphorylation promotes the interaction between NLS and importins
This model best describes the upregulating impact of phosphorylation in the vicinity of NLS,
as evidenced by the CKII-(cdk) cdc2-NLS (CcN) motif of simian virus 40 (SV40) T-antigen
(T-Ag), a tumour-inducing protein, and the CKII-CKII-NLS2 (C2N) motif of human
cytomegalovirus (HCMV) processivity factor of DNA polymerase, ppUL44 (Jans, 1995,
Alvisi et al., 2005).
The proposed mechanism of how phosphorylation in the vicinity of NLS regulates the
nucleocytoplasmic transport of viral proteins is based on the electrostatic interaction between
the positively charged basic residues of NLS and the acidic amino acid residues in the inner
side of Imp-α. Phosphorylation at a distance of approximately 5 – 25 amino acid residues
from NLS creates additional negative charges that are necessary for the interaction between
NLS and Imp-α, whereas phosphorylation inside the NLS will impair the interaction due to
electrostatic repulsion (Rihs et al., 1991, Hübner et al., 1997, Alvisi et al., 2008). The
enhanced interaction between NLS and importin eventually increases the rate of nuclear
16
import. CKII phosphorylation at S112
of SV40 T-Ag and at S413
of HCMV ppUL44 has been
shown to increase the binding of NLS to Imp-α/β complex and subsequently enhance the
nuclear accumulation of both proteins (Rihs et al., 1991, Hübner et al., 1997, Alvisi et al.,
2005).
1.3.5.2 Cytoplasmic or nuclear retention
In this model, the negative charge introduced by phosphorylation strengthens the interaction
affinity between a protein and a cytoplasmic retention factor. It has been studied at length in
the (cdk) cdc2 phosphorylation at T124
of SV40-Tag and PKC phosphorylation at T427
of
HCMV ppUL44, in which phosphorylation enhances the binding of the proteins to a
cytoplasmic retention factor named breast cancer antigen 1 (BRCA1) binding protein 2
(BRAP2) (Fulcher et al., 2010). As a result, proteins are retained in the cytoplasm and
nuclear import is inhibited.
Phosphorylation provides negative electric charge to regulate nuclear import. Substituting
kinases-recognisable residues (S/T) with negatively-charged residue, such as aspartic acid
(D), maintains the impact of phosphorylation in a protein’s function; while substitution with
neutrally-charged residue, such as glycine (G) or alanine (A), eliminates the phosphorylation
impact(s). S112
G substitution in the SV40 T-Ag and S413
G/A substitution in HCMV ppUL44
disables CK2 phosphorylation at these sites and reduces the rate of nuclear accumulation of
both proteins (Rihs et al., 1991, Alvisi et al., 2005). In terms of nuclear import inhibition by
phosphorylation, T124
A and T427
A of SV40 T-Ag and HCMV ppUL44 disables CK2 and
PKC phosphorylation, leading to reduced affinity of the proteins to BRAP2 and regular
nucleocytoplasmic transport rate (Fulcher et al., 2010). Conversely, T124
D substitution of
SV40 T-Ag and T427
D substitution of HCMV ppUL44, which were aimed to generate
phosphorylation mimicry, increases the affinity of both proteins to bind to BRAP2,
comparable with the impact of CK2 and PKC phosphorylation at T124
of SV40 T-Ag and at
T427
of HCMV ppUL44, respectively (Fulcher et al., 2010). Thus, kinase-facilitated
phosphorylation can enhance or inhibit the nucleocytoplasmic transport of a protein,
depending on the phosphorylation site, the altered structure of proteins, and the biological
pathways affected.
17
1.3.6 Regulated nucleocytoplasmic transport of viral proteins
1.3.6.1 Vesicular stomatitis virus (VSV) matrix (M) protein
VSV belongs to the genus Vesiculovirus of the family Rhabdoviridae, and is a (-)ssRNA
virus (Lyles and Rupprecht, 2007). It replicates in the cytoplasm and has its M protein
localised in the nucleus at a particular stage of infection through NLS-facilitated active
transport (Glodowski et al., 2002). The nuclear transport of VSV M protein is crucial for the
establishment of efficient infection, in that it allows VSV M to inhibit the nucleocytoplasmic
transport of molecules to and from the host nucleus, including the export of host mRNA (Her
et al., 1997, von Kobbe et al., 2000, Petersen et al., 2000). von Kobbe et al. (2000) showed
that VSV M protein strongly associates with Nup98 of the NPC, which may competitively
eliminate the ability of Ran-dependent nucleocytoplasmic transport components to use the
Nups in translocating molecules through the nuclear pore. VSV M accesses the Nup98
through an active nuclear import mechanism that is facilitated by a bipartite NLS at amino
acid 47 – 229; while VSV M association to Nup98, which leads to the inhibition of host gene
expression, is facilitated by methionine (M) at position 51 (Petersen et al., 2000, Glodowski
et al., 2002). This association prevents the nuclear export of many cellular molecules, leading
to the sequestering of host mRNA inside the nucleus and shut off of host gene expression,
including the suppression of IFN-β expression (Ahmed et al., 2003). IFN-β is a cytokine
produced to respond to viral infection and generates an ‘antiviral state’ in target cells to
restrict viral replication (Fontana et al., 2008). The failure to induce an ‘antiviral state’, as the
result of inhibition of IFN-β production, could eliminate the barrier for viral dissemination
throughout the organism. Therefore, the impact of the nucleocytoplasmic transport of VSV M
protein disrupts the host defence capacity to fight virus infection.
1.3.6.2 Dengue virus serotype 2 (DEN-2) NS5 protein
Dengue virus belongs to the genus Flavivirus of the family Flaviviridae, is a (+)ssRNA virus,
and, based on serology, is classified into dengue virus serotype 1, 2, 3, and 4 (DEN-1,-2,-3,-
4) (Henchal and Putnak, 1990). The NS5 protein is the viral RNA-dependent RNA
polymerase that performs its role in the cytoplasm in association with the viral helicase NS3.
In the much studied DEN-2, NS5 has been shown to possess two NLSs and one NES, namely
an Imp-α/β-recognised NLS (aNLS) within amino acids 369 – 389, an Imp-β1-recognised
NLS (bNLS) within amino acids 320 – 368, and a Crm1-recognised NES within amino acids
18
327 – 343, which enable it to shuttle between the nucleus and the cytoplasm (Pryor et al.,
2007, Rawlinson et al., 2009). The nuclear import is primarily facilitated by the aNLS;
whereas the bNLS overlaps with the NS3 binding site that poses a competitive binding to
Imp-β1 (Johansson et al., 2001, Pryor et al., 2007). The nucleocytoplasmic transport is
regulated by a cellular-kinase phosphorylation that affects at least four distinct serine residues
(Kapoor et al., 1995, Reed et al., 1998). Phosphorylation has been proposed to introduce
conformational changes to the cytoplasmic NS5 in order to release NS3 and unmask NLSs,
thus allowing the binding of importins; while the nuclear export may involve
dephosphorylation within the nucleus such that NES is exposed for Crm1 binding (Alvisi et
al., 2008).
The nuclear localisation of NS5 is associated with increased virus production and reduced IL-
8 synthesis, particularly during 48 – 96 hours post infection (Pryor et al., 2007, Rawlinson et
al., 2009). IL-8 is a chemotactic cytokine (chemokine) that recruits potent phagocytes, such
as neutrophils, to inflammatory sites (Mukaida et al., 1998). IL-8 function in trafficking
neutrophil migration to the infection sites for pathogen clearance; but if overexpressed, can
cause damage to the body tissues due to the cytotoxic effects of neutrophils’ granular proteins
to uninfected cells (Kindt et al., 2007), which in doing so, contributes to the development of
severe disease.
1.3.6.3 Rabies virus P protein
The neurotropic rabies virus (RABV) of the family Rhabdoviridae has a 12-kb genomic
ss(-)RNA genome that encodes nucleoprotein (N), phosphoprotein (P), matrix protein (M),
glycoprotein (G), and polymerase (L) (Lyles and Rupprecht, 2007). RABV P mRNA, in
addition to be translated into a full-length P protein (297 amino acids), is also translated into
four shorter proteins namely P2, P3, P4, and P5, due to a leaky-scanning mechanism at
internal M codons 20, 53, 69, and 83, respectively (Chenik et al., 1995). The RABV P can
localise in both nuclear and cytoplasmic compartments due to having a Crm1-dependent
amino (N)-terminal domain (NTD) NES (amino acids 49 – 58), carboxy (C)-terminal domain
(CTD) NLS (amino acids 172 – 297, active residues 211
KKYK214
and R260
), and a Crm1-
dependent protein kinase C (PKC)-activated CTD NES (amino acid 221 – 238, active
residues 227
FEQLKM232
) (Pasdeloup et al., 2005, Moseley et al., 2007). The cytoplasmic
compartmentalisation of these proteins interferes with signal transduction in the innate
19
immune response against RABV infection, by tampering with the activation of type I
interferon (IFN-α/β).
In RABV infection, the recognition of pathogen-associated molecular pattern (PAMP) viral
5’-triphosphate (ppp) ssRNA by the pattern-recognition receptor (PRR) retinoic-acid
inducible gene 1 (RIG-1) leads to a cascade of intracellular signalling that activates the type I
IFN (Faul et al., 2010). RABV P restrains the type I IFN activation in two ways. Firstly, an
internal domain of RABV P, (amino acids 176-186), masks the S386
phosphorylation of
interferon regulatory factor 3 (IRF3), which dimerisation and nuclear translocation are
required to trigger the transcription factor complex for early expression of IFN-β (Hiscott,
2007, Katze et al., 2008, Randall and Goodbourn, 2008, Takeuchi and Akira, 2008,
Mogensen, 2009). Secondly, the CTD of RABV P (amino acids 288 – 297) develops a strong
affinity to the DNA-binding domain of phosphorylated STAT1 (pSTAT1), an activator of the
transcription of interferon-stimulated genes (ISGs); thus, preventing the transcription of
products that facilitate the antiviral action of type I IFN (Vidy et al., 2005, Brzózka et al.,
2006, Vidy et al., 2007, Schnell et al., 2010). The cytoplasmic localisation of RABV P has
been shown to be regulated by the interplay between NES and NLS, and the recently
discovered PKC phosphorylation at amino acid residue S210
that activates the CTD NES
whilst silencing the NLS (Moseley et al., 2007).
1.4 The nucleocytoplasmic transport of the matrix (M) protein of RSV
1.4.1 Overview of the M protein of RSV
The RSV M protein (256 amino acids, 28.7 kDa) underlies the viral envelope, is associated
with viral nucleocapsid complex, consists mostly of basic amino acid residues, has an
increased hydrophobicity from NTD (residues 1 – 126) towards CTD (residues 140 – 255),
and has a strong tendency to self-assembly (Latiff et al., 2004, Kraus et al., 2005, Money et
al., 2009). RSV M is capable of binding to various structural viral components. RSV M
association to the G protein is influenced by the amino acid 2 (S) and 6 (D) in the NTD of
RSV G; while the binding to M2-1 protein requires the NTD of RSV M (amino acid residues
1 – 110) (Ghildyal et al., 2005b, Li et al., 2008). RSV M is also capable of independent
binding to the nuclear components via a zinc-finger domain (ZFD) contained in the residues
110 – 150; in which basic residues (K or R) at position 121, 130, 156, 157, and 170 have
been shown to be important for RNA-binding capacity (Rodriguez et al., 2004, Ghildyal et
20
al., 2005a). The interaction with various parts of the virus, added to the strong predisposition
to self-oligomerise (Kraus et al., 2005, Lamb and Parks, 2006), has made RSV M the driving
force/central organiser of the formation of new infectious particles. Further, this crucial
function has been linked to the trafficking of RSV M between the nucleus and the cytoplasm.
1.4.2 The nucleocytoplasmic transport of the M protein in infected cells
RSV M is an intracellular transport protein that is transported into the nucleus directly by
Imp-β1 and brought out of the nucleus by Crm1 (Ghildyal et al., 2005a, Ghildyal et al.,
2009). Imp-β1 recognises the bipartite NLS 154
TSKKVIIPTYLRSISVRNK172
; while Crm1
recognises a CTD NES 194
IIPYSGLLLVITV206
(Ghildyal et al., 2009). In addition, the
nuclear concentration of RSV M is also promoted by ZFD binding to nuclear component(s)
(Ghildyal et al., 2005a). The nuclear localisation of RSV M can be observed in the early
hours of infection while the cytoplasmic localisation takes place in the late stage of infection,
with the demarcating time point between the two processes is approximately 18 hours post
infection (Ghildyal et al., 2002). The nuclear export is up-regulated by a late stage CK2
phosphorylation (Alvisi et al., 2008).
1.4.2.1 Functions of the M protein in the nucleus
The nucleocytoplasmic transport of RSV M has been thought to associate with non-structural
roles of the protein since the whole replicative events, which require the structural functions
of RSV M, are cytoplasmic. One possibility is that RSV M might interfere with host genetic
machinery since its nuclear localisation has been shown to coincide with a decrease in host
transcriptional activity (Ghildyal et al., 2003). Although the precise mechanism is unclear, an
inhibition of host transcriptional activity could be facilitated by the capability of RSV M to
bind the nuclear component(s). Inhibition of host transcriptional activity might be directed to
enhanced infectious processes, as has been observed in the nuclear localisation of VSV M,
where inhibition of cellular transcription dampens the activation of the antiviral IFN-β
(Ahmed and Lyles, 1998, Ahmed et al., 2003). This suggests that RSV M nuclear localisation
could promote molecular events that lead to the generation of abundant progenies, as shown
by substitution of the positively-charged basic residues 156
KK157
of NLS into the neutrally-
charged 156
TT157
that inhibited RSV M nuclear localisation in the early stage of infection and
led to the reduced production of infectious particles (Ghildyal et al., 2009). RSV M nuclear
localisation might also facilitate efficient progeny production by delaying the
21
assembly/budding of new infectious particles until enough viral components have been
synthesised in the cytoplasm (Harrison et al., 2010). Further studies are required to elucidate
the role(s) of RSV M nuclear localisation.
1.4.2.2 Functions of the M protein in the cytoplasm and plasma membrane
RSV M cytoplasmic localisation is associated with its structural function as the driving
force/central organiser of the formation of viral progenies in the assembly/budding process.
Successful assembly/budding is important for the production of infectious particles. Virions
that cannot bud out of the infected cells are trapped inside and so the infection will not
spread. The involvement of matrix protein in the assembly/budding step can be described as
follow. Firstly, RSV M transports the nucleocapsid core from the cytoplasm to the assembly
/budding site, condenses it for incorporation to new particles and switches off viral
transcription in favour of infectious particles release; secondly, RSV M joins the
nucleocapsid complex with the glycoproteins complex arranged in the budding sites, while
developing its own hydrophobic-electrostatic interaction with the plasma membrane; and
lastly, RSV M builds infectious particles via self-aggregation followed by pinching-off of
new particles through the plasma membrane (Garoff et al., 1998, Chazal and Gerlier, 2003,
Jayakar et al., 2004, Takimoto and Portner, 2004). The matrix protein has also been shown to
mediate the cellular factors that regulate infectious particle release (Harty et al., 1999).
Considering the importance, a threshold level of functional matrix protein must be available
for the assembly/budding process as evidenced by matrix-deficient mutant strains of a
(-)ssRNA virus (rabies virus, Rhabdovirus, Rhabdoviridae, Mononegavirales) that produced
extremely low numbers of progenies to the point of near-complete defects of particle
formation (Mebatsion et al., 1999). As such, RSV M needs to be involved throughout the
assembly/budding process that takes place in the cytoplasm and plasma membrane in the late
stage of infection.
1.4.2.3 The importance of timely regulated nucleocytoplasmic transport of
RSV M
Given the transient nuclear or cytoplasmic localisation of RSV M relates to important
regulations of viral proliferation, RSV M must localise precisely and punctually in the
designated compartment in order to establish successful infection. Disruption of these
localisations has been shown to impair the production of infectious particles. Mutant RSV
22
with substitution in key basic residues of NLS (rRSV A2 M(156
KK157
156TT
157)) lost nuclear
accumulation in the early stage of infection (prior to 18 hours post infection) and underwent
reduced titre of viral proliferation in A549 cells, while mutant RSV with substitution in key
hydrophobic residues of NES (rRSV A2 M(200
LLLV203
200AAAA
203)) did not release
detectable infectious particles (Ghildyal et al., 2009). The study also found that RSV M with
200LLLV
203
200AAAA
203 did not localise in the assembly/budding sites at 24 hours post
infection in a transfection system using Vero cells. The study suggests that early stage
nuclear localisation promotes viral proliferation, although the precise mechanism is still
unknown, while positioning of RSV M in assembly/budding sites in late stage of infection is
important for production of infectious particles.
Phosphorylation is a main regulatory mechanism in the nucleocytoplasmic transport of a
protein (Jans and Hübner, 1996) The nuclear export of RSV M has been shown to be up-
regulated by CK2 phosphorylation in the late stage of infection (Alvisi et al., 2008). Potential
CK2 phosphorylation motifs have been identified along RSV M, although the precise
phosphorylation site is still under investigation (Ghildyal et al., 2006). A potential site, the
amino acid residue T205
that is positioned adjacent to the key hydrophobic residues of NES,
has been disproven as the designated site based on transfection experiments using the mutant
rRSV A2 M(T205A) (R. Ghildyal, personal communication). Further analysis revealed that
RSV mutant strain rRSV A2 M(T205A) had grown to a lower titre in cell lines expressing
IFN-α/β gene (HEp2 and A549 cell lines) compared to cell line deficient in expressing the
antiviral IFN-α/β response (Vero E6). The evidence has proposed a possibility that RSV M
might have an active role in affecting the regulation of the antiviral IFN-α/β expression
during the course of infection and, considering that RSV M is a transport protein, that the
regulation might associate with the nucleocytoplasmic transport of RSV M.
1.5 Regulation of cellular innate immunity against RSV infection
1.5.1 Overview
There are two strategies of mammalian immunity against viral infection, the innate and
adaptive immune responses. The innate immunity pre-exists prior to infection and is activated
within a short timeframe following infection, while the adaptive immunity is activated later
due to the complexity of its biological processes, but provides immunological memory that
results in more specific and faster protection on subsequent infection (Kindt et al., 2007).
23
The innate immunity is important for the first line of protection against viruses since it aims
to restrict viral spread and/or prevent disease development by inhibiting viral replication in
the early stage of infection. The immediate protection is resulted from the development of
‘antiviral state’ in infected cells that is subsequently relayed to the surrounding uninfected
cells by a signalling molecule known as cytokines, in a system known as the inflammatory
response (Arnold et al., 1994). Widely-recognised cytokines include interleukin (IL), tumour
necrosis factor (TNF), interferon (IFN), and chemotactic cytokine (chemokine) (Kindt et al.,
2007). An innate immune response generally conforms to the following cascade: recognition
of pathogen-associated molecular patterns (PAMPs) by pathogen recognition receptors
(PRRs), followed by the induction of signal transduction pathways involving transcription
factors, kinases, and adaptor molecules, which in turn regulate the expression of genes coding
for antiviral responses such as pro-inflammatory cytokines (Katze et al., 2008, Mogensen,
2009).
1.5.2 Innate immune responses against RSV infection
The hallmark of innate immunity against RSV infection in the epithelial cells of the
respiratory tract is the time- and dose-dependent secretions of two chemokines, interleukin-8
(IL-8) and regulated upon activation, normal T-cell expressed, and secreted (RANTES)
(Garofalo et al., 1996, Saito et al., 1997, Harrison et al., 1999, Domachowske et al., 2001).
Chemokines are chemotactic factors of leukocytes, with IL-8 is the chemoattractant of
neutrophils and RANTES is the chemoattractant of eosinophil (Olszewska-Pazdrak et al.,
1998, Garofalo and Haeberle, 2000). The phagocytic role and cytotoxic granule products of
leukocytes contribute to viral clearance by killing infected cells; however, their hyper-
activation, which is caused by excessive concentration of chemokines at inflammatory sites,
can affect uninfected cells and lead to tissue damage and subsequent exacerbation of illness,
which in the case of RSV includes mucus overproduction and airway epithelial cells necrosis
(bronchitis/bronchiolitis) (Sekido et al., 1993, Mukaida et al., 1998, Harrison et al., 1999,
Miller et al., 2003, Kindt et al., 2007). IL-8 and RANTES accumulation has been observed to
be higher in infants with severe RSV bronchiolitis than in those without bronchiolitis (Noah
et al., 2002, Smyth et al., 2002).
As the secretion level of IL-8 and RANTES dictates the subsequent inflammatory response
and, potentially, the eventual clinical symptoms; it is possible to use their expression as an
24
early predictor of the pathogenesis of RSV-associated disease and as a model of the immune
response and disease development against infection with an uncharacterised RSV strain. The
synthesis of IL-8 and RANTES is preceded by the activation of transcriptional factor NF-κB,
and is augmented by the presence of IFN-α/β (Mukaida et al., 1994, Garofalo et al., 1996,
Thomas et al., 1998, Genin et al., 2000, Tian et al., 2002, Spann et al., 2005). RANTES
expression is also induced by the activation of IRF3, which firstly leads to the production of
IFN-β (Bao et al., 2008).
NF-κB is natively cytoplasmic due to sequestration by the inhibitor of κB (IκB) that masks its
NLS and DNA binding domain (Bonizzi and Karin, 2004). IκB degradation by the inhibitor
of κB kinase (IKK) unmasks the NLS; therefore allowing Imp-α5 to translocate IκB into the
nucleus (Bose and Banerjee, 2003). The NF-κB binding site in RANTES gene is located in
the 5’-untranslated region, specifically at nucleotide -32 of the start codon of the first exon;
while the NF-κB binding site in IL-8 gene is situated at nucleotide -80 – (-)70 of the start
codon of exon 1 (Nelson et al., 1993, Mukaida et al., 1994).
1.5.3 Activation of NF-κB transcription factor
RSV is recognised in two ways: in the cytoplasm by the interferon (IFN)-inducible double-
stranded RNA (dsRNA)-activated serine-threonine protein kinase (PKR), and at the cellular
membrane, where RSV F protein is recognised by Toll-like receptor (TLR) 4 (Kurt-Jones et
al., 2000, Zamanian-Daryoush et al., 2000, Haynes et al., 2001, Bose and Banerjee, 2003,
Gern et al., 2003). PKR activation is replication-dependent; while TLR4 activation is
replication-independent (Fiedler et al., 1996, Olszewska-Pazdrak et al., 1998, Haeberle et al.,
2002, Miller et al., 2003). Chemokine expression is biphasic, where the weak early peak of
expression is the result of induction following viral entry, which is recognition of RSV F by
TLR4, and that the more prominent late production comes from the development of immune
response against RSV replication; which corresponds to the PKR activation (Miller et al.,
2003, Spann et al., 2005).
In PKR-activated pathway, dsRNA activates PKR by inducing dimerisation and
autophosphorylation, which in turn phosphorylates the IKK complex (Zamanian-Daryoush et
al., 2000). Activated IKK complex catalyses I-KB phosphorylation and degradation by 26S
proteasome to promote dissociation from the NF-κB subunits in order to expose the NLS and
DNA binding domain in NF-κB (Bose and Banerjee, 2003, Bonizzi and Karin, 2004). In
25
TLR4-activated pathway, the extracellular part of TLR4, leucine-rich repeat region (LRR),
initially binds to RSV F and the cytoplasmic part, Toll/interleukin-1 receptor homology (TIR)
domain, binds to an adaptor, which is the myeloid differentiation primary response gene 88
(MyD88) (Mogensen, 2009). MyD88 recruits IL-1 receptor-associated kinases (IRAKs) 4 and
1 by binding its death domain to IRAK4’s; following which the IRAK pair
autophosphorylates and IRAK1 recruits and activates TNF receptor-associated factor 6
(TRAF6) (Mogensen, 2009). Phosphorylated (p)IRAK1 dissociates the heterodimer IRAK-
MyD88 complex by forming a new complex consisting of IRAK1, TRAF6, TGF-β-activated
kinase 1 (TAK1), TAK1-binding protein 2 (TAB2) and TAK1-binding protein 3 (TAB3);
then the newly-formed complex translocates to the cytoplasm and TAK1 activates the IKK
complex (Takeda and Akira, 2004).
1.5.4 Activation of IRF3 transcription factor
IRF3 is activated following recognition of RSV 5’-triphosphate (ppp) ssRNA by retinoic-acid
inducible gene I (RIG-I) (Faul et al., 2010). The RIG-I DEx(D/H) box RNA helicase domain
binds to the RNA, while its N-terminal caspase recruitment domain (CARD) binds to the
CARD of an adaptor, IFN-β promoter stimulator 1 (IPS-1; also known as virus-induced
signalling adaptor (VISA) or mitochondrial antiviral signalling protein (MAVS) or Cardif)
(Randall and Goodbourn, 2008, Takeuchi and Akira, 2008, Mogensen, 2009). The IPS-I
mediates an association with TNF-receptor-associated factor 3 (TRAF3), which then recruits
TANK-binding kinase I (TBK-I) and inducible IκB kinase (IKKϵ) (Hiscott, 2007). The
kinases activate IRF3 at C-terminal region, driving its dimerization and nuclear translocation
to join a transcription factor complex consisting of NF-κB, p300/CBP, and ATF-2/c-Jun
heterodimer to promote the transcription of the early-expressed IFN-β (Hiscott, 2007, Katze
et al., 2008, Randall and Goodbourn, 2008, Takeuchi and Akira, 2008, Mogensen, 2009).
The release of early IFN-β signals for the production of IFN-α/β in neighbouring cells by
associating with their receptors, the interferon α receptor 1 and 2 (IFNAR1/2), to activate
receptor-associated JAK1 and Tyk2 tyrosine-kinases (Platanias, 2005, Randall and
Goodbourn, 2008). Tyk2 phosphorylates Y466
of IFNAR1 in order to create docking site(s)
for the incoming STAT2, as well as phosphorylates Y690
of STAT2 in preparation for
association with STAT1. Meanwhile, JAK1 phosphorylates Y701
of STAT1 (Platanias, 2005,
Randall and Goodbourn, 2008). The phosphorylated (p)STAT1 and pSTAT2 form a stable
26
heterodimer, in which the Imp-α5-dependent NLS in STAT1 is exposed and NES in STAT2
is inactivated (Platanias, 2005, Vidy et al., 2005, Randall and Goodbourn, 2008). The
pSTAT1/pSTAT2 complex detaches from the IFNAR1/IFNAR2 oligomer and binds to the
DNA binding protein p48 (IFN regulatory factor 9 (IRF9)) to form the IFN-stimulated
growth factor 3 (ISGF3) (Platanias, 2005, Randall and Goodbourn, 2008). The ISGF3 then
translocates to the nucleus and binds to IFN-stimulated response elements (ISREs) that is
located in the promoters of IFN-stimulated genes (ISGs); therefore activating the
transcription of pro-inflammatory mediators (Platanias, 2005, Randall and Goodbourn, 2008).
1.6 RSV vaccine
A licensed RSV vaccine is currently not available due to a major difficulty. The population
having the highest risk of developing severe LRTIs is infants younger than 3 months old, who
may not respond adequately to the vaccination due to suppression of immune response by
maternal antibody or immaturity of the immune system (Dudas and Karron, 1998). In
addition, it is important to vaccinate other age groups to control the transmission of RSV as
these act as the reservoir of infection, such that the introduction of infection to the main target
group can be limited (Haydon et al., 2002).
Other problems that slow the progression of RSV vaccine as described by Collins and
Murphy (2002) include the following. Firstly, the presence of maternally-derived antibody
presents hurdles in evaluating the safety and efficacy of the vaccine, since it is impossible to
find infants who are RSV-naïve. Secondly, RSV does not replicate efficiently in vitro and is
structurally frail, such that cautious handling is required to maintain its infectivity.
In addition to difficulties in handling RSV, significant obstacle to developing RSV vaccines
has been the experience with formalin-inactivated (FI) RSV, which elicits partial immunity,
as reviewed in Dudas and Karron (1998). FI-inactivated RSV induced exaggerated clinical
symptoms in infants who were RSV-naïve before vaccination since formalin inactivation
selectively altered the epitopes in the F and G surface glycoproteins. This alteration resulted
in inadequate levels of neutralising antibodies in serum and induced the production of IL-4,
IL-5, and IL-10, followed by the influx of lymphocytes and eosinophil, and the release of
additional mediators; the combination of which caused inflammation and airway constriction
that leads to bronchiolitis.
27
Further, an ideal RSV vaccine would be in the form of live-attenuated virus that is
administered via the intranasal route. Live-attenuated vaccine is expected to induce an
immune response that closely resembles the response to natural infection, without causing
severe disease. Intranasal administration can induce both local mucosal and systemic
immunity; therefore is expected to overcome the suppressive effects of maternal serum
antibodies (Collins and Murphy, 2005).
Technology used to achieve attenuation must serve the purpose that the best vaccine would
be highly immunogenic, such that it can be given to people from all age groups, but must not
cause disease following natural infection. Attenuated strains are generally less immunogenic
than clinical isolates, because of, in part, reduced replication and antigen production in vivo
(Collins and Murphy, 2005). Therefore, it is important to ensure that the attenuation
technology can fulfil the requirements of suitable RSV vaccine.
Conventional methods to attenuate RSV have been based on strain RSV A/Australia/A2/1961
via combination of extensive in vitro passaging in multiple temperatures and chemical
mutagenesis to result in cold passaged (cp) and temperature sensitive (ts) mutants (Dudas and
Karron, 1998). The mutants are expected to be restricted at human body temperature (37o C),
but replicate efficiently at suboptimal temperature. However, the best candidate has not been
discovered yet, with in vivo characterisation of these mutants revealing either under or over
attenuation (Karron et al., 1997b).
The progression in molecular biology techniques, known as reverse genetics, allows for a
method to generate recombinant RSV strain A2 (rRSV A2) containing attenuating mutations.
This technique allows for prediction of attenuation and fine-tunes the degree of attenuation at
the stage of designing the recombinant virus. Attenuation can be achieved by point mutation,
gene deletion, targeting a specific protein and replacing charged amino acids with a non-
charged amino acid, or changing the gene order (Collins and Murphy, 2002). Recombinant
RSV is generated from cDNA by expression in Vero cells, which are approved for vaccine
production according to the principle that the attenuating mutation should not reduce virus
yield in vitro since RSV does not replicate to a high titre and reduction of replication
efficiency in vitro could interfere with vaccine manufacture (Collins and Murphy, 2005).
28
1.7 Human metapneumovirus (hMPV)
The other human virus that is taxonomically closest to RSV is human metapneumovirus
(hMPV). Although hMPV was only recently identified in 2001 from a 20-year collection of
respiratory secretions obtained from children who suffered from respiratory tract infection,
seroprevalence analysis indicated that it has been circulating in human population at least
since 1958 (van-den-Hoogen et al., 2001). hMPV infects all age groups and commonly
causes ILI, although the prevalence and disease pathogenesis is heightened in the population
of infants/very young children (less than 2 years old), the elderly, and the
immunocompromised (van-den-Hoogen et al., 2001, Boivin et al., 2002, van-den-Hoogen et
al., 2004b, Williams et al., 2004, Falsey et al., 2006, Sarasini et al., 2006, Liao et al., 2012).
hMPV particles are thought to be spread by large respiratory secretions and fomites, are
stable at room temperature for 6 hours, and can resist 10 freeze-thaw cycles (Broor et al.,
2008, Papenburg and Boivin, 2010, Tollefson et al., 2010). The development of a hMPV
vaccine is complicated by the fact that the population most at risk of developing severe
clinical manifestations is the least able to develop a sufficient immune response.
1.7.1 Epidemiology
Globally, hMPV circulates all year round, but in the temperate regions it has a seasonal peak
that tends to be approximately 2 months later than that of RSV, occurring mostly during late
winter to early spring (Falsey et al., 2003, Esper et al., 2004, Gray et al., 2006, Kahn, 2006,
Sloots et al., 2006, Feuillet et al., 2012). RSV and hMPV co-infection, which is enabled by
the overlapping temporal distribution, has been attributed as the risk factor in the
development of severe RSV-associated bronchiolitis (Greensill et al., 2003, Semple et al.,
2005). As a single aetiology, hMPV is the second most common cause of bronchiolitis-
associated hospitalisation in infants and young children after RSV (Kahn, 2006, Gaunt et al.,
2009). Global annual epidemic of hMPV may be caused by many different strains in separate
regions; which differs from influenza virus, where the yearly epidemic is caused by 2 – 3
strains that circulate simultaneously worldwide (Esper et al., 2004, Williams et al., 2004,
Kahn, 2006). Re-infection is common, very possibly reflecting short-term immunity due to
the high genetic variability of the hMPV and the rapid evolution of the circulating genotypes
that overcome pre-existing immunity in the population (Williams et al., 2004).
29
1.7.2 hMPV infection
The main replication site of hMPV is the ciliated epithelial cells of the respiratory tract,
which span from the nasal cavity to bronchioles; however hMPV also replicates in the type 1
pneumocytes (Kuiken et al., 2004). Viral replication eliminates ciliation, causes the
sloughing-off of necrotic epithelial cells, recruits neutrophils, and induces the rare formation
of syncytia (Kuiken et al., 2004). The clinical symptoms of infection range from mild upper
respiratory tract illnesses, which encompass fever, cough, rhinorrhoea (runny nose), and
rhinitis (nasal inflammation), to the more severe lower respiratory tract illnesses including
bronchitis and pneumonia (van-den-Hoogen et al., 2001, Esper et al., 2004, Williams et al.,
2004). Additional symptoms include wheezing, myalgia (muscle pain), diarrhoea, and
vomiting (van-den-Hoogen et al., 2001, Crowe-Jr. and Williams, 2011). Overall, the
characteristics of hMPV infection are quite indistinguishable from RSV infection; although
hMPV infections are likely to be milder among immunocompetent adults, less associated
with pneumonia, inclined to be associated with hospitalisation in an older age group (median
age 6 – 12 months (hMPV) vs 6 months (RSV)), and more associated with exacerbation of
asthma (Boivin et al., 2002, Falsey et al., 2003, Williams et al., 2004, Papenburg and Boivin,
2010, Li et al., 2012). The severe hMPV-associated illness in infants/children less than 2-year
old and in the elderly, very likely is the consequence of deficient immune responses (van-
den-Hoogen et al., 2004b, Liao et al., 2012). In addition, the degree of disease severity has
been associated with nasopharyngeal viral load, with a higher viral titre is linked to the
development of LRTIs and results in hospitalisation (Bosis et al., 2008).
In cell culture, hMPV replicates more slowly than RSV, undergoes a delayed CPE
development, is trypsin-dependent, and tends to grow to a lower peak titre (van-den-Hoogen
et al., 2001, Tollefson et al., 2010, Crowe-Jr. and Williams, 2011). These characteristics are
often claimed as the cause of the recent discovery of hMPV despite its long-term circulation
in the population. CPEs are generally observed at days 10 –14 post infection, and include the
formation of syncytia, followed by rapid internal disruption of the cells and subsequent
detachment of the cells from the surface of culture plate (van-den-Hoogen et al., 2001, Boivin
et al., 2002, van-den-Hoogen et al., 2004b, Williams et al., 2004). hMPV can be propagated
in epithelial cells, such as Vero, Vero E6, A549, and LLC-MK2 cell lines; the most
permissive cell lines being the trypsin-resistant LLC-MK2 of Macaca mulatta kidney and
30
BEAS-2B of human bronchus (van-den-Hoogen et al., 2001, Tollefson et al., 2010). Syncytia
formation is cell-type dependent (Deffrasnes et al., 2005).
1.7.3 Taxonomy
Based on the relatedness of nucleotide sequences, hMPV is classified into the family
Paramyxoviridae, subfamily Pneumovirinae, and genus Metapneumovirus (van-den-Hoogen
et al., 2001, van-den-Hoogen et al., 2002). Based on the variability in structural genes,
including N, P, L, F, and G, hMPV is consistently classified into two genetic lineages: the
genotypes A and B, with subgenotypes A1, A2, B1, and B2 inside each genotype (Peret et al.,
2002, van-den-Hoogen et al., 2004a). hMPV is the taxonomically closest human pathogen to
RSV; the other member of the Metapneumovirus genus is an animal virus, the avian
metapneumovirus (aMPV) previously known as turkey rhinotracheitis virus (TRTV) or avian
pneumovirus (APV).
1.7.4 The structure of an hMPV particle (the virion)
The morphology of hMPV resembles RSV, i.e pleomorphic, spherical, or filamentous, in
which a nucleocapsid structure is contained within a space that is sheathed by an envelope
containing projections of glycoproteins (Peret et al., 2002).
The hMPV genome is a nonsegmented (-)ssRNA of 13.3 kb, is equipped with noncoding 3’-
leader and 5’-trailer sequences, contains 8 genes namely 3’-N-P-M-F-M2-SH-G-L-5’ that
code for 9 proteins, which in order of the gene sequence are the nucleoprotein (N),
phosphoprotein (P), matrix (M) protein, fusion (F) protein, transcription elongation factor
(M2-1), RNA synthesis regulatory factor (M2-2), small hydrophobic surface protein (SH),
major attachment glycoprotein (G), and large polymerase subunit (L) (van-den-Hoogen et al.,
2002). Each gene is flanked by a well conserved GS and a less conserved GE sequences
(Bastien et al., 2003).
In hMPV-infected cells, the N, P, and L proteins constitute a structure central for genome
replication known as ribonucleoprotein (RNP) complex, which appears as the inclusion
bodies in the cytoplasm, with the N and P protein association being the core of the inclusion
(Collins and Crowe Jr., 2007, Derdowski et al., 2008). Unlike in RSV, hMPV M2-1 is not
included in the RNP complex, does not bind to N, is not required for in vitro growth–
although is required for the optimum in vivo replication, and does not function as the
31
antitermination of transcriptional read-through (Herfst et al., 2004, Buchholz et al., 2005,
Derdowski et al., 2008).
Consistent with the general structure of Paramyxoviruses, the replication complex is sheathed
by a lipid bilayer envelope that is derived from the plasma membrane of the host cell, and
contains the F, G, and SH glycoproteins (Lamb and Parks, 2006). The homotrimeric-arranged
F protein is the hMPV major neutralisation antigen, is first synthesised as an F0 precursor that
is subsequently cleaved into the F1 and F2 subunits, functions in the viral attachment and
fusion, determines cell tropism, and induces syncytia formation (Crowe-Jr. and Williams,
2011). hMPV F cleavage site is unique, consisting of a R-Q-X-R motif compared to the
common R/K-X-R/K-R furin cleavage site found in the Paramyxovirus F, and is possibly not
cleaved by furin protease such that trypsin supplementation is required for in vitro growth
(van-den-Hoogen et al., 2002, Bastien et al., 2003, Collins and Crowe Jr., 2007). The hMPV
G and SH are not required for in vitro growth; but, the hMPV G is involved in the activation
of NF-κB and IRF3 in vitro (Biacchesi et al., 2004, Bao et al., 2008).
The hMPV M protein and its biology has so far been deduced from the biology of RSV M,
such as being located between the RNP complex and the envelope glycoproteins, binding to
the cytoplasmic tail of the envelope glycoproteins, and having a significant role in virion
assembly by binding to the nucleocapsid complex and ceasing RNA synthesis (Collins and
Crowe Jr., 2007, Crowe-Jr. and Williams, 2011). However, hMPV M has been hypothesised
to induce the secretion of proinflammatory mediators in vitro by promoting the maturation of
dendritic cells (Bagnaud-Baule et al., 2011).
There is not much known about hMPV M2 gene products, but they are predicted to control
RNA synthesis based on the roles of M2-1 and M2-2 proteins of RSV, where RSV M2-1
promotes the read-through of intergenic junction during transcription and M2-2 acts as the
controlling switch between RNA replication and transcription (Hardy and Wertz, 1998,
Bermingham and Collins, 1999). hMPV M2-2 has been shown to upregulate RNA
transcription, and together with M2-1, is not important for in vitro growth although is
required for optimal in vivo replication (Buchholz et al., 2005).
32
1.7.5 The life cycle of hMPV
The replicative cycle of hMPV has not been studied in detail, but is very likely to be similar
to that of RSV (Crowe-Jr. and Williams, 2011). Replication processes take place in the
cytoplasm and encompass the adsorption/entry, RNA synthesis, and assembly/budding
processes. Notable differences are that the kinetics of hMPV infection are slower compared
to other Paramyxoviruses: the peak of hMPV intracellular protein expression is 48 – 72 hours
post infection while Paramyxoviruses commonly complete a growth cycle in 14 – 30 hours
(Lamb and Parks, 2006). Compared to RSV, the CPE is less prominent and the yield of
infectious particles is approximately 5 – 10 fold lower (Collins and Crowe Jr., 2007).
1.7.6 The role of N protein in virus replication
The N protein of nonsegmented (-)ssRNA viruses associates directly with the genomic/(-) or
antigenomic/(+) strands of RNA in infected cells as well as within virus particles
(Ball, 2007). The known functions of N-RNA interaction include: physical protection for the
RNA, regulation of template fidelity, formation of viral replication complex, and RNA
packaging in preparation of virion assembly/budding and maturation.
1.7.6.1 RNA protection
The N protein protects RNA against ribonuclease degradation and high concentration of salts,
as well as maintains the rigidity of RNA structure following cycles of
folding/dissociation/refolding during replication cycle (Lynch and Kolakofsky, 1978, Mir and
Panganiban, 2006, Raymond et al., 2010). The N subunits self-associate, are aligned in a
continuous tunnel, and cover the full length of viral RNA, such that the closed conformation
prevents accesses to RNA except for a very short time when the conformation is altered to
allow copying by RNA polymerase (Green et al., 2011).
1.7.6.2 Regulation of template fidelity
The N protein functions in regulating the fidelity of viral genome by maintaining the ratio of
association between N subunits and ribonucleotides, which serves as a screening mechanism
to eliminate pseudo genomes containing non-templated insertions from the genome pool
(Barr and Fearns, 2010). Little is known about the N:ribonucleotide ratio in hMPV; known
stoichiometry include one N molecule binds to nine ribonucleotides (1:9) in Rhabdoviridae,
33
1:12 in Bunyaviridae, and 1:6 in Paramyxoviridae (Calain and Roux, 1993, Mohl and Barr,
2009). In Paramyxoviruses, pseudo genomes that arise from the copying errors of RNA
polymerase and do not follow the N:ribonucleotides stoichiometry, decrease RNA synthesis
by approximately 50 – 100 fold (Kolakofsky et al., 1998).
1.7.6.3 Formation of replication complex/ ribonucleoprotein (RNP) complex
The N protein is the driving force for the formation of RNP complex due to the propensity to
self-associate and because N possesses binding sites for other RNP components, including
RNA, P, and L (Becker et al., 1998, Coronel et al., 2001, DiCarlo et al., 2007, Raymond et
al., 2010, Green et al., 2011). N has an inherent ability to interact with P and this interaction
is particularly important because N-RNA association is not specific, while N-P conformation
confers specificity for encapsidation such that N-P associated RNA is the form that can be
recognised by RNA polymerase (Baric et al., 1988, Howard and Wertz, 1989, Myers et al.,
1999, Yoo et al., 2003, Nayak et al., 2009). In hMPV, the N-P interaction is also required to
prevent N condensation due to self-oligomerisation (Derdowski et al., 2008). This interaction
has been shown to result in the very early formation of cytoplasmic inclusion bodies in
hMPV and RSV, once the first molecules of N and P proteins have been translated (García-
Barreno et al., 1996, Derdowski et al., 2008).
1.7.6.4 RNA packaging in assembly, budding, and particle release
The packaging signal in N activate the packaging signal in RNA by masking the polymerase
recognition site that overlaps with RNA packaging signal during the late stage of infection,
i.e once enough N protein has accumulated (Fosmire et al., 1992, Rein et al., 1994).
Following genome packaging, N binds to M in order to stabilise the replication complex and
facilitates interaction with plasma membrane for the release of new particles (Stricker et al.,
1994, Coronel et al., 2001, Dancho et al., 2009). M-N interaction is specific, that is, M only
binds to N that encapsidates full-length RNA; thus ensuring the released progeny virions are
infectious and have a fully-functioning genome (Narayanan et al., 2000).
hMPV N is coded by a 1185-bp gene and contains 395 amino acids with a molecular mass of
43.5 kDa (Bastien et al., 2003, Petraityte-Burneikiene et al., 2011). hMPV N has important
function for the success of viral proliferation as shown by transcriptional silencing assay
34
using siRNA, where the absence of hMPV N decreases viral load in vitro and in vivo (Darniot
et al., 2012).
1.7.7 The nucleocytoplasmic transport of nucleocapsid proteins
Viruses with (-)ssRNA genome naturally replicate in the cytoplasm; therefore there is a need
for the N protein to localise in the cytoplasm as it forms the central part of viral replication
complex. However, the N protein of several cytoplasmic-replicating viruses has been shown
to localise in the nucleus or nucleolus of infected cells during certain stages of infection. The
N nuclear/nucleolar localisation usually takes place early in the infectious cycle as soon as it
is translated, possibly to play non-structural functions, and then returns in the cytoplasm in
the late stage of infection to participate in assembly (Tijms et al., 2002, Yoo et al., 2003).
1.7.7.1 Examples of viruses in which N protein undergoes nucleocytoplasmic
transport
The N protein of equine arteritis virus (EAV), porcine reproductive and respiratory
syndromes virus (PRRSV), severe acute respiratory syndrome coronavirus (SARS Co-V),
infectious bronchitis virus (IBV), Borna disease virus (BDV), and some Paramyxoviruses:
canine distemper virus (CDV), rinderpest virus (RPV), and measles virus (MV) has been
shown to localise in the nucleus or nucleolus during infection.
The EAV N nuclear import has not been clearly understood, but the nuclear export is
facilitated by Crm1 (Tijms et al., 2002). The PRRSV N possesses NLS, NoLS, and NES
motifs that facilitate independent nucleocytoplasmic transport and accumulation in the
nucleoli of infected cells, where it binds to the 18S and 28S rRNAs and associates to
fibrillarin, an RNA-associated nucleolar protein that plays a role in assembly of ribosomes
and regulation of cell cycle (Rowland et al., 1999, Yoo et al., 2003). The nuclear/nucleolar
localisation does not affect the capability to produce infectious particles, but attenuates the
amount of the released particles and in vivo pathogenesis (Lee et al., 2006). The IBV N also
interacts with, and even reorganises fibrillarin distribution, where the interaction and/or
redistribution are associated with delay in cell growth (Hiscox et al., 2001, Chen et al., 2002).
There are three putative NLSs and a Crm1-independent NES that have been identified in
SARS Co-V N (You et al., 2005). The NES is the predominant signal and SARS Co-V N
cytoplasmic localisation has been shown to be facilitated by 14-3-3 (tyrosine 3-
35
monooxygenase/tryptophan 5-monooxygenase activation protein) that recognised
phosphorylated serine residues within RSXS*XP motif (Macara, 2001, Surjit et al., 2005).
The genome of BDV is replicated in the nucleus and the N protein is equipped with NLS and
Crm1-associated NES, such that N can facilitate the nuclear import of RNP complex for
replication and nuclear export to the cytoplasm for virion assembly (Kobayashi et al., 2001).
The N proteins of CDV and RPV possess conserved NLS and NES motifs, where the NES,
although a leucine-rich motif, is Crm-1 independent (Sato et al., 2006). MV is a virus whose
inclusion bodies were produced in the nucleus of infected cells. More importantly, the
nuclear translocation of MV N protein also affects the host immune response, which is done
by inhibiting the nuclear translocation of STATs without interfering the nuclear transport
factor of STATs, Imp-α5; therefore causing disruption of the IFN-α/β and IFN-γ signalling
pathways (Takayama et al., 2012).
1.7.7.2 The possible functions of nuclear and/or nucleolar localisation of N
The nuclear/nucleolar translocation of the N protein of viruses that replicate in the cytoplasm
is mostly associated with optimisation of replication. For example IBV N redistributes
fibrillarin and delays cytokinesis to divert biosynthetic resources from the dividing nucleus to
the cytoplasm, where viral replication takes place (Chen et al., 2002).
Other possibilities include ‘accidental’ translocation schemes, which include the molecular
mimicry of nuclear or nucleolar localisation signal (NLS/NoLS), the association with
ribosomal proteins, and passive diffusion. In molecular mimicry, the K/R-rich RNA binding
domains may resemble NLS/NoLS motifs, allowing the N protein to be transported into the
nucleus by a transport factor (Rowland and Yoo, 2003). Association with ribosomal proteins
in cytoplasm might result in N being coincidentally translocated into the nucleolus, where the
ribosomal proteins associate with rRNA to form ribosomal subunits (Tijms et al., 2002). The
NPC allows for passive diffusion for proteins that size less than 50 kDa or 5 nm (Wurm et al.,
2001). The accidental localisation might serve a purpose by maintaining the balance of
N:ribonucleotides stoichiometry in the cytoplasm in order to produce functional genomes,
since N can be overproduced and transient nuclear/nucleolar localisation can be a means to
keep the appropriate concentration of N molecules in the cytoplasm (Rowland and Yoo,
2003).
36
1.8 Hypotheses and aims
The need for a safe and immunogenic live-attenuated RSV vaccine has led to the use of
reverse genetics to facilitate efficient vaccine production. Reverse genetics allows for
achieving attenuation from altering the genomic composition of RSV, which is done by
introducing mutation in the viral genome, followed by rescuing a recombinant virus in Vero
cells transfected with cDNAs of the mutated genome. The mutation targets in the genome are
regions, or sites, that control important functions for RSV replication.
The nucleocytoplasmic transport of structural proteins facilitates the replication processes in
many viruses; making the nucleocytoplasmic transport of viral proteins a viable target for
attenuation. In RSV M, timely and appropriate nucleocytoplasmic transport of the M protein
has been shown to be important for determining the success of replication. Disruption of the
nuclear import leads to decreased particle production and disruption of the nuclear export
ceases the infection process. As a result, the RSV M nucleocytoplasmic transport can be
targeted for attenuation. One mechanism is to alter the components of nucleocytoplasmic
transport in the structure of RSV M, such as the NLS, NES, or regulatory phosphorylation
sites.
The success of RSV M nucleocytoplasmic transport is determined by the functionality of the
NLS, NES, and the regulatory sites. A potential CK2 phosphorylation site was identified
adjacent to the NES, M205(T). Since RSV M nucleocytoplasmic transport is regulated by a
phosphorylation mechanism (Alvisi et al., 2008), the role of RSV M205 in virus replication
can be assessed by mutating the positively charged threonine (T) residue into the neutrally
charged alanine (A)
The same approach can also be applied to attenuate other viruses where nucleocytoplasmic
transport of a structural protein serves a function in replication. Therefore, this study attempts
to assess the potential of attenuating hMPV, a virus closely related to RSV, by interfering
with the nucleocytoplasmic transport of the N protein. Previous work in the laboratory has
shown that hMPV N protein can localise in the nucleus in the very late stage of infection (day
5 post infection) (Ghildyal, R & Gahan, M; unpublished); therefore, it is important to gather
evidence in order to facilitate further study.
37
The hypotheses of this study are:
1. Mutation in a CK2 phosphorylation motif adjacent to the NES reduces the production
of infectious progeny and dampens the activation of pro-inflammatory responses.
2. The hMPV N protein has inherent nuclear transport capability.
The aims of this study are:
1. To determine the impact of mutation at a CK2 phosphorylation motif adjacent to the
NES on virus replication and on the activation of pro-inflammatory responses in cell
culture.
2. To examine the nuclear localisation of hMPV N in transfected cells.
38
39
CHAPTER 2
MATERIALS AND METHODS
2 Materials and Methods
40
41
2.1 Materials
2.1.1 Cell lines
Table 2-1 Cell lines used in this study
2.1.2 Viruses
Table 2-2 Viruses used in this study Virus Strain Description Reference
Respiratory syncytial
virus
rRSV A2 Recombinant RSV strain A2 (Collins et al.,
1995)
rRSV A2 M(T205A) Recombinant RSV strain A2 with TA
substitution at M 205*
(Ghildyal et al.,
2009)
Human
metapneumovirus
hMPV CAN97-83 Clinical isolate from a patient with
respiratory diseases in Quebec, Canada
GenBank accession number AY297749
(Peret et al., 2002)
*NES-associated-CK2-phosphorylation-motif of the M protein
2.1.3 Plasmids and bacterial cultures
Table 2-3 Plasmid/bacteria used in this study
Plasmid/bacteria Source Description pDONR®207 [Invitrogen, Carlsbad, USA] Entry vector
pEPI-DESTC [R. Ghildyal, University of Canberra, Canberra, Australia]
(Ghildyal et al., 2005a)
Expression vector
E. coli ElectroMAXTM
DH10BTM
[Invitrogen, Carlsbad, USA] Competent cells
Cell line Source ATCC®
Number
Description
Vero E6 Epithelial cells from the kidney of
Cercopithecus aethiops (African green
monkey)
CRL-1586TM Lack functional type I interferon
response
A549 Carcinoma epithelial cells from human lungs CCL-185TM Active type I interferon
signalling pathway
Cos-7 Cells from the kidney of Cercopithecus
aethiops (African green monkey) that are transformed with SV40 T-antigen
CRL-1651TM Transfection host cell line
42
2.1.4 Primers
Table 2-4 Primers used in this study Use Primer name Template Sequence (5’ 3’) Reference Supplier
Sequencing pEPIseqFWD pEPI-DESTC GCC CCG TGC TGC TGC CCG (Ghildyal et
al., 2005a)
[GeneWorks,
Hindmarsh, Australia]
pEPIseqREV pEPI-DESTC GTG AAA AGA AAG TAT ACC
DONRFWD pDONR®207. TCG CGT TAA CGC TAG CAT GGA TCT C
DONRRev pDONR®207 GTA ACA TCA GAG CTT TTG AGA CAC
HMPV83Nf_inner hMPV.N TGA TGC ACT CAA AAG ATA CCC This study
HMPV83Nr_inner hMPV.N GTT TGA CCA GCA CCA TAA GC
PCR/cloning attB1RSVM1 RSV M *GGG GAC AAG TTT GTA CAA AAA AGC
AGG CTT GGA AAC ATA CGT GAA CAA GC
(Ghildyal et
al., 2005a)
attB2RSVM256 RSV M *GGG GAC CAC TTT GTA CAA GAA AGC
TGG GTT AAT CTT CCA TGG GTT TGA TTG C
HMPV83NattB1 hMPV N *GGG GAC AAG TTT GTA CAA AAA AGC
AGG CTC ATC TCT TCA AGG GAT TCA CCT
GAG
This study
HMPV83NattB2 hMPV N *GGG GAC CAC TTT GTA CAA GAA AGC
TGG GTC TTA CTC ATA ATC ATT TTG ACT
GTC GTC AC
hMPVN_f_attB1 hMPV N *GGG GAC AAG TTT GTA CAA AAA AGC AGG CTT GAT GCA CTC AAA AGA TAC CC
hMPVN_r_attB2 hMPV N *GGG GAC CAC TTT GTA CAA GAA AGC TGG GTG TTT GAC CAG CAC CAT AAG C
hMPVN_f-
rev_attB2
hMPV N *GGG GAC CAC TTT GTA CAA GAA AGC
TGG GTG GGT ATC TTT TGA GTG CAT CA
Mycoplasma
test
Myco F1 Mycoplasma sp. ACA CCA TGG GAG CTG GTA AT
Myco R1 Mycoplasma sp GTT CAT CGA CTT TCA GAC CCA AGG CAT
Myco F2 Mycoplasma sp GTT CTT TGA AAA CTG AAT
Myco R2 Mycoplasma sp GCA TCC ACC AAA AAC TCT
*bold characters = attB sequences
43
2.1.5 Enzymes/buffers for molecular biology assays
Table 2-5 Enzymes used in this study
Enzymes Manufacturer Mango TaqTM DNA polymerase [Bioline, Alexandria, Australia]
Gateway® BP ClonaseTM II Enzyme Mix [Invitrogen, Carlsbad, USA]
Gateway® LR ClonaseTM II Enzyme Mix [Invitrogen, Carlsbad, USA]
2.1.6 Antibodies
Table 2-6 Antibodies used in this study
Antibodies Target Manufacturer Use Mouse anti-RSV fusion protein
monoclonal antibody
RSV F [Millipore, Billerica,
USA]
RSV titration
Ms X Hu Metapneumovirus hMPV nuclear
protein
[Millipore, Billerica,
USA]
hMPV titration
Goat anti-mouse IgG-alkaline
phosphatase
Mouse IgG [Sigma-Aldrich, St.
Louis, USA]
RSV and hMPV
titration
2.1.7 Kits
Table 2-7 Kits used in this study
Kits Manufacturer Vector® Black Alkaline Phosphatase Substrate Kit II [Vector Laboratories Inc., Burlingame, USA]
cDNA Synthesis Kit [Bioline, Alexandria, Australia]
Wizard® SV Gel and PCR Clean-Up System [Promega, Madison, USA]
Human RANTES ELISA Development Kit [Peprotech, Rocky Hill, USA]
Human IL-8 ELISA Development Kit [Peprotech, Rocky Hill, USA]
QIAprep® Spin Miniprep Kit [Qiagen, Hilden, Germany]
QIAGEN Plasmid Midi Kit [Qiagen, Hilden, Germany]
44
2.1.8 Buffers, media, and solutions
Table 2-8 Culture media used in this study
Media Composition
Cell culture Methyl cellulose 2% methyl cellulose [Sigma-Aldrich, St. Louis, USA]; 15% HBSS [Sigma-
Aldrich, St. Louis, USA]; 2% FBS [Bovogen, Melbourne, Australia]; 80.9%
Dulbeccos’s Modified Eagle Medium (DMEM) [Sigma-Aldrich, St. Louis,
USA]; 0.1% 1000X antibiotic mix: penicillin+streptomycin+neomycin (PSN)
Vero E6 growth medium (RSV) DMEM [Sigma-Aldrich, St. Louis, USA] + 5% FBS [Bovogen, Melbourne, Australia]
Vero E6 growth medium (hMPV) OPTI-MEM® Reduced Serum Medium (1X) [GibcoTM, Invitrogen Corp.,
Carlsbad, USA] + 5% FBS [Bovogen, Melbourne, Australia]
A549 growth medium F12K Nutrient Mixture Kaighn’s Modification (1X) [Invitrogen, Carlsbad,
USA] + 10% FBS [Bovogen, Melbourne, Australia]
Cos-7 growth medium DMEM [Sigma-Aldrich, St. Louis, USA] + 5% FBS [Bovogen, Melbourne,
Australia] + 1X PSN
Bacterial culture Luria Bertani (LB) 10 g/l tryptone [Sigma-Aldrich, St. Louis, USA]; 5 g/l yeast extract [Oxoid,
Basingstoke, England]; 10 g/l NaCl [Univar APS, Seven Hills, Australia]
LB agar LB with 2% bacterial agar [Oxoid, Basingstoke, England]
SOB -Mg medium 20 g/l tryptone [Sigma-Aldrich, St. Louis, USA]; 5 g/l yeast extract [Oxoid,
Basingstoke, England]; 10 mM NaCl [Univar APS, Seven Hills, Australia];
2.5 mM KCl [Univar APS, Seven Hills, Australia]
LB + gentamycin LB + 7 μg/ml Gentamycin [Sigma-Aldrich, St. Louis, USA]
LB + kanamycin LB + 100 μg/ml Kanamycin [Sigma-Aldrich, St. Louis, USA]
LB agar + gentamycin LB agar + 7 μg/ml Gentamycin [Sigma-Aldrich, St. Louis, USA]
LB agar + kanamycin LB agar + 100 μg/ml Kanamycin [Sigma-Aldrich, St. Louis, USA]
Table 2-9 List of commercial chemicals used in this study
Buffers/solutions Manufacturer Foetal bovine serum (FBS) [Bovogen, Melbourne, Australia]
Hank’s Balanced Salt Solution (HBSS) [Sigma-Aldrich, St. Louis, USA]
Trizol® reagent [Invitrogen, Carlsbad, USA]
(5X) DNA loading dye, blue [Bioline, Alexandria, Australia]
Agarose [Amresco, Solon, USA]
GelRedTM fluorescent nucleic acid gel stain, 10,000X in H2O [Biotium, Hayward, USA]
LipofectamineTM 2000 Transfection Reagent [Invitrogen, Carlsbad, USA]
2, 2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)
(ABTS liquid substrate)
[Sigma-Aldrich, St. Louis, USA]
RNAse-free water [Qiagen, Hilden, Germany]
HyperLadderTM I DNA ladder [Bioline, Alexandria, USA]
HyperPage Protein ladder [Bioline, Alexandria, USA]
Gentamycin [Sigma-Aldrich, St. Louis, USA]
Kanamycin [Sigma-Aldrich, St. Louis, USA]
Bovine serum albumin (BSA) [Sigma-Aldrich, St. Louis, USA]
Dimethyl sulfoxide [Sigma-Aldrich, St. Louis, USA]
Glycerol [Unilab APS, Seven Hills, Australia]
45
Table 2-10 List of in-house prepared buffers and solution
Buffers/solutions Composition 10X phosphate-buffered saline (PBS) 1.36 M NaCl [Univar APS, Seven Hills, Australia]; 101.44
mM Na2HPO4 [ChemSupply, Gilman, Australia]; 26.83 mM
KCl [Univar APS, Seven Hills, Australia]; 16.90 mM
KH2PO4; pH 7.5
Trypsin/EDTA 2.5% Trypsin [Sigma-Aldrich, St. Louis, USA]; 26.5 mM
EDTA [Unilab APS, Seven Hills, Australia]
Trypan blue 0.16% trypan blue [Sigma-Aldrich, St. Louis, USA]; 0.85%
NaCl [Univar APS, Seven Hills, Australia]; distilled H2O
1000X PSN 30.072 g/l Penicillin G [ICN-MP Biomedicals, Solon, USA];
50 g/l Streptomycin sulphate [Sigma-Aldrich, St. Louis, USA];
50 g/l Neomycin sulphate [Sigma-Aldrich, St. Louis, USA]
50X Tris-acetic acid ethylenediamine tetraacetic acid (TAE)
2 M Tris [Amresco, Solon, USA], 1 M acetic acid [Fronine, Taren Point, Australia], 0.05 M EDTA [Unilab APS, Seven
Hills, Australia]
Developing solution for Vector® Black
Alkaline Phosphatase Substrate Kit II
0.1 M Tris-HCl [Amresco, Solon, USA]; 0.1 M NaCl [Univar
APS, Seven Hills, Australia]; 0.05 M MgCl2 [Unilab APS,
Seven Hills, Australia]; pH 9.5
Block solution (immunostaining plaque assay) 5% skim milk powder [Diploma, Mt. Waverley, Australia];
PBS
Wash solution (immunostaining plaque assay) 0.5% Tween-20 [Amresco, Solon, USA]; PBS
1X TE buffer 10 mM Tris-HCl [Amresco, Solon, USA] pH 7.5; 1 mM
EDTA [Unilab APS, Seven Hills, Australia] pH 8.0
Block solution (ELISA) 1% BSA [Sigma-Aldrich, St. Louis, USA]; PBS
Wash solution (ELISA) 0.05% Tween-20 [Amresco, Solon, USA]; PBS
Diluent (ELISA) 0.05% Tween-20 [Amresco, Solon, USA]; 0.1% bovine serum
albumin (BSA) [Sigma-Aldrich, St. Louis, USA]; PBS
30% LB-Glycerol 30% glycerol [Unilab APS, Seven Hills, Australia]; LB
46
2.2 Methods
2.2.1 Cell culture
2.2.1.1 Routine culturing
Vero E6 cells were grown in DMEM and OPTI-MEM® Reduced Serum Medium (1X), both
supplemented with 5% (v/v) heat-inactivated FBS. A549 cells were grown in F-12K Nutrient
Mixture, Kaighn’s Modification (1X), supplemented with 10% (v/v) heat-inactivated FBS.
Cos-7 cells were grown in DMEM supplemented with 10% (v/v) heat-inactivated FBS and
1X PSN. The cells were maintained in a humidified 37o C incubator supplied with 5% CO2
and subcultured 2 – 3 times per week. Trypsin/EDTA solution was used to detach cells from
the culture flask. All media, buffers, and solutions were pre-warmed to 37o C before use.
2.2.1.2 Freezing and thawing cells
5 X 106 – 1 X 10
7 cells/ml were resuspended in freezing medium (growth medium
supplemented with 10% (v/v) dimethyl sulfoxide) and 1 ml aliquots frozen slowly overnight
in a Cryo 1o C freezing container [Nalgene
TM, Rochester, USA]. Frozen cell stocks were
prepared for each cell line and stored at -80o C or in liquid nitrogen for later use.
When cells reached a high passage number (> 35), a vial of frozen cells was retrieved from
storage and thawed quickly in a 37o C waterbath. The cell suspension was added to 9 ml of
growth medium, resuspended, and cells pelleted by centrifugation at 400x g (Techcomp
CT15RT) [Techcomp Ltd., Kwai Chung, Hong Kong], at room temperature, for 5 minutes.
The pellet was then resuspended in 5 ml of growth medium and cells cultured in a 25 cm2
flask [Corning Inc., Lowell, USA].
2.2.1.3 Mycoplasma test
Cells were tested using polymerase chain reaction (PCR) for contamination by Mycoplasma
sp. Firstly, cells were cultured without antibiotics. For cells cultured in a 25 cm2 flask
[Corning Inc., Lowell, USA], the monolayer was washed once with PBS, detached with
trypsin/EDTA solution, and resuspended in 2 ml of growth medium. 250 µl of the cell
suspension was pelleted at 10,000x g and 4o C for 10 minutes. The cell pellet was washed
twice using 1 ml PBS followed by centrifugation at 10,000x g and 4o C for 10 minutes, and
resuspended in 240 µl of nuclease-free water. 20 µg/µl of Proteinase K was added to the cell
47
suspension, followed by mixing and 1 hour incubation at 50o C. Cells were then lysed by
heating at 100o C for 10 minutes, and then pelleted by centrifugation at 13,000x g for 5
minutes at room temperature. Supernatant was collected and used directly as the PCR
template or stored at -20o C until further use. The PCR mixture, detailed in table 2-11, was
prepared on ice.
Table 2-11 Components of PCR for Mycoplasma sp. detection
Component Final concentration (5X) Mango Taq Colored Reaction Buffer 1 X
50 mM MgCl2 1.5 mM
2 mM dNTPs 0.2 mM
5 pmoles/μl MycoF1 primer 0.25 pmoles
5 pmoles/μl MycoR1 primer 0.25 pmoles
5 U/μl MangoTaq DNA polymerase 0.075 U
DNA template 1 µl
Nuclease-free water to 20 µl
PCR cycling conditions were 94o C for 3 minutes, followed by 30 cycles of 94
o C for 30
seconds, 54o C for 2 minutes, and 72
o C for 1 minute, and terminated by a final extension step
of 72o C for 5 minutes in a Mastercycler
® gradient thermal cycler model 5533 [Eppendorf,
Hamburg, Germany].
The positive control was Mycoplasma sp. contaminated cell culture and the negative control
was nuclease-free water. PCR results were viewed using agarose gel electrophoresis, as
described in section 2.2.8.2, by running 15 µl of PCR product mixed with 3 µl of (5X) DNA
loading dye. If the PCR results were negative, 1 µl of the PCR product was used as the
template for a nested PCR using the pair of MycoF2 and MycoR2 primers (Table 2-4) and
similar mixture and cycling conditions as described in this section.
2.2.1.4 Trypan blue exclusion assay
The test is based on the principle that live cells possess intact cell membranes that exclude the
trypan blue dye, while dead cells do not. A viable cell should present a clear cytoplasm while
a nonviable cell should appear completely blue. Trypan blue was prepared by mixing 200 µl
of 0.2% (w/v) trypan blue in distilled water with 50 µl of 4.25% (w/v) NaCl in distilled
water. Diluted trypan blue and cell culture in growth medium were mixed in a 1:1 ratio and
applied to a haemocytometer. Cells were observed using an inverted light microscope
48
[Olympus, Mt. Waverley, Australia], and viable cell number determined using the following
equation:
Viable cell count (cells/ml) =
⁄
2.2.1.5 Heat inactivation of FBS
500 ml of frozen FBS was slowly thawed overnight at 4o C. The FBS and a temperature
control consisting of an FBS bottle filled with 500 ml of water and inserted with a
thermometer were slowly heated to 37o C in a waterbath, and then transferred to a 56
o C
waterbath. The heat inactivation was carried out for 30 minutes at 56o C, starting from once
the thermometer inserted into the control reached 56o C.
2.2.1.6 Preparation of 2% methyl cellulose
Methyl cellulose powder (10 g) was slowly added into 75 ml of boiled HBSS, with the
solution mixed through stirring. The solution was autoclaved at 121o C for 37 minutes, and
then cooled to 37o C on a stirrer. 40 ml of HBSS, 10 ml of FBS, 0.5 ml of 1,000X PSN, and
409.5 ml of serum-free DMEM was added to the methylcellulose-HBSS mix. The solution
was then shaken vigorously, stirred again for 30 minutes at room temperature, transferred to
4o C for 1 hour, while being continuously stirred, until the solution reached a consistent
thickness. The 2% methyl cellulose was stored at 4o C and pre-heated to 37
o C before each
use.
2.2.2 Virus propagation
2.2.2.1 Cell preparation
Vero E6 cells were prepared as 80%-confluent monolayer in a 150 cm2 flask [Corning Inc.,
Lowell, USA], which was approximately 8 X 106 cells. For RSV propagation, cells were
grown in DMEM + 5% FBS; while for hMPV propagation, cells were grown in OPTI-MEM®
+ 5% FBS.
49
2.2.2.2 Virus inoculation
2.2.2.2.1 RSV
The culture medium was removed and Vero E6 monolayer washed with HBSS. Cells were
infected with recombinant RSV in 8 ml DMEM, at a multiplicity of infection (MOI) of 0.1 –
0.5, depending on the titre of available virus stock. Virus was adsorbed for 1 hour in a
humidified 37o C incubator supplied with 5% CO2; following which 6 ml of DMEM + 5%
FBS was added. A flask that was mock infected served as the control for the infection.
Infected cells were incubated at 37o C, 5% CO2 until approximately 50% syncytia had
developed evenly across the flask, accompanied by minimal cell death.
2.2.2.2.2 hMPV
Vero E6 monolayer was first rinsed with HBSS, followed by a second rinse with
OPTI-MEM® + 5 μg/ml trypsin. Cells were infected with hMPV in MOI of 0.5 in 6 ml of
OPTI-MEM® + 5 μg/ml trypsin and adsorbed for 2 hours in a 37
o C incubator supplied with
5% CO2. After adsorbtion, 6 ml of OPTI-MEM® + 5% FBS was added and infected cells
were incubated at 37o C, 5% CO2. hMPV was harvested when 25 – 50% CPE was observed
evenly across the flask and cell death was minimal.
2.2.2.3 Harvest
RSV and hMPV were harvested using a similar technique. Firstly, infection medium was
removed while leaving 2.5 – 3 ml in the flask and infected cells detached using a scraper
[Corning Inc., Lowell, USA]. Infectious particles contained within cells were released using
five times 75% amplitude sonication [Sonics®, Newton, USA]. Each sonication lasted five
seconds, with a two-minute incubation on ice between each sonication. The virus was
separated from cell debris by centrifugation at 1200x g and 40 C for 7 minutes. The virus-
containing supernatant was collected in cryovials [GREINER Cryo.sTM
, Kaysville, USA] and
stored at -800 C.
2.2.2.4 Virus titration
RSV and hMPV was titrated to determine viral infectivity as previously described (Tripp,
2006, Tripp, 2010). Slight modifications were made to these procedures and these are
detailed below.
50
Briefly, 1 X 105 Vero E6 cells were seeded in each well of a 24-well plate in 0.5 ml of growth
medium, which was DMEM + 5% FBS for RSV and OPTI-MEM® + 5% FBS for hMPV, and
grown overnight (less than 24 hours) to obtain 80% confluent monolayer on the day of virus
inoculation.
10-fold serial dilutions of virus in serum-free DMEM (RSV) or OPTI-MEM® + 5 μg/ml
trypsin (hMPV) were used for quadruplicate inoculation. The growth medium was removed
from the well, cells briefly washed with HBSS, and 200 μl of diluted virus was added into
each well. Serum-free DMEM or OPTI-MEM® + 5 μg/ml trypsin alone was used as a control
(mock) infection for RSV and hMPV, respectively. Virus was adsorbed to the cells for 1 hour
at 37o C, 5% CO2, following which the infection was overlaid with 1 ml of 2% methyl
cellulose (RSV) or 200 μl of OPTI-MEM® + 5% FBS (hMPV) and the plate incubated at 37
o
C, 5% CO2 until day 6 post infection.
The culture medium was removed using suction and the cells washed with PBS and fixed
using ice-cold acetone-methanol (6:4) for 10 minutes. Following fixation, monolayers were
washed with PBS and air-dried. Fixed cells were blocked with PBS + 5% skim milk powder
for 30 minutes at room temperature for RSV titration, and for 1 hour at 37o C for hMPV.
Block solution was then replaced by 200 µl of anti-RSV F protein monoclonal antibody clone
131-2A (1:1000 dilution in block solution) for RSV titration or Ms X Hu Metapneumovirus
(1:1000 dilution in block solution) for hMPV titration, followed by 2 hours incubation at 37o
C. Cells were rinsed and washed twice with PBS + 0.5% Tween-20, 5 minutes each, on a
rocker [Labnet, Woodbridge, USA]. 200 µl of the secondary antibody, goat anti-mouse IgG
conjugated to alkaline phosphatase (diluted 1:1000 in block solution), was added and cells
incubated for 1 hour at 37o C, then washed as described previously. Vector
® Black Alkaline
Phosphatase Substrate Kit II was prepared according to the manufacturer’s protocol shortly
before use and 200 µl added to each well. Staining was developed for 20 minutes and stained
cells washed with water to stop the reaction. The plate was then air-dried and stained
immunoplaques counted using an inverted light microscope [Olympus, Mt. Waverley,
Australia]. Virus titre (plaques/ml) was determined using the formula
dilution factor 5,
where N is the number of immunoplaques in each well.
51
2.2.3 Replication kinetics assay
The replication kinetics assay was done in two cell lines, Vero E6 and A549. The cells were
prepared as 80%-confluent monolayer in 500 µl of growth medium, which was
approximately 1 X 105 cells per well in a 24-well plate. A control (mock infection) consisting
of serum-free media alone was included. RSV was diluted to an MOI of 0.03 and 3.0
PFU/cell in 200 µl of serum-free DMEM for Vero E6 cells and F12K Nutrient Mixture
Kaighn’s Modification for A549 cells. The growth medium was removed from the well, cell
monolayers were briefly washed with HBSS, and the diluted RSV added in quadruplicate.
Virus was adsorbed to cells for 1 hour in a 37o C incubator supplemented with 5% CO2. The
infecting medium was replaced with 600 µl of 1:1 mix of serum-free medium and cell growth
medium, and then plates were incubated at 37o C, 5% CO2 until the specified endpoints. The
endpoints of single cycle growth infection (MOI 3.0) were 0 hour (H), 18 H, 24 H, 30 H, 36
H, and 48 H; while the end points of multi-cycle infection (MOI 0.03) were day (D) 0, D1,
D2, D3, D4, and D5. At the specified end point, 450 μl of the 600 μl infection medium was
collected as the supernatant of the samples and the remaining 150 μl was used to generate cell
suspension by scraping off monolayer. Both supernatant and cell suspension were stored at -
80o C until further use. The supernatant was used to measure the secretion level of cellular
IL-8 and RANTES via ELISA, as described in section 2.2.4. The cell suspension was used
for measuring viral titre at each end point of infection via immunostaining plaque assay
described in section 2.2.2.4.
Viral titres were calculated in pfu/ml. A linear graph was built based on the time point of
termination of infection (x-axis) and log10 of viral titres (y-axis), virus titres were represented
by the means of quadruplicate titres from independent assays with a standard error. The
nonparametric Mann-Whitney U test (2-tailed) was used to compare the mean titres between
viruses. All calculation and graph building were done using GraphPad (Prism) v.5 software
(GraphPad, La Jolla, USA).
52
2.2.4 Enzyme-linked immunofluorescence assay (ELISA)
2.2.4.1 Plate-based optical density measurement
The concentrations of IL-8 and RANTES in RSV-infected cells were measured using the
ELISA development kit according to the manufacturer’s protocol. The ELISA plate [Thermo
Scientific, Roskilde, Denmark] was first prepared by overnight coating with 100 µl of capture
antibody (0.5 µg/ml) in PBS at room temperature. Thereafter, the liquid was aspirated and
plate washed 4 times, each with 300 µl of wash buffer (PBS + 0.05% Tween-20). 300 µl of
block buffer (PBS + 1% BSA) was applied to the wells and incubated for 1 hour at room
temperature, following which the plate was washed as described.
The kit-provided standard was two-fold serially diluted in diluent (0.1% BSA, 0.05% Tween-
20, PBS). The RANTES standard curve ranged from 2 ng/ml to 0 ng/ml and IL-8 from 1
ng/ml to 0 ng/ml. Samples (see section 2.2.3) were diluted 1:2 in diluent. 100 µl of diluted
samples and standards were added to the plate in triplicate. The reaction was incubated for 2
hours at room temperature and then washed. Detection antibody was diluted 0.25 µg/ml in
diluent, 100 µl added per well, incubated for another 2 hours at room temperature, and then
the plate was washed. 100 µl of avidin-HRP conjugate in diluent (1:2000) was applied into
wells, the plate incubated for 30 minutes at room temperature, and washed. As the final step,
100 µl of ABTS liquid substrate solution was added and colour development monitored with
a Benchmark-Plus ELISA plate reader [Bio-Rad Laboratories Inc., Hercules, USA] at 405
nm, with wavelength correction set at 650 nm.
2.2.4.2 Enumeration of IL-8 and RANTES concentration (pg/ml)
The standard curve was built based on the optical density values of the standard dilutions,
which were plotted on an xy graph, with the optical density values as the dependent variable
(y) and the concentration of the standard dilutions as the independent variable (x). A
regression analysis of scatterplots in the standard curve was done using Microsoft Excel
software [Microsoft Corporation]. The scatterplot exhibited a quadratic regression and a
polynomial regression curve and its function in was determined. The
IL-8 and RANTES concentrations in supernatant of infected cells were calculated using the
equation, based on the optical density values of the diluted samples. The kinetics of
chemokine expression was then evaluated based on the mean values of the concentration of
53
the chemokine in the samples, with variability of the values expressed as standard error, and a
graph was built based on the means value of the titres with the standard error of the means.
The nonparametric Mann-Whitney U test (2-tailed) was used to compare the mean values of
chemokine expression induced by the two viruses. All calculation and graph building were
done using GraphPad (Prism) v.5 software (GraphPad, La Jolla, USA).
2.2.5 hMPV N constructs
2.2.5.1 RNA extraction
hMPV was first propagated in Vero E6 cells that were cultured in a 25 cm2 flask [Corning
Inc., Lowell, USA] and the N gene isolated from RNA, which was extracted using TRIzol®
reagent as per manufacturer’s instruction. The growth medium was replaced with 2.5 ml of
TRIzol® reagent, and flask incubated for 5 minutes at room temperature. The suspension was
transferred into a 10 ml conical tube [Cellstar®, Greiner Bio One, Frickenhausen, Germany],
mixed vigorously with 0.5 ml chloroform, and incubated for 3 minutes at room temperature.
Suspension was centrifuged at 12,000x g for 15 minutes at 4o C. The aqueous phase was
transferred into a new 10 ml conical tube, and RNA precipitated with 1.25 ml of isopropyl
alcohol for 10 minutes at room temperature, followed by centrifugation at 12,000x g and 4o C
for 10 minutes. The RNA pellet was then washed by vigorous mixing with 2.5 ml of 75%
ethanol, followed by centrifugation at 7,500x g and 4o C for 5 minutes. As the final step, the
RNA was air dried, resuspended in nuclease-free water, and stored at -80o C until further use.
2.2.5.2 Reverse transcription and polymerase chain reaction (RT-PCR)
The cDNA of hMPV RNA was generated using the cDNA synthesis kit. RNA concentration
was first measured using NanoDropTM
1000 UV/Vis spectrophotometer [NanodropTM
,
Wilmington, USA]. Firstly, reaction mix I (table 2-12) was first prepared on ice.
Table 2-12 The reaction mix I to generate cDNA from hMPV RNA
Component Final concentration RNA 1 µg
270 ng/μl Oligo(dT)10 270 ng
10 mM dNTP Mix 2 mM
DEPC-treated water To a final volume of 10 µl
The reaction was incubated for 10 minutes at 65o C, immediately chilled on ice for 2 minutes,
and added to reaction mix II (table 2-13).
54
Table 2-13 The reaction mix II to generate cDNA from hMPV RNA
Component Final concentration (5X) RT Buffer 2X
10 U/μl RNase Inhibitor 10 U
200 U/µl Reverse Transcriptase 5 U
DEPC-treated water To a final volume of 10 µl
The final reaction was incubated for 45 minutes at 45o C, and reverse transcription terminated
by 15 minutes incubation at 70o C. cDNA was stored at -80
o C until further use.
The hMPV N gene was amplified via PCR using the cDNA of hMPV genome as template. In
addition to amplification, the PCR also equipped the N gene with attB sequences that
function as site-specific recombination required for generating the entry clone that is
described in section 2.2.6.1. The PCR is also the means to generate the truncated ORF
constructs of hMPV N gene, which was done by adjusting the specific primer pair to the
desired ORF. The PCR components are detailed in table 2-14 and the primer pairs that were
used to generate the hMPV N constructs are listed in table 2-15.
Table 2-14 The PCR components to generate hMPV N constructs
Component Final concentration (5X) Mango Taq Colored Reaction Buffer 1X
50 mM MgCl2 1.5 mM
2 mM dNTPs 0.2 mM
5 pmoles/μl forward primer 0.25 pmoles
5 pmoles/μl reverse primer 0.25 pmoles
5 U/μl MangoTaq DNA polymerase 0.025 U
cDNA 5 μl
Nuclease-free water To a final volume of 40 µl
Table 2-15 List of primer pairs used to generate hMPV N construct
ORF Primer pair hMPV N HMPV83NattB1 + HMPV83NattB2
hMPV N – fragment A HMPV83NattB1+hMPVN_r_attB2
hMPV N – fragment B HMPV83NattB1+hMPVN_f-rev_attB2
hMPV N – fragment C hMPVN_f_attB1+HMPV83NattB2
PCR cycling conditions were 95o C for 7 minutes, followed by 35 cycles of 96
o C for 30
seconds, 58o C for 45 seconds, and 72
o C for 1 minute. PCR results were viewed via agarose
gel electrophoresis as described in section 2.2.8.2, from 5 µl of PCR product that was mixed
with 1 µl of (5X) DNA loading dye.
2.2.5.3 DNA clean up
PCR product was purified from agarose after separation via electrophoresis. DNA
purification was carried out to eliminate the excess salts and primers that might interfere with
55
the recombination reaction. Briefly the total volume of PCR product was mixed with DNA
loading dye in 5:1 ratio and electrophoresed in 1% agarose gel, as described in section 2.2.9.
The desired DNA fragment was visualised using UV light from a transilluminator and cut
from the gel using a clean scalpel. The DNA was then purified using Wizard® SV Gel and
PCR Clean-Up System [Promega, Madison, USA]. Excised gel was first dissolved with
Membrane Binding Solution in a ratio of 1 µl solution for 1 mg gel via incubation at 65o C
for 10 minutes. The solution was then transferred to an SV Minicolumn and incubated for 1
minute at room temperature. The column was centrifuged at 19,000x g for 1 minute and the
membrane washed twice with Membrane Wash Solution. The first wash used 700 µl of
solution and the second wash used 500 µl of solution. The column was centrifuged at 19,000x
g for 1 minute in each wash. An additional centrifugation was carried out to remove the
residual wash solution, then the column transferred to a 1.5 ml microfuge tube and the DNA
eluted with 30 µl of nuclease-free water. The purified DNA was stored at -80o C until further
use.
2.2.6 Cloning of hMPV N gene using GatewayTM
Technology
The hMPV N protein was expressed in mammalian cells as an expression clone, by being
inserted into an expression vector. The expression clone was prepared using the Gateway®
recombination cloning. This technology makes use of the specific att recombination sites
present in E. coli bacterial chromosome (attB) and bacteriophage lambda DNA (attP) that
ensures the correct directionality of the recombinant. The Gateway® technology consists of
two steps: the construction of an entry clone and the construction of an expression clone. The
entry clone was generated by inserting the PCR product of hMPV N gene into the entry
vector pDONR®207 via BP recombination. The expression clone was generated by transport
the entry clone into the destination vector, pEPI-DESTC, via LR recombination.
2.2.6.1 Entry clone construction
Table 2-16 The components of BP recombination reaction
Component Final concentration attB-tagged ORF (section 2.2.5.3) 16 – 40 ng
attP-containing pDONR®207 150 ng
Gateway ® BP Clonase® Enzyme Mix 2 µl
TE buffer pH 8.0 To 10 µl
The reaction was incubated at 25o C for 18 hours and terminated with addition of 2 µl of
Proteinase K in 10-minutes incubation at 37o C. 1 – 2 µl of the reaction was transformed into
56
ElectroMAXTM
DH10BTM
Cells (as described in section 2.2.10.2). 100 µl of the transformed
E. coli suspension was plated on LB agar supplemented with 7 µg/ml gentamycin and
incubated overnight at 37o C. Positive colonies were picked up and cultured in 5 ml LB
supplemented with 7 µg/ml of gentamycin, at 37o C, 220 rpm, for plasmid isolation (section
2.2.6.3).
2.2.6.2 Expression clone construction
Table 2-17 The components of LR recombination reaction
Component Final concentration attP containing pDONR®207.hMPV N construct (section 2.2.6.1) 50 – 150 ng
attB containing pEPI-DESTC 150 ng
LR Clonase® 2 µl
TE buffer pH 8.0 To 10 µl
The reaction was incubated 25o C for 18 hours and terminated with addition of 2 µl of
Proteinase K in 10 minutes incubation at 37o C. 1 – 2 µl of the reaction was transformed into
ElectroMAXTM
DH10BTM
Cells (as described in section 2.2.10.2). 100 µl of the transformed
E. coli suspension was plated on LB agar supplemented with 100 µg/ml kanamycin and
incubated overnight at 37o C. Positive colonies were picked up and cultured in 5 ml LB
supplemented with 100 µg/ml of kanamycin at 37o C, 220 rpm, for plasmid isolation (section
2.2.6.3).
2.2.6.3 Plasmid isolation
The desired plasmid, obtained from BP or LR recombination reaction, was isolated using the
the QIAprep® Spin Miniprep or Midiprep Kits [Qiagen].
2.2.6.3.1 Plasmid isolation using Miniprep Kit (Qiagen)
Bacterial culture grown overnight in 5 ml of LB supplemented with appropriate antibiotics
(section 2.2.6.1 and 2.2.6.2) was first pelleted through centrifugation at 4,100x g and 4o C for
20 minutes, and resuspended in 250 µl of buffer PI. 250 µl of buffer P2 was added and mixed
thoroughly by 4 – 6 times inversion. 350 µl of buffer N3 was added and mixed in the same
way. The solution was then centrifuged at 16,500x g for 10 minutes and supernatant loaded
on to a QIAprep® spin column. The column was briefly centrifuged at 16,500x g, washed
with 0.5 ml of buffer PB in a brief centrifugation at 16,500x g, followed by a secondary wash
with 0.75 ml of buffer PE. An additional centrifugation was carried out to remove the
residual wash buffers. Finally, the column was transferred to a 1.5 ml microfuge tube and the
57
plasmid eluted with 30 µl of RNase-free water. The plasmid was stored at -20o C until further
use.
2.2.6.3.2 Plasmid isolation using Midiprep Kit (Qiagen)
A starter culture was first prepared by growing the desired bacterial colonies (section 2.2.6.1
and 2.2.6.2) in 5 ml of LB supplemented with appropriate antibiotics at 37o C, 300 rpm, for 8
hours. A bacterial culture was made by re-culturing 50 μl of the starter culture in 25 ml of LB
supplemented with appropriate antibiotic solution at 37o C, 300 rpm, overnight. The bacteria
were harvested by centrifugation at 6000x g and 4o C for 15 minutes, then resuspended in 4
ml of buffer P1. 4 ml of buffer P2 was added and mixed by 4 – 6 times tube inversion, and
incubated at room temperature for 5 minutes. 4 ml of buffer P3 was then added and mixed by
4 – 6 times tube inversion, followed by 15 minutes incubation on ice. The tube was
centrifuged at 21,800x g and 4o C for 30 minutes; then supernatant transferred into a new
15-ml conical tube and re-centrifuged at 21,800x g and 4o C for 15 minutes. Subsequently,
the supernatant was applied to a QIAGEN-tip 100. The tip was washed twice with 10 ml of
buffer QC and plasmid eluted in 5 ml of buffer QF. The plasmid was precipitated using 3.5
ml isopropanol accompanied by centrifugation at 18,000x g and 4o C for 18 minutes. The
DNA pellet was washed with 2 ml of 70% room-temperature ethanol, followed by
centrifugation at 18,000x g for 10 minutes. As the final step, the pellet was air dried for 10
minutes and plasmid redissolved in 500 μl of RNase-free water. The plasmid was stored at -
80o C until further use.
2.2.7 Confirmation of the hMPV N gene inserted into vectors using PCR and
DNA sequencing method
2.2.7.1 PCR
The second method to verify entry and expression clones was via PCR using the same primer
pair used to generate the ORF of interest. The components of the PCR are listed in table 2-18.
58
Table 2-18 PCR components to confirm the presence of hMPV N insert in cloning vectors.
PCR cycling conditions were 95o C for 7 minutes, followed by 35 cycles of 96
o C for 30
seconds, 58o C for 45 seconds, and 72
o C for 1 minute. PCR results were viewed via agarose
gel electrophoresis as described in section 2.2.7.6, from 5 µl of PCR product that was mixed
with 1 µl of (5X) DNA loading dye.
2.2.7.2 DNA sequencing
The sequence of ORF generated via PCR was confirmed via DNA sequencing. The
sequencing reaction is described table 2-19.
Table 2-19 Components of DNA sequencing reaction, with PCR product as template
Component Final concentration PCR product 30 – 75 ng
5 pmoles/μl primer* 0.5 pmoles
Nuclease-free water To a final volume of 12 μl
*the primers used were listed in table 2-15.
The results of each Gateway® recombination reaction were also verified via DNA
sequencing, using sequencing primers listed in table 2-4. The sequencing reaction is
described in table 2-20.
Table 2-20 Components of DNA sequencing reaction, with ds plasmid as template
Component Final concentration ds plasmid 600 ng
Primer 0.5 pmoles
Nuclease-free water To a final volume of 12 μl
DNA sequencing was outsourced to the Australian Genome Research Facility
(www.agrf.org.au), and the reaction was prepared according to the company’s requirement.
The chromatograms were analysed using BioEdit 7.0.4 (Hall, 2005, Ibis Biosciences)
software.
Component Final concentration (5X) Mango Taq Colored Reaction Buffer 1X
50 mM MgCl2 1.5 mM
2 mM dNTPs 0.2 mM
5 pmoles/µl forward primer 0.25 pmoles
5 pmoles/µl reverse primer 0.25 pmoles
5 U/μl MangoTaq DNA polymerase 0.025 U
DNA template (entry or expression clones) 5 μl
Nuclease-free water To a final volume of 40 µl
59
2.2.8 Measurement of nucleic acid concentration
DNA/RNA quantitation was carried out using the NanoDropTM
1000 UV/Vis
spectrophotometer [NanodropTM
, Wilmington, USA]. Briefly, 2 μl of DNA/RNA sample was
loaded on the pedestal and DNA/RNA concentration was measured by absorbance at 260 nm
(A260), A280, and A230. The ratios of A260/A280 and A260/A230 were analysed by the
NanoDropTM
v3.7.1 software. The spectrophotometer was blanked with the solution in which
the nucleic acid was resuspended.
2.2.9 Agarose gel electrophoresis
Agarose gel (1%) was prepared from 0.5 g agarose powder in 50 ml 1X TAE, which was
dissolved by heating the mix in a microwave for 1 minute, and adding 50 µl GelRedTM
nucleic acid gel stain (1:10) into the mix. The DNA was mixed with (5X) DNA loading dye
(1 part DNA loading dye + 5 parts DNA) and the mix loaded into gel wells. The
electrophoresis was carried out at 80 volts for 80 minutes and the result viewed using GelDoc
[Biorad] UV transilluminator and analysed with Quantity One® v4.6.5 software [Bio-Rad
Laboratories Inc., Hercules, USA].
For confirmation of hMPV N constructs (Figures 5-3 and 5-4), agarose gel images were
digitally modified using Adobe Photoshop CS5 software [Adobe Systems Incorporated] by
rearranging the lanes containing confirmation using PCR and uncut plasmid, for the purpose
of presentation clarity.
2.2.10 Plasmid storage in glycerol solution
Plasmids (entry and expression clones) were stored in transformed ElectroMAXTM
DH10BTM
competent E. coli. The bacteria were grown overnight on LB agar plates supplemented with
appropriate antibiotics at 37o C. The colonies that grew all over the agar surface were made
into a bacterial suspension by adding 1 ml of LB + 30%-glycerol and scraping off the
colonies using a glass spreader. The suspension was pipetted into cryovials [GREINER
Cryo.sTM
, Kaysville, USA] and stored at -80o C.
60
2.2.11 hMPV N protein expression
2.2.11.1 Cell transfection
Cos-7 cells (3 X 105 cells in 2 ml growth medium) were seeded overnight in advance, on
glass coverslips (diameter = 24 mm) [ProSciTech, Thuringuwa, Australia] that were placed in
a 6-well plate to obtain 80%-confluent monolayer at the time of transfection.
LipofectamineTM
2000 Transfection Reagent was diluted 1:25 in serum-free DMEM. Green
fluorescent protein (GFP) – tagged ORFs in pEPI-DESTC was diluted in serum-free DMEM
to a concentration of 2 µg per well. Diluted LipofectamineTM
2000 Transfection Reagent and
diluted plasmid was mixed in 1:1 (v:v) ratio and incubated 20 minutes at room temperature.
The monolayer was washed with PBS, then 200 µl of LipofectamineTM
2000 Transfection
Reagent/plasmid solution added in a dropwise fashion, and incubated at 37o C, 5% CO2 for
20 minutes. 2 ml growth medium was added and transfected cells were maintained at 37o C,
5% CO2, until a specified time point, when the expression of green fluorescent protein (GFP)-
tagged protein was viewed and recorded using confocal laser scanning microscopy (CLSM)
as described in section 2.2.11.2.
2.2.11.2 Confocal laser scanning microscopy (CLSM)
For live cells imaging, 24 or 48 hours after transfection the coverslip was placed on a ring-
shaped coverslip holder and cells covered with 37o C PBS. A drop of immersion oil was
placed on top of the objective of Nikon Eclipse T [Nikon Instruments Inc., Melville, USA]
to layer between the lens and coverslip.
The NIS-Elements 64 bit 3.21.00 software [Nikon Instruments Inc., Melville, USA] captured
and recorded the cell images. First, the wavelength of imaging light was adjusted to the
fluorescent dye used to label cells in the ‘channel’ menu of the scan setting window; the most
generally used was 488 nm, which recognises the light emitted by the GFP. To select the cells
whose images were to be recorded, the menu from NIS-Elements 64 bit 3.21.00 software was
shifted to ‘widefield’, and the sight field scrutinised while the lens focus was being adjusted.
To capture and record the images, the menu was shifted to ‘confocal’, the desired cells
localised in the ‘scan area’ function of the navigation window, and lens focus manually
adjusted during ‘live’ viewing. The frozen images were captured using 4X averaging of the
‘XY’ navigation and saved in JPEG 2000 Image format (.jp2). The pixel density of
61
fluorescence in nucleus (Fn) and in cytoplasm (Fc) were obtained using the ImageJ 1.45M
software (Ferreira & Rasband, 2011) and the fluorescence ratio determined using the formula
Fn/c =
, where Fn is the pixel density fluorescence in nucleus, Fc is the pixel density of
fluorescence in the cytoplasm, and Fb is the pixel density of background fluorescence.
The values of nuclear and cytoplasmic fluorescence ratio (Fn/Fc) of subcellular localisation
of hMPV N constructs were determined according to section 2.2.11.3. The mean value of
Fn/Fc ratio for each construct was determined from total cells observed (generally > 10), and
presented as a bar, with variability expressed as the standard error of the mean (SEM). The
statistical difference of Fn/Fc ratio between each of hMPV N construct was assessed using
the nonparametric Mann-Whitney U test (2-tailed). All calculation and graph building were
done using GraphPad (Prism) v.5 software (GraphPad, La Jolla, USA).
2.2.12 Bacterial culture
2.2.12.1 Preparation of competent E. coli
The ElectroMAXTM
DH10BTM
Cells [Invitrogen] are specifically designed to facilitate
recombinational cloning using GatewayTM
technology. From the initial frozen culture, a loop
of ElectroMAXTM
DH10BTM
Cells [Invitrogen] was streaked on to an antibiotic-free LB agar
plate at 37o C. A single colony was selected and grown overnight in 5 ml of SOB –Mg
medium at 37o C in a shaker incubator [Bioline]. Four ml of the culture was then re-cultured
in 200 ml of SOB –Mg medium for 2 – 3 hours at 37o C until OD600 ranged between 0.8 and
1.0. The suspension was pelleted at 2500x g centrifugation at 4o C for 10 minutes, the pellet
resuspended in 100 ml of 10% pre-chilled glycerol, and re-pelleted by centrifugation for 10
minutes at 2500x g and 4o C. The supernatant was carefully removed and the pellet
resuspended in 200 ml of 10% pre-chilled glycerol and centrifuged 2500x g at 4o C for 10
minutes. As the final step, bacterial pellet was resuspended in approximately 2 ml of the
remaining liquid, aliquoted in 50 µl volume, and stored at -80o C until further use.
2.2.12.2 Cell transformation
On ice, 1 – 10 µl plasmid that was generated from BP or LR reaction was mixed with 50 µl
of ElectroMAXTM
DH10BTM
competent E. coli cell suspension and inserted into a pre-chilled
1 ml cuvette [Bio-Rad Laboratories Inc., Hercules, USA]. The plasmid was transformed into
bacterial cells via electroporation using GenePulser® II electroporator [Bio-Rad Laboratories
62
Inc., Hercules, USA] with the following conditions: 25 µF capacitance, 200 Ω resistance, 2.0
kV voltage, then 450 μl of LB medium was added. Transformed bacteria were incubated at
37o C and 220 rpm in a shaker incubator [Bioline, Alexandria, Australia] for an hour. 100 –
150 µl of cell suspension was cultured overnight on an LB agar plate supplemented with
appropriate antibiotic at 37o C. Surviving colonies were either inoculated in 5 ml LB for
further assay or re-cultured on LB agar plate for storage.
2.2.12.3 Long term storage of E. coli
Transformed or non-transformed E. coli were preserved in 30% glycerol solution as
described in section 2.2.10.
63
CHAPTER 3
IN VITRO REPLICATION EFFICIENCY
OF rRSV A2 M(T205A)
3 Replication Efficiency of RSV in Cell Culture
64
65
3.1 Introduction
The replication efficiency, or fitness, measured in cell culture can correlate with the clinical
outcome of infections and host-to-host transmission in whole organism (Dietzschold et al.,
1985, Dietzel et al., 2011). Replication efficiency includes two components: the capabilities
of the virus to, firstly, establish infection and, secondly, to transmit the infection. Viral
infectivity can be studied through a replication kinetics assay wherein a high ratio of virus
particles to cells (multiplicity of infection (MOI)) is used and observing infectious particle
production in a short period following infection (typically hours), which is essentially a single
cycle growth curve. Viral transmissibility can be studied by conducting replication kinetics
assay using a low MOI and observing viral growth over a longer period following infection
(typically days); therefore studying viral growth over multiple replication cycles.
Studying viral growth in cell cultures (in vitro) allows determination of viral fitness over a
period of time in a system where host antiviral mechanisms are well characterised, compared
to the more complex antiviral network in a whole organism (in vivo). The replication
efficiency of RSV was studied in two cell lines: Vero E6 and A549 cells. Vero E6 is a cell
line that originates from the kidney epithelium of the African green monkey (Cercopithecus
aethiops) and lacks the structural genes of the antiviral IFN-α/β (Emeny and Morgan, 1979),
therefore is highly susceptible to infection and in this study acted as the control system for the
IFN-α/β antiviral response. A549 is a cell line that originates from the human respiratory
epithelial cells and possesses the functional structural genes of IFN-α/β; and therefore are a
relevant model for studying RSV infection, in a system that resembles the natural site of
infection (Stoltz and Klingström, 2010). IFN-α/β is a strong innate immune response against
viral infection and in RSV infection contributes to virus clearance as well as directs the
biology of the subsequent pro-inflammatory responses, which often determine the disease
outcome (Noah et al., 2002, Smyth et al., 2002).
The wild type and mutant viruses are both well-characterised recombinants and have identical
genomes, except for threonine into alanine substitution (TA) at RSV M205. The wild type
virus was generated using reverse genetics technology based on the genome of RSV strain A2
and confers the CK2 phosphorylatable phenotype of RSV M 205 (T). The mutation of M205
(TA) in the mutant virus was designed on the basis of previous work that showed that CK2
phosphorylation regulates nuclear export of RSV M (Ghildyal et al., 2006). The recombinant
66
RSV were produced by our collaborators in University of South Florida (Dr Michael Teng
and Ms Kim Tran) who performed full genome sequencing for rRSV A2 and rRSV A2
M(T205A) prior to us importing the virus into Australia. It was expected that the difference
in viral titres would represent the impact of this single amino acid substitution on the
replication efficiency. Previously, the impact of M(T205) substitution has been studied using
expression in transfected cells and in viral infection, but the results have not yet been
published (R. Ghildyal, personal communication).
In this study, both rRSV A2 and rRSV A2 M(T205A) were passaged multiple times in Vero
E6 cells prior to replication kinetics assay. Multiple passaging of a recombinant mutant virus
confers a possibility that selection, which favours the wild type genome, may cause the
mutant phenotype to revert into the wild type. In this study, the multiple passaging of rRSV
A2 M(T205A) could by chance mutate the M(A205) into the wild type M(T205), and in
doing so, the result obtained for the mutant may not be the true phenotype. Therefore, the
genotypes of the rRSV M constructs were confirmed using DNA sequencing after the
multiple passaging.
This study aimed to investigate the impact of mutation of a putative CK2 kinase
phosphorylation site within the M protein on the replication efficiency of RSV in an
environment that exerts the antiviral activity of IFN-α/β, as is found in the human respiratory
tract. The investigation was done by comparing the replication fitness of a wild type
(rRSV A2) and a mutant (rRSV A2 M(T205A)) virus in a single cycle replication assay. The
impact of RSV M (T205A) mutation on viral transmissibility was also studied in a multiple
cycle replication assay. Investigation of the spread of the virus in the monolayer of cell
culture, which is a closed population with a controlled environment, could give insights into
important determinants of transmissibility in a more complex system, i.e a whole organism.
3.2 Methods
3.2.1 Cells and virus
Vero E6 and A549 cells were maintained in culturing medium as described in section 2.2.1.
The wild type (rRSV A2) and mutant (rRSV A2 M(T205A)) virus were grown in Vero E6,
followed by immunostaining plaque assay to determine the titre of infectious virus particle
(pfu/ml) as described in section 2.2.2.
67
3.2.2 Single cycle growth curve
Quadruplicate monolayers of Vero E6 and A549 cells were infected with recombinant wild
type (rRSV A2) or mutant (rRSV A2 M(T205A)) RSV at an MOI of 3, in 200 µl of serum-
free medium. Two hours after inoculation, the medium was replaced with 600 µl of infecting
medium, and infection developed until specified end points, which were 18, 24, 30, 36, and
48 hours post infection as described in section 2.2.3. At each specified time point, 450 µl of
culture supernatant was collected for measuring chemokine expression (Chapter 4) and the
remaining 150 µl was used to collect cell lysates by scraping the well bottom. The cells were
lysed by three freeze-thaw cycles, followed by a brief centrifugation, to release the virus
particles. The count of infectious viral particles was estimated from the clarified lysates by
immunostaining plaque assay (2.2.2.4).
3.2.3 Multiple cycle growth curve
Quadruplicate monolayers of Vero E6 and A549 cells were infected with recombinant wild
type (rRSV A2) or mutant (rRSV A2 M(T205A)) RSV at an MOI of 0.03, in 200 µl of
serum-free medium. Two hours after inoculation, the medium was replaced with 600 µl of
infecting medium, and infection developed until specified end points, which were 0, 1, 2, 3,
4, and 5 days post infection as described in section 2.2.3. At each specified time point, 450 µl
of culture supernatant was collected for measuring chemokine expression (Chapter 4) and the
remaining 150 µl was used to collect cell lysates by scraping the well bottom. The cells were
lysed by three freeze-thaw cycles, followed by a brief centrifugation, to release the virus
particles. The count of infectious viral particles was estimated from the clarified lysates by
immunostaining plaque assay (2.2.2.4).
3.2.4 Data analysis
The RSV titres at each end point were plotted on a log scale (log pfu/ml) against time point of
collection. The mean ± standard error of the mean (SEM) from four replicates of each sample
were used. Statistical differences between the titres of rRSV A2 and rRSV A2 M(T205A) at
each time point were determined by the nonparametric Mann-Whitney U test (GraphPad
(Prism) v.5 software (GraphPad, La Jolla, USA), and a difference was considered significant
if P<0.05.
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3.2.5 Confirmation of the stability of rRSV M constructs
rRSV M constructs were obtained by isolating total RNA from infected Vero E6 cells,
followed by generating cDNA using oligo(dT)10, and capturing the M gene using the primer
pair attB1RSVM1 and attB2RSVM256 in PCR. The amplicons obtained in PCR were then
agarose gel-purified and sequenced in the forward and reverse direction using the primer
attB1RSVM1 (forward) and attB2RSVM256 (reverse). The raw sequence data was examined
using BioEdit 7.0.4 (Hall, 2005; Ibis Biosciences).
3.3 Results
3.3.1 Single cycle replication kinetics
Figure 3-1 represents the single cycle growth curve of the rRSV A2 and rRSV A2 M(T205A)
in Vero E6 (A) and A549 cells (B).
Figure 3-1 A shows that the rRSV A2 had exponential growth during 18 – 30 hours post
infection and a lag phase or levelling-off afterwards. However, the rRSV A2 M(T205A) did
not follow the pattern since there were no exponential or lag phases observed. Instead, the
growth curve fluctuated from an initial high titre (18 h.p.i), level off (24 h.p.i), a decrease
from 24 to 36 h.p.i, and then another increase (48 h.p.i). The different shape of the growth
curve was supported by the significantly different titres between the two rRSV at 18, 30, and
36 hours post infection. At 18 h.p.i, the rRSV A2 had a lower titre (mean titre = 2.7 X 103
pfu/ml) than rRSV A2 M(T205A) (mean titre 1.1 X 105 pfu/ml). However, at 30 and 36 h.p.i,
the rRSV A2 had a higher titre (mean titres = 3.5 X 105 pfu/ml at 30 h.p.i, 8.6 X 10
5 pfu/ml at
36 h.p.i) than rRSV A2 M(T205A) (mean titres = 6.2 X 104 pfu/ml at 30 h.p.i, 5.2 X 10
4
pfu/ml at 36 h.p.i). At 24 h.p.i, the rRSV A2 had a higher titre than rRSV A2 M(T205A)
although this was not statistically significant. At 48 h.p.i, there was no statistically significant
difference between rRSV A2 and rRSV A2 M(T205A); however, the titre of rRSV A2 (7.7 X
105 pfu/ml) was lower than rRSV A2 M(T205A) (1.9 X 10
6 pfu/ml).
Figure 3-1 B details the replication of the recombinant RSV in A549 cells. The rRSV A2 had
an exponential phase from 18 – 30 h.p.i and then lagged toward 48 h.p.i., where virus
production slowly decreased. The rRSV A2 M(T205A) proliferated exponentially only up to
24 h.p.i, then lagged in a decreasing fashion for the next 24 hours.
69
The rRSV A2 M(T205A) yielded lower amounts of infectious particles at each time point
observed, compared to rRSV A2 (Figure 3-1 B). Its exponential peak was represented by a
lower titre of infectious particle compared to the amount produced by rRSV A2 at the same
time point (24 h.p.i., mean titres = 6.0 X 104 pfu/ml (rRSV A2) and 1.6 X 10
4 pfu/ml (rRSV
A2 M(T205A)). At the time rRSV A2 reached the exponential peak (30 h.p.i), the rRSV A2
M(T205A) has started decreasing, and it was reflected by the significantly different amounts
70
Figure 3-1 The growth curve of single cycle replication kinetics of rRSV A2 and rRSV A2
M(T205A) in (A) Vero E6 and (B) A549 cells. Quadruplicate monolayers of cells were infected at
a multiplicity of infection (MOI) of 3. Whole cell lysates were collected at the indicated time
points and titrated using immunostaining plaque assay described in section 2.2.2.4. Shown is the
means of log10 viral titres (pfu/ml) ± standard error, n ≥ 4, with error bars indicating standard error
of the means. Continuous black line (– ) represents rRSV A2. Continuous pink line represents
rRSV A2 M(T205A). Dotted line indicates limit of detection by immunostaining plaque
assay (50 pfu/ml). Asterisk (*) denotes that the viral titres are significantly different between the
two viruses (P < 0.005) based on the nonparametric Mann-Whitney U-test (GraphPad (Prism), La
Jolla, USA).
71
of infectious particles produced at that time point. The mean titre of rRSV A2 was 2.3 X 105
pfu/ml, while the mean titre of rRSV A2 M(T205A) was 6.3 X 103 pfu/ml.
The rRSV A2 M(T205A) was capable of producing a relatively comparable amount of virus
particles to rRSV A2 at 48 h.p.i in IFN-α/β-free environment (Figure 3-1 A), but it did not do
so in the presence of IFN-α/β (Figure 3-1 B). The rRSV A2 produced a significantly higher
viral titre (mean titre = 9.4 X 104 pfu/ml) than rRSV A2 M(T205A) (mean titre = 2.1 X 10
3
pfu/ml) at 48 h.p.i.
Comparison between the rRSV A2 growth in a non-IFN-α/β-expressing environment (Vero
E6 cells, Figure 3-1 A) and in an IFN-α/β-expressing environment (A549 cells, Figure 3-1 B)
shows that the exponential phase lasted during 18 – 30 h.p.i and the lag phase took place 30 –
48 h.p.i in both cells. The rRSV A2 grown in A549 cells peaked at a significantly lower titre
(mean = 2.3 X 105 pfu/ml) than rRSV A2 grown in Vero E6 cells (mean = 3.5 X 10
5 pfu/ml).
While rRSV A2 grown without the presence of IFN-α/β continued to produce more particles
in the lag phase, rRSV A2 grown in IFN-α/β expressing environment was affected by the
IFN-α/β such that the production of infectious particles declined during the lag phase. This
was shown by the significantly different amount of infectious particles produced at 48 h.p.i
(mean titre: rRSV A2 A549 = 9.4 X 104 pfu/ml; rRSV A2 Vero E6 = 7.7 X 10
5 pfu/ml).
rRSV A2 M(T205A) replicated at significantly lower titres in A549 cells (Figure 3-1 B) than
in Vero E6 cells (Figure 3-1 A), at 18, 30, and 48 h.p.i. At 18 h.p.i., the mean titres were 1.1
X 105 pfu/ml (Vero E6) and 3.6 X 10
2 pfu/ml (A549). At 30 h.p.i., the mean titres were 6.2 X
104 pfu/ml (Vero E6) and 6.3 X 10
3 pfu/ml (A549). At 48 h.p.i, the mean titres were 1.9 X
106 pfu/ml (Vero E6) and 2.1 X 10
3 pfu/ml (A549). This result shows that overall, the
replication of rRSV A2 M(T205A) was significantly inhibited by the antiviral action of IFN-
α/β.
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3.3.2 Multiple cycle replication kinetics
Figure 3-2 represents the multiple cycle growth curve of the rRSV A2 and rRSV A2
M(T205A) in Vero E6 (A) and A549 cells (B).
In the IFN-α/β-free environment, both the recombinant RSV grew by following the
exponentiallag phases of virus replication curve (Figure 3-2 A). The exponential peak took
place at day 2 post infection, and the lag phase followed until the endpoint of observation
(day 5 post infection). The lag phase of rRSV A2 had a trend toward a slight increase in the
number of infectious particle production; while the lag phase of rRSV A2 M(T205A) showed
a decrease in the number of infectious particle production. The rRSV A2 M(T205A)
proliferated into significantly lower number of progeny than the rRSV A2, as evidenced at
the days 2, 4, and 5 post infection, where the mean titres of rRSV A2 were 9.7 X 105 pfu/ml
(D2), 1 X 106 pfu/ml (D4), and 3.7 X 10
6 pfu/ml (D5) while the mean titres of rRSV A2
M(T205A) were 4.9 X 104 pfu/ml (D2), 4 X 10
4 pfu/ml (D4), and 5.7 X 10
3 pfu/ml (D5).
In an environment that exerted the antiviral IFN-α/β pressure (A549 cells, Figure 3-2 B),
rRSV A2 grew exponentially, peaking at day 2 post infection, and then had a lag phase with a
slight increase in particle production. Although showing a similar pattern of growth kinetics,
the amount of infectious particles produced in A549 cells (Figure 3-2 B) were significantly
lower than the amount produced in Vero E6 cells (Figure 3-2 A) at each time point of
observation.
The titres of rRSV A2 M(T205A) in A549 cells were below the detection limit of the
immunostaining plaque assay (50 pfu/ml, dotted line) at each time point (Figure 3-2 B).
73
Figure 3-2 The growth curve of multiple-step replication kinetics of rRSV A2 and rRSV A2
M(T205A) in (A) Vero E6 and (B) A549 cells. Quadruplicate monolayers of cells were
infected at a multiplicity of infection (MOI) of 0.03. Whole cell lysates were collected at the
indicated time points and titrated using immunostaining plaque assay described in section
2.2.2.4. Shown is the means of log10 viral titres (pfu/ml) ± standard error, n ≥ 4, with error
bars indicating standard error of the means. Continuous black line (– ) represents rRSV A2.
Continuous pink line represents rRSV A2 M(T205A). Dotted line indicates limit of
detection by immunostaining plaque assay (50 pfu/ml). Asterisk (*) denotes that the viral titres
are significantly different between the two viruses (P < 0.005) based on the nonparametric
Mann-Whitney U-test (GraphPad (Prism), La Jolla, USA).
74
3.3.3 Confirmation of sequence of rRSV A2 and rRSV A2 M(T205A)
The codon at amino acid 205 was shown to be ACA, which codes for threonine (T) for the
wild type rRSV A2, and GCT, which codes for alanine (A) for the mutant rRSV A2
M(T205A) (Figure 3-3). This shows that the rRSV M constructs were stable throughout all
passages and experimental procedures.
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Figure 3-3 Chromatogram of DNA sequencing of the matrix gene of rRSV A2 and rRSV A2
M(T205A) showing the difference between the threonine and alanine codons at amino acid 205.
76
3.4 Discussion
Reverse genetics technology, which was used to generate recombinant RSVs, ensures that the
mutant and wild type viruses being compared have identical genomes except at the sites
where mutation was performed. Therefore, the effects of the compensatory mutations in other
genes, which may appear due to natural selection in naturally-circulating strains, are not
present, allowing a direct study of the effect of a single mutation on the replication efficiency
of a virus.
RSV M205 (T) was initially selected for mutation due to its proximity to the NES
(194
IIPYSGLLLVITV206
) and its potential as a CK2 phosphorylation site. The nuclear export
of RSV M has been proposed to be regulated by a phosphorylation mechanism (Alvisi et al.,
2008); therefore RSV M205 (T) was suggested to have a possible role in the regulation of
nuclear export. In this study, the rRSV A2 M(T205A) was able to produce a relatively similar
titre to the rRSV A2 in a single cycle of replication (Vero E6 cells, Figure 3-1 A). This
suggests that amino acid residue 205 (T) is not a site that is critical for the success of RSV M
nuclear export to facilitate assembly of ribonucleoproteins and glycoproteins to produce new
infectious particles. Furthermore, although not directly tested, T205 may not be the
regulatory phosphorylation site that promotes the nuclear export of RSV M, as suggested by
the fact that the assembly of progeny virus did not appear to be affected (Vero E6 cells,
Figure 3-1 A). Regulated, timely-mannered localisation of the M protein is essential for
optimal RSV replication and assembly (Ghildyal et al., 2009), and the results obtained in this
study, from the single cycle replication assay, suggest that M is present in the cytoplasm to
facilitate particle assembly after 24 h.p.i.
Many factors regulate the capability of a virus to spread, including the inherent properties of
the virus, such as the structural proteins. In this study, the impact of M(T205A) substitution
on the capability of RSV to spread the infection from cell to cell was investigated by
conducting multiple-step replication kinetics assay with a low virus to cell ratio (MOI =
0.03). Multiple cycle replication assay is more sensitive than single cycle replication assay to
identify different properties between two virus because it can magnify the impact of small
differences (Wang and Bushman, 2006). When the initial virus-to-cell ratio is low (multiple
cycle replication assay, MOI 0.03), viral particles need more than one replication cycle to
77
maximally infect all cells, and this requires the capability of the virus to reinitiate infection in
uninfected cells.
In this study, the multiple cycle replication assay showed that rRSV A2 M(T205A) has a
defect in spreading the infection from infected to uninfected cells. In Vero E6 cells, the
multiple cycle replication assay resulted in a lower amount of infectious progeny in the rRSV
A2 M(T205A) compared to the rRSV A2 (Figure 3-2 A), unlike in the single cycle
replication assay where the rRSV A2 M(T205A) proliferated more than the rRSV A2 (Figure
3-1 A). This suggests that without the confounding effect of the antiviral IFN-α/β, rRSV A2
M(T205A) managed to produce progeny but suffered from a restricted ability to spread the
infection. This finding corroborates the observation that the processes in the replication that
associate with particle spread was likely to be affected by M205 (TA) substitution, or
M205 (T) plays an important role in the budding or release of RSV. In addition, it is
important to note the fact that rRSV A2 M(T205A) produced a higher titre than rRSV A2, in
single cycle replication assay at 18 h.p.i, may be attributed to a high starting titre in Vero E6
cells possibly the result of unremoved inoculum.
The lower titres of rRSV A2 in A549 cells (Figure 3-1 B and 3-2 B) compared to Vero E6
cells (Figure 3-1 A and 3-2 A) suggests that the products of the antiviral IFN-α/β gene restrict
the replication efficiency of RSV. This finding corroborates a previous study describing that
the absence of IFN-α/β in Vero E6 cells allows for a higher level of viral replication
compared to other cell lines such as A549 and HUVEC (Kraus et al., 2004). In the case of
rRSV A2 M(T205A), the IFN-α/β activity severely restricts its spread to the extent that viral
titre could not be detected by the immunostaining plaque assay (Figure 3-2 B). Thus, RSV M
could have an important role for IFN-α/β suppression to facilitate the spread of infection and
that M205 (T) might be functional in this role. Overall, the M(T205A) substitution confers a
defect in biological processes of RSV replication that prevent the virus to re-initiate infection
and to suppress the antiviral activity of IFN-α/β. The actual mechanism of how the single-site
M(T205A) substitution reduces RSV capability to inhibit the antiviral activity of IFN-α/β
cannot be assessed in detail based on the result obtained in this study. Likewise, whether
RSV M functions independently or in cooperation with other cellular or viral components
still requires further study.
The results obtained in this study clearly represent the characterisation of the mutant rRSV
A2 M(T205A) in comparison to the wild type rRSV A2. This was confirmed by performing
78
DNA sequencing of the M constructs of rRSV, which showed that the amino acid residue
threonine (T) in M205 of rRSV A2 was coded by the codon ‘ACA’ and the amino acid
residue alanine (A) in M205 of rRSV A2 M(T205A) was coded by the codon ‘GCT’.
Therefore, the two rRSV M constructs were stable and the viral titres, and immune responses
(detailed in Chapter 4), observed during this study truly represent the phenotypes of the
mutant rRSV A2 M(T205A) and the wild type rRSV A2 in in vitro growth.
3.5 Summary
There are some points that can be inferred based on the findings presented in this chapter.
Firstly, the M protein has a substantial role in ensuring the success of viral egress in RSV
replication. Secondly, the M protein might have a function in counteracting the antiviral
activity of IFN-α/β at the level of replication in infected cells and in the spread of infection.
Thirdly, the M205 (T) is potentially a site that is critical for M to facilitate the egress of
progeny, as well as to suppress the antiviral activity of IFN-α/β. In summary, this study
shows that the M protein is crucial for the replication efficiency of RSV.
79
CHAPTER 4
IN VITRO EXPRESSION OF PRO-
INFLAMMATORY MEDIATORS IN
RESPONSE TO INFECTION BY rRSV
A2 M(T205A)
4 Pro-Inflammatory Response in RSV Infection
80
81
4.1 Introduction
Inflammatory mediators, such as chemokines (chemotactic cytokines), are of particular
relevance in RSV infection because the inflammatory responses triggered by RSV infection
contribute largely to RSV-induced bronchiolitis and pneumonia (Garofalo et al., 1996, Dudas
and Karron, 1998, Casola et al., 2001a, Noah et al., 2002). Chemokines function in
trafficking leukocyte migration to the infection sites for pathogen clearance; but if
overexpressed, can cause damage to the body tissues due to cytotoxicity (Kindt et al., 2007).
IL-8 and RANTES are two chemokines that are activated prominently in response to RSV
infection, with IL-8 the attractant for neutrophils and RANTES the attractant of eosinophils
(Collins and Graham, 2008). Overexpression of IL-8 and RANTES during RSV infection has
been shown to associate with the development of severe bronchiolitis in infants (Mukaida et
al., 1998, Genin et al., 2000).
The expression of IL-8 and RANTES in infected cells is regulated by the activation of
transcriptional regulatory proteins. The main regulators for RANTES expression following
RSV infection are NF-κB and IRF3 (Casola et al., 2001b). Viral infection activates IRF3 by
inducing phosphorylation at serine/threonine residues in the CTD (S385, 386, 396, 398, 402,
405, and T404), which causes IRF3 nuclear translocation to promote the transcription of
interferon-stimulated genes (ISGs), including RANTES, and IFN-α/β that in turn can induce
RANTES expression via JAK-STAT signalling (Bose and Banerjee, 2003). IRF3 can also
induce RANTES activation directly, bypassing the JAK-STAT signalling pathway (Lin et al.,
1999). The induction of RANTES gene transcription by virus infection requires cooperation
between the IRF3 and NF- κB. Disruption of either pathway abolishes the ability of the other
to activate RANTES expression; however, only IRF3 is targeted by RSV, whereas NF- κB
plays more of a cofactor role (Lin et al., 1999, Genin et al., 2000, Casola et al., 2001b).
The transcriptional activators of IL-8 expression following RSV infection consist of NF- κB
and NF-IL-6, with IRF-1 acts as a cofactor (Mukaida et al., 1994, Garofalo et al., 1996,
Casola et al., 2000). The combinational activation of NF-κB and NF-IL-6 binding sites in the
5’-flanking region of the gene is required for the transcription of IL-8 gene following
sensitisation by RSV infection; neither site alone is sufficient for gene activation (Mukaida et
al., 1994, Mastronarde et al., 1996).
82
The secretion of IL-8 and RANTES has been shown to dictate the subsequent inflammatory
response and the eventual clinical symptoms in RSV disease (Olszewska-Pazdrak et al.,
1998, Miller et al., 2003). Therefore, it is of great value to study their expression in cell
culture, particularly with the aim of assessing their activation as the potential early predictor
of the severity of RSV-associated illness.
This chapter aimed to assess the impact of M(T205A) substitution on the production of pro-
inflammatory mediators RANTES and IL-8 in RSV infection, with and without the influence
of the antiviral action of IFN-α/β. The pro-inflammatory response directed to rRSV A2
M(T205A) was studied in Vero E6 cells that have a genetic defect in the IFN-β locus and
thus cannot express type-1 IFN (IFN-α/β) (Emeny and Morgan, 1979), and in A549 cells that
express the strong antiviral IFN-α/β signalling pathway. In doing so, the experimental results
from Vero E6 cells can serve as the control for expression of the IFN-α/β–mediated pro-
inflammatory response. In addition, Vero E6 cells are useful to study NF-κB-mediated
immune response without the confounding effects of IFN-α/β activation.
4.2 Methods
4.2.1 Cells and virus
Vero E6 and A549 cells were maintained in culturing medium as described in section 2.2.1.
The wild type (rRSV A2) and mutant (rRSV A2 M(T205A)) virus were passaged in Vero E6
cells, followed by immunostaining plaque assay to determine the titre of infectious virus
particle (pfu/ml) as described in section 2.2.2.
4.2.2 Single cycle replication assay
Quadruplicate monolayers of Vero E6 and A549 cells were infected with rRSV A2 or rRSV
A2 M(T205A) at an MOI of 3, in 200 µl of serum-free medium. Mock infection was also
performed using just serum-free medium. Two hours after inoculation, the medium was
replaced with 600 µl of infecting medium, and infection developed until specified end points,
which were 18, 24, 30, 36, and 48 hours post infection as described in section 2.2.3. At each
specified time point, 450 µl of culture supernatant was collected.
83
4.2.3 Multiple cycle replication assay
Quadruplicate monolayers of Vero E6 and A549 cells were infected with rRSV A2 or rRSV
A2 M(T205A) at an MOI of 0.03, in 200 µl of serum-free medium. Mock infection was also
performed using just serum-free medium. Two hours after inoculation, the medium was
replaced with 600 µl of infecting medium, and infection developed until specified end points,
which were 0, 1, 2, 3, 4, and 5 days post infection as described in section 2.2.3. At each
specified time point, 450 µl of culture supernatant was collected.
4.2.4 Enzyme-linked immunosorbent assay (ELISA)
Chemokine measurement using ELISA was performed as detailed in section 2.2.4.1. Briefly,
ELISA plate was coated with 100 µl of capture antibody (0.5 µg/ml in PBS) at room
temperature overnight. Plate was then washed four times with PBS + 0.05% Tween-20,
blocked with 300 µl PBS + 1% BSA for an hour at room temperature, and washed again as
described. IL-8 and RANTES standards and the culture supernatants were two-fold-serially-
diluted in diluent (PBS + 0.1% BSA+ 0.05% Tween-20). 100 µl diluted standard and samples
were added into the plate in triplicate and incubated for two hours at room temperature. The
plate was washed, 100 µl of 0.25 µg/ml detection antibody in diluent added, and incubated at
room temperature for 2 hours. The plate was washed again, then 100 µl of avidin-HRP
conjugate in diluent (1:200) was applied and plate incubated for 30 minutes at room
temperature, then washed. 100 µl of ABTS liquid substrate solution was added and optical
density (OD) values collected based on absorbance of light with wavelength set at 405 nm in
ELISA plate reader.
The concentration of chemokine (pg/ml) was calculated as explained in section 2.2.4.2.
Briefly, the optical density values of the standard (y-axis) were plotted against time point (x-
axis). A regression analysis of the scatterplots was done to determine the function of the
polynomial regression curve. The chemokine concentration was determined by entering the
OD values into the function. The values of chemokine concentration were then normalised
against that of the mock, and presented as the mean value from triplicate reactions with
variability expressed as the standard error of the mean.
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4.2.5 Data analysis
Data was analysed using the nonparametric Mann-Whitney U-test (GraphPad (Prism) v5, La
Jolla, USA), as explained in section 2.2.13.2. Results were considered statistically significant
if P<0.05.
4.3 Results
4.3.1 Single cycle replication assay
The chemokine expression in single cycle replication assay was investigated to understand
the initial pro-inflammatory response directed towards infection by rRSV A2 M(T205A) in
only one cycle of viral replication.
4.3.1.1 Expression of IL-8
Following infection by either rRSV A2 or rRSV A2 M(T205A), IL-8 was produced at only a
very low concentration in Vero E6 cells (Figure 4-1 A), but was expressed higher in A549
cells (Figure 4-1 B). In A549 cells, but not Vero E6 cells, rRSV A2 M(T205A) induced a
significantly higher concentration of IL-8 than rRSV A2 at 30, 36, and 48 h.p.i (Figure 4-1
B). It was shown that IL-8 had a trend toward decreased expression after 30 h.p.i in rRSV
A2-infected A549 cells; however, the trend was toward increasing expression in rRSV A2
M(T205A)-infected A549 cells (Figure 4-1 B).
85
Figure 4-1 Expression of interleukin 8 (IL-8) in cells infected with rRSV A2 or rRSV A2
M(T205A) in single cycle replication assay. (A) Expression of IL-8 in Vero E6 cells. (B)
Expression of IL-8 in A549 cells. Cells were infected with RSV at a multiplicity of infection = 3
and supernatant collected at 18, 24, 30, 36, and 48 hours post infection to measure the chemokine
concentration secreted overtime using enzyme-linked immunoassay (ELISA). The concentration
of cell-secreted chemokines is plotted as a function of time after infection. Shown are the mean
values of three separate optical density readings that were then converted into concentration
(pg/ml), with standard error of the means. Black line (– ) represents rRSV A2. Pink line
represents rRSV A2 M(T205A). Asterisk (*) denotes that the chemokine concentrations are
significantly different between the two viruses (P<0.05) based on the nonparametric Mann-
Whitney U-test (GraphPad (Prism), La Jolla, USA).
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4.3.1.2 Expression of RANTES
In contrast to IL-8, RANTES was produced in both the Vero E6 and A549 cells that were
infected by either rRSV A2 or rRSV A2 M(T205A) (Figure 4-2).
The production of RANTES in Vero E6 cells infected with rRSV A2 was significantly higher
than in cells infected with rRSV A2 M(T205A) at 18 and 48 h.p.i (Figure 4-2 A). In rRSV A2
M(T205A)-infected Vero E6 cells, RANTES expression was low, with the mean
concentration below 250 pg/ml, except at 24 h.p.i (344 pg/ml); as compared to the expression
in rRSV A2-infected Vero E6 cells, which was over 250 pg/ml in all the time points of
observation.
In A549 cells, however, there was no significant difference in RANTES expression in cells
infected by either rRSV A2 or rRSV A2 M(T205A) (Figure 4-2 B). RANTES appeared to be
induced by rRSV A2 in a biphasic fashion, where two peaks of production were observed at
30 and 48 h.p.i. The biphasic production of RANTES was not observed in rRSV A2
M(T205A)-infected cells.
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Figure 4-2 Expression of regulated upon activation, normal T-cell expressed, and secreted
(RANTES) in cells infected with respiratory syncytial virus (RSV) in single cycle replication
assay. (A) Expression of RANTES in Vero E6 cells. (B) Expression of RANTES in A549 cells.
Cells were infected with RSV at a multiplicity of infection = 3 and supernatant collected at 18,
24, 30, 36, and 48 hours post infection to measure the chemokine concentration secreted
overtime using enzyme-linked immunoassay (ELISA). The concentration of cell-secreted
chemokines is plotted as a function of time after infection. Shown are the mean values of three
separate optical density readings that were then converted into concentration (pg/ml), with
standard error of the means. Black line (– ) represents rRSV A2. Pink line represents rRSV
A2 M(T205A). Asterisk (*) denotes that the chemokine concentrations are significantly
different between the two viruses (P<0.05) based on the nonparametric Mann-Whitney U-test
(GraphPad (Prism), La Jolla, USA).
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4.3.2 Multiple cycle replication assay
The chemokine expression in multiple cycle replication assay was investigated to understand
the pro-inflammatory response directed towards infection by rRSV A2 M(T205A) in the
condition of more than one cycle of viral replication.
4.3.2.1 Expression of IL-8
There are three notable observations from IL-8 production in multiple cycle replication assay.
Firstly, IL-8 production in multiple cycle replication assay, in agreement with the single cycle
replication assay (Figure 4-1), was observed only in rRSV-infected A549, and not in Vero E6
cells (Figure 4-3). In A549 cells, a significantly higher titre of IL-8 was observed in cells
infected with rRSV A2 compared to rRSV A2 M(T205A) at days 0 – 4 post infection (Figure
4-3 B). This pattern is in contrast to IL-8 production in rRSV-infected A549 cells in single
cycle replication assay, where rRSV A2 M(T205A)-infected A549 cells secreted a
significantly higher titre of IL-8 than rRSV A2-infected A549 cells (Figure 4-1 B).
Secondly, to compare between IL-8 expression in rRSV A2-infected A549 cells in single
cycle replication assay (Figure 4-1 B) and multiple cycle replication assay (Figure 4-3 B)
during a similar period of observation (48 h.p.i (single cycle) or 2 d.p.i (multiple cycle)), the
values of IL-8 concentration in single cycle replication assay were less variable than in
multiple cycle replication assay. In single cycle replication assay, the lowest concentration of
secreted IL-8 was 34.72 pg/ml, the highest concentration was 1031.07 pg/ml, and the mean
value was 703.13 pg/ml. In multiple cycle replication assay, the lowest concentration was 0
pg/ml, the highest concentration was 1044.75 pg/ml, while the mean value was 417.97 pg/ml.
Thirdly, the rRSV A2 M(T205A)–infected A549 cells produced a lower IL-8 concentration in
multiple cycle replication assay than in single cycle replication assay in a period of 2 d.p.i (48
h.p.i) (Figure 4-1 B and 4-3 B). In single cycle replication assay, the mean value was 915.9
pg/ml; while in multiple cycle replication assay, the mean value was 27.16 pg/ml.
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Figure 4-3 Expression of interleukin 8 (IL-8) in cells infected with respiratory syncytial virus
(RSV) in multiple cycle replication assay. (A) Expression of IL-8 in Vero E6 cells. (B)
Expression of IL-8 in A549 cells. Cells were infected with RSV at a multiplicity of infection =
0.03 and supernatant collected at day 0, 1, 2, 3, 4, and 5 post infection to measure the
concentration of chemokines expressed overtime using enzyme-linked immunoassay (ELISA).
The concentration of cell-secreted chemokines is plotted as a function of time after infection.
Shown are the mean values of three separate optical density readings that were then converted
into concentration (pg/ml) with standard error of the means. Black line (– ) represents rRSV A2.
Pink line represents rRSV A2 M(T205A). Asterisk (*) denotes that the chemokine
concentrations are significantly different between the two viruses (P<0.05) based on the
nonparametric Mann-Whitney U-test (GraphPad (Prism), La Jolla, USA).
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4.3.2.2 Expression of RANTES
RANTES was produced in Vero E6 and A549 cells in multiple cycle replication assay
(Figure 4-4), but only rRSV A2-infected cells expressed RANTES at significantly higher
concentration than rRSV A2 M(T205A)-infected cells. In addition, Vero E6 cells expressed
an overall higher concentration of RANTES than A549 cells. The mean titre of RANTES
expression in rRSV A2-infected Vero E6 cells was 952.5 pg/ml and in A549 cells was 532.5
pg/ml. In rRSV A2 M(T205A)-infected cells, the mean titre was 184.8 pg/ml (Vero E6) and
49.15 pg/ml (A549).
In rRSV A2-infected Vero E6 and A549 cells, expression of RANTES peaked at day 4 post
infection, where the mean titre in Vero E6 cells was 2210.41 pg/ml and the mean titre in
A549 cells was 1275 pg/ml. The rRSV A2 M(T205A)-infected cells, however, produced peak
RANTES expression at day 1 post infection (24 h.p.i), albeit in much lower titre. The mean
titre in Vero E6 cells was 66.80 pg/ml and the mean titre in A549 cells was 196.64 pg/ml.
Based on the findings, two differences were observed between RANTES expression in
multiple cycle and single cycle replication assays. Firstly, rRSV A2-infected Vero E6 and
A549 cells always produced a higher titre of RANTES in multiple cycle replication assay; in
contrast to in single cycle replication assay, where rRSV A2 M(T205A)-infected A549 cells
managed to secrete a relatively similar titre of RANTES during a period of 48 h.p.i.
Secondly, in multiple cycle replication assay overall RANTES expression in rRSV A2-
infected cells was higher in Vero E6 (mean titre = 952.50 pg/ml) than in A549 cells (mean
titre = 448.57 pg/ml), while in single cycle replication assay the overall titre was lower in
Vero E6 (878.70 pg/ml) than in A549 cells (1177.09 pg/ml).
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Figure 4-4 Expression of regulated upon activation, normal T-cell expressed, and secreted
(RANTES) in cells infected with respiratory syncytial virus (RSV) in multiple cycle
replication assay. (A) RANTES expression in Vero E6 cells. (B) RANTES expression in
A549 cells. Cells were infected with RSV at a multiplicity of infection = 0.03 and
supernatant collected at day 0, 1, 2, 3, 4, and 5 post infection to measure the concentration
of chemokines expressed overtime using enzyme-linked immunoassay (ELISA). The
concentration of cell-secreted chemokines is plotted as a function of time after infection.
Shown are the mean values of three separate optical density readings that were then
converted into concentration (pg/ml) with standard error of the means. Black line (– )
represents wild type rRSV A2. Pink line represents CK2 phosphorylation mutant
rRSV A2 M(T205A). Asterisk (*) denotes that the chemokine concedntrations are
significantly different between the two viruses (P<0.05) based on the nonparametric Mann-
Whitney U-test (GraphPad (Prism), La Jolla, USA).
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4.4 Discussion
IL-8 was expressed in rRSV-infected A549 cells, but not in Vero E6 cells, in both single
cycle and multiple cycle replication assays. There are two possible mechanisms to explain
this observation. Firstly, IL-8 production in RSV-infected respiratory epithelial cells (A549
cells) has been shown to be controlled by the combinational activation of NF-κB and NF-IL-6
transcriptional factors (Mastronarde et al., 1996). Vero E6 cells have a functional NF-κB
transcriptional factor, but no studies have confirmed the presence of NF-IL-6; therefore, this
potentially partial activation might lead to the lack of IL-8 expression in Vero E6 cells.
Secondly, the IFN-α/β expression in A549 cells might provide a positive feedback, as a
soluble mediator that can signal the ‘antiviral state’, resulting in the amplification of IL-8
expression, particularly during the secondary activation (Bose and Banerjee, 2003).
The concentration of IL-8 in the rRSV A2 M(T205A)-infected A549 cells was significantly
higher than that of the rRSV A2-infected A549 cells in single cycle replication assay (Figure
4-1 B), but it was the opposite way in multiple cycle replication assay (Figure 4-3 B). Based
on this finding, there are two points that can be inferred. Firstly, M(T205A) substitution
might potentially dampen the ability of rRSV in suppressing the IL-8 production, allowing
the secretion of a high IL-8 concentration during the initial recognition. However, continuing
IL-8 production requires particle proliferation, particularly to activate the NF-κB activation.
(Fiedler et al., 1996, Garofalo et al., 1996). Therefore, the rRSV A2 M(T205A) infection in
multiple cycle replication assay resulted in muted IL-8 production because the rRSV A2
M(T205A) lacked the ability to spread (Figure 3-2 B). Secondly, IL-8 expression has been
shown to be dependent on a certain threshold of the initiating inoculum titre (Arnold et al.,
1994), probably representing the number of infected cells. As a result, the rRSV A2
M(T205A) induced a higher concentration of IL-8 in single cycle replication assay where
cells were initially infected with 100 times greater virus to cell ratio (MOI of 3), but not in
multiple cycle replication assay, where cells infected with an MOI of 0.03.
M (T205A) substitution was shown to also affect RANTES, with expression only occurring
in an infection system that exerts the antiviral IFN-α/β (Figure 4-2 B). The expression of
RANTES is regulated by IRF3, which is the transcriptional factor of IFN-α/β, independently
or with the assistance of NF-κB (Lin et al., 1999, Casola et al., 2001b, Chew et al., 2009).
Both the transcriptional factors are present in Vero E6 cells, but the IFN-α/β-facilitated
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JAK/STAT signalling, that is present only in A549 cells, amplifies chemokine production by
introducing an ‘antiviral state’ in the population of cells (Bose and Banerjee, 2003). In doing
so, the redirection might represent the effect of IFN-α/β in elevating RANTES production.
By taking the fact that rRSV A2 M(T205A) induced the production of a higher titre of
RANTES in single cycle replication (MOI = 3) (Figure 4-2 B) than in multiple cycle
replication (MOI = 0.03) (Figure 4-4 B) into consideration, it can be inferred that viral
replication is required for RANTES expression. Indeed, it has been shown that RANTES
secretion in RSV-infected cells requires replicating particles in infected cells (Miller et al.,
2003). One importance of the viral replication for RANTES expression is by inducing the
production of the IFN-α/β, which functions to amplify RANTES production. The
transcriptional factor of IFN-α/β, IRF3, is activated only by the recognition of dsRNA, which
in the case of (-) ssRNA virus like RSV, is produced as the genomic intermediate during viral
replication (Stetson and Medzhitov, 2006). Therefore, the disrupted viral replication can
eliminate the optimal IFN-α/β-facilitated signalling pathway. As such, the disrupted
proliferation ability of rRSV A2 M(T205A) likely contributed to the low induction of
RANTES production despite the presence of IFN-α/β.
The suggestion that M(T205A) substitution potentially dampen the ability of rRSV in
suppressing the IL-8 production to result in a higher IL-8 concentration during the initial
recognition, might work for the RANTES activation as well. RANTES expression has been
shown to be elevated in 24 h.p.i (Figures 4-2 A, 4-4 A, and 4-4 B), but depleted afterwards,
consistent with the requirement of viral replication for RANTES expression. As such, it can
be inferred that M(T205A) substitution might result in the suppression of pro-inflammatory
mediators, but the phenotype was not expressed since the rRSV A2 M(T205A) lacks the
ability to spread.
The results presented in this chapter suggest that RSV M has a significant role in the
regulation of the expression of pro-inflammatory mediators in the innate immune response.
This role is suggested to be associated with the proliferating ability of RSV, since replication
processes have been shown to be important for the production of RANTES and IL-8 in RSV
infection (Fiedler et al., 1996, Miller et al., 2003). RSV proteins that have been shown to alter
the expression of IL-8 and RANTES, as well as their transcriptional factors IRF3 and NF-
κB, are NS1 and NS2 (Spann et al., 2005). Therefore, it is interesting to propose a possible
cooperation between RSV M, NS1, and NS2 in regulating the expression of IL-8 and
94
RANTES. Further study is needed to elucidate the exact role of the M protein in inducing
pro-inflammatory mediators in RSV infection.
4.5 Summary
The findings presented in this chapter shows that the M(T205A) substitution led to a lower
chemokine production in infected cells, very likely associated with the reduced ability of the
mutant to undergo proliferation. One mechanism underlying this observation is that the
proliferation defect leads to the weakened induction of IFN-α/β that has a role in amplifying
chemokine expression by introducing an ’antiviral state’ among the population of cells.
95
CHAPTER 5
THE SUBCELLULAR LOCALISATION
OF hMPV N PROTEIN IN
TRANSFECTED CELLS
5 The Subcellular Localisation of hMPV N Protein in Transfected Cells
96
97
5.1 Introduction
The nucleocapsid (N) protein of several (-)ssRNA viruses has been shown to undergo
independent nucleocytoplasmic transport in order to perform non-structural functions in the
nucleus. For example, the nuclear localisation of infectious bronchitis virus (IBV) N is
associated with the reorganisation of fibrillarin distribution in an attempt to delay cell growth
(Hiscox et al., 2001, Chen et al., 2002), while the nuclear localisation of measles virus (MV)
N protein blocks the nuclear translocation of STATs (Takayama et al., 2012). Preliminary
investigation showed that the hMPV N localises to the nucleus in the late stage of infection
(day 5 post infection) (Ghildyal R & Gahan M, personal communication). Putative NES and
NLS motifs were identified in the ORF of hMPV Can97-83 N gene (GenBank accession
number AY145278). The putative NES motif 1MSLQGIHLSDLSYKH
15, was identified by
submitting the hMPV Can97-83 N amino acid sequence to EXPASY
(http://prosite.expasy.org/), a resource portal that provides access to databases and software
tools in motif recognition. This motif conforms to the common NES motif LXXXLXXLXL,
which is rich in hydrophobic residues (la-Cour et al., 2004). A putative bipartite NLS-like
motif 185
RRANRVLSDALKRYPR200
was manually identified (R. Ghildyal), and resembles
the RSV M NLS 154
TSKKVIIPTYLRSISVRNK172
(Ghildyal et al., 2006). As a first step
towards determining the functionality of these motifs, and as a prelude to elucidating the
possible nuclear functions of hMPV N, the subcellular localisation of hMPV N was studied in
transfected mammalian cells.
The functionality of the NES and NLS was studied in the context of full-length hMPV N and
as independent transport motifs. Identifying the functionality of a structural motif as a part of
a protein, and as an individual motif, is important because of the following reasons. Firstly,
motifs that are exposed on the surface are more likely to be recognised by nuclear transport
factors; secondly, the functionality of a motif might be assisted by unique flanking
sequence(s) (Silver, 1991). The subcellular localisation of full-length hMPV N and the
transport motif was studied by expressing full-length and deletion constructs generated based
on hMPV N gene in a transfection host.
To study the subcellular localisation, hMPV N constructs were expressed as green fluorescent
protein (GFP)-fusion proteins, in which GFP is positioned in the N-terminal end of each
construct. GFP is a green-light emitting marker for gene expression that has stable
98
fluorescence, even after formaldehyde treatment, does not have a toxic effect, and does not
interfere with cell growth and function (Chalfie et al., 1994). The localisation of hMPV N
constructs in transfected cells can be traced via GFP fluorescence upon excitation by the blue
laser (395 – 490 nm).
Cloning in an expression vector is one approach to generate a GFP-tagged ORF without
interfering with the function of the ORF itself. The GatewayTM
recombination cloning (RC)
technology was used to clone the hMPV N constructs into an expression vector. The
GatewayTM
RC technology is highly accurate in generating expression clones, being
facilitated by two specific recombination steps and stringent negative and positive selection,
to specifically maintain the directionality of the ORF (Hartley et al., 2000). The first
recombination step is to produce an entry clone and the second recombination is to generate
the expression clone. Both recombination are facilitated by specific recombination sites,
which is a pair of attB sites in hMPV N constructs and the complementary pair of attP sites in
the vector, to allow for conservative (no nucleotides are added or lost), directional
(orientations of segments in new clones are fixed according to the orientations of the att
sites), and concerted (all strand exchanges occur in protein-DNA complexes, no free ends are
available for side reactions) cloning (Hartley et al., 2000, Hopkins et al., 2012). The selection
for each clone is facilitated by the negative selection marker, ccdB gene, and the antibiotic
resistance gene in each vector. ccdB gene encodes a cell-killing protein, which affects
bacterial gyrase, an enzyme that facilitates DNA unwinding during replication (Reyrat et al.,
1998). Importantly, the 5’-end recombination site of the GatewayTM
RC, attB1, contains the
translation signals for E. coli and mammalian cells, making the technology versatile for
protein expression in both prokaryotic and mammalian cells (Hartley et al., 2000).
Transfection is a method of introducing foreign genetic material into a cell with the aim of
analysing gene expression. The use of mammalian cells give the advantage of assisting
peptide folding, providing posttranslational modifications compared to prokaryotic cells
(E. coli), and allowing the expression of protein in the proper intracellular compartments
(Kingston, 2003, Brondyk, 2009, Hopkins et al., 2012). Additionally, transfection has the
advantage of protein overexpression, allowing the researcher to observe the protein separately
from other components of the cells. Also, cells used in transfection have generally been well
characterised such that no cellular parameters interfere with the investigation. Transient
transfection has the advantage of not requiring integration of the gene of interest into host
99
chromosomal DNA; therefore can allow quick observation of the subcellular localisation of
hMPV N constructs (24 – 48 hours post transfection) (Liew et al., 2007).
This study aimed to investigate the nucleocytoplasmic transport of hMPV N in transiently
transfected Cos-7 cells, by using the GatewayTM
RC technology to generate an expression
clone containing GFP-tagged hMPV N constructs.
5.2 Methods
5.2.1 Generation of constructs and recombination cloning
Presented is a brief overview of generating hMPV N constructs and recombinational cloning
using Gateway technology. Total RNA was first extracted from hMPV-infected Vero E6 cells
(5.2.1.1) and reverse transcribed into cDNA (5.2.1.2); then the hMPV N constructs were
generated via PCR using the cDNA as template and specific attB-tagged primer pairs (table
2-15) (5.2.1.3). The PCR products, were purified (5.2.1.4), and each construct was then
inserted into the donor vector (pDONR®207) by recombination between attB1/attP1 and
attB2/attP2 sites, catalysed by BP ClonaseTM
(5.2.1.5). The hMPV N construct was inserted
into the expression clone by recombination between the entry clone and the expression vector
pEPI-DESTC (recombination between attL1/attR1 and attL2/attR2 sites), catalysed by LR
ClonaseTM
(5.2.1.5). The products of the BP and LR recombination reactions were
transformed into electrocompetent E. coli to generate the entry and expression clone
(5.2.1.6).
5.2.1.1 Extraction of total RNA from hMPV-infected cells
80%-confluent Vero E6 monolayer was infected with hMPV at an MOI of 1 and incubated at
37o C for 24 hours (section 2.2.2.2). The infection medium was replaced with 2.5 ml of
TRIzol® reagent and flask incubated for 5 minutes at room temperature to lyse the cells. Cell
suspension was transferred into a 10 ml conical tube, mixed with 0.5 ml chloroform,
incubated for 3 minutes at room temperature, and centrifuged at 12,000x g for 15 minutes at
4o C. The aqueous phase (clear) was transferred into a new 10 ml conical tube. From this
aqueous phase, RNA was precipitated with 1.25 ml of isopropyl alcohol for 10 minutes at
room temperature, and pelleted by centrifugation at 12,000x g and 4o C for 10 minutes. RNA
was washed with 2.5 ml of 75% ethanol, and re-pelleted by centrifugation at 7,500x g and 4o
100
C for 5 minutes. The RNA was air dried and resuspended in nuclease-free water. The RNA
extraction is described in detail in section 2.2.6.1.
5.2.1.2 Reverse transcription
1 µg hMPV RNA was mixed with 270 ng of Oligo(dT)10, 2mM dNTP, and DEPC-treated
water in a final volume of 10 µl, then incubated at 65o C for 10 minutes. The reaction was
added into a mixture of 2X RT buffer, 10 U RNase inhibitor, 5 U reverse transcriptase, and
DEPC-treated water (to a final volume of 10 µl). The final reaction was incubated at 45o C
for 45 minutes. The reverse transcription was terminated by incubation at 70o C for 15
minutes.
5.2.1.3 Polymerase chain reaction (PCR)
PCR was done to generate the hMPV N constructs, by using cDNA as template and a specific
primer pair (Table 2-15) for each construct. 5 µl of cDNA was mixed with 1X Mango Taq
reaction buffer, 1.5 mM MgCl2, 0.2 mM dNTP, 0.25 pmoles of each forward and reverse
primer, 0.025 U Mango Taq DNA polymerase, and nuclease-free water to a final volume of
40 µl. The reaction was subjected to cycling conditions consisting of 95o C incubation for 7
minutes, followed by 35 repeats of 96o C for 30 seconds, 58
o C for 45 seconds, and 72
o C for
1 minute.
5.2.1.4 Gel purification of PCR products
The detailed description of gel purification of PCR products is explained in section 2.2.5.3.
Briefly, whole PCR product was mixed with DNA loading dye (5:1) and electrophoresed in
1% agarose gel (section 2.2.9). The gel was visualised using UV light and the desired DNA
band excised and dissolved in Membrane Binding Solution (1 µl solution for 1 mg gel) via
incubation at 65o C for 10 minutes. The dissolved gel was transferred to an SV Minicolumn
and incubated for 1 minute at room temperature. The column was centrifuged at 19,000x g
for 1 minute and the membrane washed twice with Membrane Wash Solution: the first wash
used 700 µl and the second wash used 500 µl, with 19,000x g and 1 minute centrifugation for
each wash. An additional centrifugation was carried out to remove the residual wash solution,
then the purified DNA eluted with 30 µl of nuclease-free water via 19,000x g for 1 minute
centrifugation.
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5.2.1.5 DNA ligation
To generate the entry clones, purified PCR products of a hMPV N construct, pDONR®207,
BP ClonaseTM
, and TE buffer (pH 8.0) were incubated at 25o C overnight, then transformed
into ElectroMAXTM
DH10BTM
competent E. coli (2.2.12.2) and plasmid DNA isolated using
the Miniprep or Midiprep kit (Qiagen) (section 2.2.6.3).
To generate the expression clones, entry clones (pDONR®207.hMPV N construct), pEPI-
DESTC, LR ClonaseTM
, and TE buffer (pH 8.0) were incubated at 25o C overnight, then
transformed into ElectroMAXTM
DH10BTM
competent E. coli (2.2.12.2) and plasmid DNA
isolated using the Miniprep or Midiprep kit (Qiagen) (section 2.2.6.3).
Selection for the growth of entry clone- or expression clone-containing bacteria was done by
plating bacterial suspension on antibiotic-supplemented LB agar, with gentamycin for
selection of entry clone and kanamycin for selection of expression clone.
5.2.1.6 Transformation
1 – 10 µl of BP or LR reaction was mixed with 50 µl of ElectroMAXTM
DH10BTM
competent
E. coli cell suspension, inserted into a pre-chilled 1 ml cuvette, and transformed into the
bacterial cells via electroporation using 25 µF capacitance, 200 Ω resistance, and 2.0 kV
voltage. 450 μl of LB medium was added, then the transformed bacteria incubated at 37o C
and 220 rpm for 1 hour. 100 – 150 µl of the bacterial suspension was plated on an antibiotic-
supplemented LB agar plate, and cultured overnight at 37o C.
5.2.2 Transient transfection into Cos-7 cells
5.2.2.1 Plasmid preparation
The expression clones (pEPI.DESTC - hMPV N constructs) were firstly selected in LB agar
by the lethal marker ccdB gene product and antibiotic selection, gentamycin for entry clone
and kanamycin for expression clone, then plasmid DNA isolated as described in section
2.2.6.3. Plasmids were isolated using Miniprep Kit (Qiagen), or Midiprep Kit (Qiagen) if a
larger amount of DNA was required.
For Miniprep plasmid isolation, selected colonies from antibiotic-supplemented LB agar were
re-cultured in 5 ml of antibiotic-supplemented LB medium (37o C, 220 rpm, 24 h). Bacterial
102
cells were pelleted through centrifugation at 4,100x g and 4o C for 20 minutes, and
resuspended in 250 µl of buffer PI. 250 µl of buffer P2 was added, followed by 350 µl of
buffer N3. The solution was centrifuged at 16,500x g for 10 minutes and supernatant loaded
onto a QIAprep® spin column. The column was centrifuged at 16,500x g, washed with 0.5 ml
of buffer PB via centrifugation at 16,500x g, and washed again with 0.75 ml of buffer PE and
centrifugation at 16,500x g. An additional centrifugation was carried out to remove the
residual wash buffers, then the plasmid eluted with 30 µl of RNase-free water.
For Midiprep plasmid isolation, a starter culture was prepared by growing the selected
bacterial colonies in 5 ml of antibiotic-supplemented LB medium at 37o C, 300 rpm, for 8
hours. 50 μl of the starter culture was cultured in 25 ml of antibiotic-supplemented LB at 37o
C, 300 rpm, overnight. Bacterial cells were pelleted by centrifugation at 6000x g and 4o C for
15 minutes, then resuspended in 4 ml of buffer P1. 4 ml of buffer P2 was added, the solution
incubated at room temperature for 5 minutes, then 4 ml of buffer P3 added, and solution
incubated for 15 minutes on ice. The tube was centrifuged at 21,800x g and 4o C for 30
minutes; then supernatant transferred into a new 15-ml conical tube and re-centrifuged at
21,800x g and 4o C for 15 minutes. Subsequently, the supernatant was applied to a QIAGEN-
tip 100, which was washed twice with 10 ml of buffer QC. Plasmid was then eluted in 5 ml of
buffer QF, precipitated using 3.5 ml isopropanol, and pelleted by centrifugation at 18,000x g
and 4o C for 18 minutes. The DNA was washed with 2 ml of 70% room-temperature ethanol
via centrifugation at 18,000x g for 10 minutes. The DNA was air dried for 10 minutes and
redissolved in 500 μl of RNase-free water.
The concentration of plasmid was determined using NanoDropTM
1000 UV/Vis
spectrophotometer (section 2.2.8). Briefly, 2 μl of DNA sample was loaded on the pedestal
and DNA concentration was measured by absorbance at 260 nm (A260), A280, and A230. The
ratios of absorbance at A260/A280 and A260/A230 determine the purity of DNA. The DNA
concentration was presented in ng/µl.
5.2.2.2 Transfection
The transient transfection of Cos-7 cells was performed using cationic-lipid (liposome)
platform of Lipofectamine 2000 (Invitrogen), as described in detail in section 2.2.11.1.
Briefly, 3 X 105 of Cos-7 cells were seeded on 24-mm glass coverslips placed in a 6-well
plate, then incubated at 37o C, with 5% CO2 supplementation, for a maximum of 24 hours. 2
103
µg of expression clones containing GFP alone or GFP–tagged hMPV N construct was
prepared in serum-free DMEM and mixed in 1:1 (µg:ml) ratio with LipofectamineTM
2000
Transfection Reagent diluted in serum-free DMEM (1:25), and incubated for 20 minutes at
room temperature. The transfection mix was dropped onto the monolayer, incubated at 37o C,
5% CO2 for 20 minutes, then 2 ml of DMEM + 10% FBS was added. The transfected cells
were maintained at 37o C, 5% CO2 and cells observed at 24 and 48 hours post tranfection.
5.2.3 Confocal laser scanning microscopy (CLSM)
5.2.3.1 Image analysis
The detail steps of cell imaging using CLSM are presented in section 2.2.11.2. The coverslip
was placed on a ring-shaped coverslip holder, covered with 37o C PBS. A drop of immersion
oil was placed on top of the objective of Nikon Eclipse T to layer between the lens and
coverslip, and cells were directly observed.
Cell images were captured and recorded in a JPEG2000 format using the NIS-Elements 64 bit
3.21.00 software. Digital images were analysed with ImageJ 1.45M software (Ferreira &
Rasband, 2011). The pixel density of fluorescence in the nucleus (Fn) and in the cytoplasm
(Fc) were measured and the ratio of nuclear and cytoplasmic fluorescence was calculated
based on the formula Fn/c =
, where Fn is the pixel density of nuclear fluorescence, Fc
is the pixel density of cytoplasmic fluorescence, and Fb is the pixel density of background
fluorescence.
5.2.3.2 Statistical analysis
The mean value of Fn/Fc ratio for each construct was determined from total cells observed
(≥ 10), and presented in a bar chart, with variability expressed as the standard error of the
mean (SEM). The value of 1.0 (in live cells) and 1.4 (in fixed cells) was used to draw a line,
where Fn/Fc greater than 1.0 or 1.4 denotes nuclear localisation and Fn/Fc less than 1.0 or 1.4
denotes cytoplasmic localisation. The statistical difference of Fn/Fc ratio between each
hMPV N construct was assessed using the nonparametric Mann-Whitney U test (2-tailed)
(GraphPad (Prism) v.5 software (GraphPad, La Jolla, USA)).
104
5.3 Results
5.3.1 The generation of hMPV N constructs
Four constructs were generated by RT-PCR from total RNA isolated from hMPV-infected
Vero E6 cells as template and the specific attB-tagged primer pairs (Figure 5-1). The four
constructs were the full-length hMPV N, fragment A (hMPV N(1-250)) that contains both the
NES and NLS motifs, fragment B (hMPV N(1-199)) that contains the NES motif and part of
the NLS motif, and fragment C (hMPV N(192-394)) that lacks the NES and contains part of
the NLS motif (Figure 5-1). The constructs were confirmed as correct by PCR (Figure 5-2)
and sequencing (Appendix).
105
Figure 5-1 Schematic representation of hMPV N constructs generated in this study. The
constructs were designed to accommodate independent investigation of nuclear transport. PCR
was conducted as described in section 2.2.5, using cDNA of total RNA from hMPV-infected
Vero E6 cells as template and specific pair of primers for each construct. NES stands for nuclear
export signal. NLS stands for nuclear localisation signal. Sequence of the putative NES and
NLS is shown with residues in red denoting essential residues within the motif. Right arrow
indicates forward direction of primer. Left arrow indicates reverse direction of primer. Full
length hMPV N (residue 1 – 394) contains both NES and NLS. Fragment (fr) A hMPV N
(residue 1 – 250) contains both NES and NLS, but without the carboxy end of the protein.
Fragment (fr) B hMPV N (residue 1 – 199) contains intact NES, but partial NLS. Fragment (fr)
C (residue 192 – 394) contains partial NLS. attB1 and attB2 sequences attached to the forward
and reverse primers specify the attB recombination sites that are complementary to the attP
recombination sites in the entry vector to facilitate the recombinational cloning.
106
Figure 5-2 Human metapneumovirus N (hMPV N) gene constructs and vectors used
to investigate the subcellular localisation of the constructs. A. Lane 1 - HyperLadderTM
DNA molecular weight marker, lane 2 – full-length hMPV N, lane 3 – pDONR® 207
(empty entry vector), lane 4 – pEPI.DESTC (empty expression vector). hMPV N gene
was obtained from reverse transcription polymerase chain reaction (RT-PCR) of total
RNA isolated from hMPV-infected Vero E6 cells and then gel-purified as described in
section 2.2.5. pDONR®207 and pEPI-DESTC were isolated from re-culturing the
glycerol stock as described in section 2.2.6.3. B. Lane 1 - HyperLadderTM
DNA
molecular weight marker, lane 2 – fragment A (hMPV N(1-250)), lane 3 – fragment B
(hMPV N(1-199)), lane 4 – fragment C (hMPV N(192-394)). The truncated constructs
of hMPV N gene were generated using PCR with cDNA as template, together with
appropriate primer pairs (table 2-15, figure 5-1), and then gel purified as described in
section 2.2.5. 1 μl of DNA was loaded on to 1% agarose gel stained with 0.01%
GelRedTM
and electrophoresed at 80 volts for 90 minutes. Numbers on the left indicate
molecular weight in base pairs (bp). The length of each PCR product is indicated on
the right.
107
5.4 The GatewayTM
recombination cloning (RC) technology system
5.4.1 BP recombination reaction
The success of BP recombination was assessed to identify the correct transfer of hMPV N
constructs into pDONR®207
using PCR and DNA sequencing methods (section 2.2.7).
The confirmation of the hMPV N constructs recombination into the entry vector
pDONR®207 using PCR is presented in Figure 5-3. The size of DNA (hMPV N constructs)
identified by PCR was as follows: full length hMPV N (Figure 5-3 A, Lane 1) 1185 bp,
fragment A (Figure 5-3 B, Lane 2) 770 bp, fragment B (Figure 5-3 C, lane 2) 596 bp, and
fragment C (Figure 5-3 D, Lane 2) 609 bp. The size of PCR products is as expected and
confirms the correctness of the insert.
Uncut plasmid containing each of the hMPV N construct was also run on an agarose gel and
the size was as follows: pDONR®207.full length hMPV N (Figure 5-3 A, Lane 2) 4553 bp,
pDONR®207.fragment A (Figure 5-3 B, Lane 1) 4110 bp, pDONR
®207.fragment B (Figure
5-3 C, lane 1) 3964 bp, and pDONR®207.fragment C (Figure 5-3 D, Lane 1) 3977 bp. The
size of entry clones shown in the agarose gel may not in accordance with their predicted size
because a plasmid can move in an agarose gel in the form of coiled DNA that does not allow
for the preview of the actual base-pair (bp) measurement.
The presence of the hMPV N constructs inserted into the pDONR®207 was also confirmed
using DNA sequencing according to a method described in section 2.2.7.2, and as shown in
Appendix. In the hMPV N constructs, there are some nucleotide changes compared to the N
gene of Can97-83. The similar changes occurred in all constructs generated in this study, but
do not change the amino acid sequence. Therefore, the presence of each of hMPV N
construct in the entry clone was confirmed.
108
Fig
ure
5-3
Scr
eenin
g f
or
E.
coli
co
lonie
s co
nta
inin
g t
he
pD
ON
R®207 –
hM
PV
N c
onst
ruct
s u
sing
PC
R a
s d
escr
ibed
in s
ect
ion
2.2
.7.
Pla
smid
was
extr
acte
d f
rom
ind
ivid
ual
colo
nie
s an
d u
sed a
s te
mp
late
fo
r P
CR
usi
ng t
he
pair
s o
f pri
mer
use
d t
o g
ener
ate
each
const
ruct
. 1 µ
l o
f p
lasm
id D
NA
or
5 µ
l o
f P
CR
pro
duct
was
lo
aded
on t
o t
he
well
s o
f 1%
agar
ose
gel st
ained
wit
h 0
.01%
GelR
edT
M a
nd e
lect
rophore
sis
per
form
ed u
sing 8
0V
ele
ctri
c vo
ltag
e. I
mag
e w
as t
aken
wit
h U
V i
llu
min
atio
n.
L –
Hyp
erL
adder
TM
DN
A m
ole
cula
r w
eig
ht
mar
ker
. (A
) L
ane
1 –
co
nfi
rmat
ion o
f hM
PV
N i
n p
DO
NR
®2
07
.hM
PV
N v
ia P
CR
(si
ze 1
18
5 b
p),
Lane
2 –
uncu
t pD
ON
R®20
7.h
MP
V N
. (B
) L
ane
1 –
uncu
t pD
ON
R207.f
rA,
Lan
e 2 –
co
nfi
rmat
ion o
f fr
agm
ent
A (
hM
PV
N (
1-2
50))
in
pD
ON
R®207.f
rA v
ia P
CR
(si
ze 7
70 b
p).
(C
) L
ane
1 –
uncu
t pD
ON
R®20
7.f
rB,
Lan
e 2 –
co
nfi
rmat
ion o
f fr
agm
ent
B (
hM
PV
N (
1-
199))
in p
DO
NR
®207.f
rB v
ia P
CR
(si
ze 5
96 b
p).
(D
) L
ane
1 –
uncu
t pD
ON
R®207.f
rC,
Lane
2 –
co
nfi
rmat
ion o
f fr
agm
ent
C
(hM
PV
N (
200
-394))
in p
DO
NR
®207.f
rC v
ia P
CR
(si
ze 6
09 b
p).
109
5.4.2 LR recombination reaction
The success of LR recombination was assessed via PCR and DNA sequencing to identify the
correct transfer of hMPV N constructs into pEPI-DESTC using PCR and DNA sequencing
methods (section 2.2.7).
The confirmation of hMPV N constructs recombination into the expression vector pEPI-
DESTC using PCR is presented in Figures 5-4. The size of DNA (hMPV N constructs)
identified by PCR was as follows: full length hMPV N (Figure 5-4 A, Lane 2) 1185 bp,
fragment A (Figure 5-4 B, Lane 2) 770 bp, fragment B (Figure 5-4 C, lane 2) 596 bp, and
fragment C (Figure 5-4 D, Lane 2) 609 bp. The size of PCR products is as expected and
confirms the correctness of the insert.
Uncut plasmid containing each of the hMPV N construct was also run on an agarose gel and
the size was as follows: pEPI-DESTC.full length hMPV N (Figure 5-4 A, Lane 1) 7945 bp,
pEPI-DESTC.fragment A (Figure 5-4 B, Lane 1) 7530 bp, pEPI-DESTC.fragment B (Figure
5-4 C, lane 1) 7415 bp, and pEPI-DESTC.fragment C (Figure 5-4 D, Lane 1) 7369 bp. The
size of entry clones shown in the agarose gel may not in accordance with their predicted size
because a plasmid can move in an agarose gel in the form of coiled DNA that does not allow
for the preview of the actual base-pair (bp) measurement.
The presence of the hMPV N constructs inserted into the pEPI-DESTC was also confirmed
using DNA sequencing according to a method described in section 2.2.7.2, and as shown in
Appendix. In the hMPV N constructs, there are some nucleotide changes compared to the N
gene of Can97-83. The similar changes occurred in all constructs generated in this study, but
do not change the amino acid sequence. Therefore, the presence of each of hMPV N
construct in the expression clone was confirmed.
110
Fig
ure
5-4
Scr
eenin
g f
or
E.
coli
co
lonie
s co
nta
inin
g t
he
pE
PI-
DE
ST
C –
hM
PV
N c
onst
ruct
s usi
ng P
CR
as
des
crib
ed i
n
sect
ion 2
.2.7
. P
lasm
id w
as e
xtr
acte
d f
rom
ind
ivid
ual co
lonie
s and u
sed a
s te
mp
late
fo
r P
CR
usi
ng t
he p
air
s o
f pri
mer
use
d
to g
ener
ate
each
co
nst
ruct
. 1 µ
l o
f p
lasm
id D
NA
or
5 µ
l o
f P
CR
pro
duct
was
lo
aded
on t
o t
he
well
s o
f 1%
agar
ose
gel
stai
ned
wit
h 0
.01%
Gel
Red
TM
and e
lect
rophore
sis
per
form
ed u
sing 8
0V
ele
ctri
c vo
ltag
e. I
mag
e w
as t
aken
wit
h U
V
illu
min
atio
n.
L –
Hyper
Lad
der
TM
DN
A m
ole
cula
r w
eig
ht
mar
ker
. (A
) L
ane
1 –
uncu
t pE
PI-
DE
ST
C.h
MP
V N
, L
ane
2 –
confi
rmat
ion o
f hM
PV
N i
n p
EP
I-D
ES
TC
.hM
PV
N v
ia P
CR
(si
ze
1185
bp).
(B
) L
ane
1 –
uncu
t pE
PI-
DE
ST
C.f
rA,
Lan
e 2
– c
onfi
rmat
ion o
f fr
agm
ent
A (
hM
PV
N (
1-2
50))
in p
EP
I-D
ES
TC
.frA
via
PC
R (
size
770 b
p).
(C
) L
ane
1 –
uncu
t pE
PI-
DE
ST
C.f
rB,
Lan
e 2 –
co
nfi
rmat
ion o
f fr
agm
ent
B (
hM
PV
N (
1-1
99))
in p
EP
I-D
ES
TC
.frB
via
PC
R (
size
596 b
p).
(D
)
Lane
1 –
uncu
t pE
PI-
DE
ST
C.f
rC,
Lane
2 –
co
nfi
rmat
ion o
f fr
agm
ent
C (
hM
PV
N (
192
-394))
in p
EP
I-D
ES
TC
.frC
via
PC
R (
size
609 b
p).
111
5.5 Expression of hMPV N constructs in transfection cells
5.5.1 Expression of full-length hMPV N (1-394) observed in live Cos-7 cells
Subcellular localisation of GFP-tagged full-length hMPV N was studied using CLSM, with
GFP alone used as control. GFP was shown to diffuse easily across the nuclear envelope and
localise equally in the cytoplasm and the nucleus of transfected cells (Figure 5-5), showing
the transfection was efficient.
GFP-tagged full-length hMPV N was present in both the nucleus and cytoplasm of
transfected Cos-7 cells at 24 hours post transfection (Figure 5-5 A, right image), as
determined by green fluorescence in live cells that is similar to the distribution of GFP alone
(Figure 5-5 A, left image). There was no change in the localisation of either GFP alone, or
GFP-tagged full-length hMPV N, at 48 hours post transfection (Figure 5-5 A, lower images),
with both equally present in the nucleus and cytoplasm.
The fluorescence intensity within the nucleus (Fn) and cytoplasm (Fc) of Cos-7 cells
transfected with pEPI.DESTC-GFP and pEPI.DESTC-hMPV N was measured to generate the
nuclear to cytoplasmic ratio (Fn/Fc) (Figure 5-5 B), as detailed in section 2.2.11.3. GFP
accumulated equivalently in the cytoplasm and in the nucleus at both 24 (Fn/Fc = 1.21) and
48 hours post infection (Fn/Fc = 1.03), as did GFP-tagged hMPV N (Fn/Fc 24H = 1.07,
Fn/Fc 48H = 1.04). The values of Fn/Fc ratio support the cytoplasmic vs nuclear
accumulation observed from fluorescence in cell images (Figure 5-5 A). Based on this
finding, it can be inferred that the hMPV N protein localised in both the cytoplasm and in the
nucleus of transfected cells in the period of 48 hours after transfection, and the equal
distribution of hMPV N in the cytoplasm and in the nucleus was stable over the time of the
experiment.
112
Figure 5-5 Subcellular localisation of GFP-tagged full-length hMPV N (1-394) protein observed in
Cos-7 cells. Cos-7 cells were transfected with an expression clone containing full-length hMPV N
gene, as described in section 2.2.11.1 and images were taken from live cells 24 and 48 hours post
transfection using confocal laser scanning microscopy (CLSM) as described in section 2.2.11.2. A
Subcellular localisation of GFP and GFP-tagged hMPV N observed in live Cos-7 cells 24 and 48
hours post infection. B Quantitative analysis of CLSM imaging is presented as the ratio between
nuclear and cytoplasmic concentration (Fn/Fc) of hMPV N, which were determined from pixel
density in the image and the means of the ratio ± SEM presented.
113
5.5.2 Expression of hMPV N deleted constructs observed in Cos-7 cells
Cos-7 cells transfected with GFP alone or GFP fused to full-length hMPV N or its truncated
constructs, were observed using CLSM at 24 hours after transfection (Figure 5-6 A). Full
length hMPV N (1-394) localised in a similar manner as GFP in transfected cells, which is
equally localised in both the cytoplasm and in the nucleus, 24 hours after transfection. This
similar localisation was supported by the mean value of Fn/Fc, which are 1.110 (GFP) and
1.109 (hMPV N) (Figure 5-6 B), and the statistical analysis (nonparametric Mann-Whitney
U-test) that showed no statistical difference (P = 5.194; Figure 5-6 C).
Fragment A (hMPV N (1-250)), which contains both NLS and NES, was distributed in both
the nucleus and cytoplasm (Figure 5-6 A). This observation was also supported by Fn/Fc
ratio average of 1.018 (Figure 5-6 B), which resulted in no statistically different localisation
compared to full-length hMPV N (P = 0.2076; Figure 5-7 C). In other words, fragment A was
distributed similarly as hMPV N.
Fragment B (hMPV N (1-199)), which contains intact NES and partial NLS, presented with
more cytoplasmic localisation compared to the other hMPV N constructs and GFP (Figure 5-
6 A). The determination of Fn/Fc ratio also supports the cell image analysis that fragment B
localised predominantly in the cytoplasm 24 hours after transfection, with the mean of Fn/Fc
values 0.7008 (Figure 5-6 B). The cytoplasmic localisation of fragment B was significantly
different to the nucleocytoplasmic distribution of full length hMPV N (P < 0.0004; Figure 5-6
C).
The localisation of fragment C (hMPV N (192-394)) containing C-terminal domain (CTD)
NLS resembled that of the full length hMPV N and fragment A (hMPV N (1-250)), which
was equally distribution in both the cytoplasm and the nucleus (Figure 5-6 A). This
observation was supported by a mean value of Fn/Fc = 0.1057 (Figure 5-6 B). However, a
statistical difference to full length hMPV N was observed (P = 0.0249).
114
Fig
ure
5-6
Subce
llu
lar
loca
lisa
tio
n o
f G
FP
-tag
ged
hM
PV
N c
onst
ruct
s in
liv
e C
os-
7 c
ell
s. (
A)
Co
s-7 c
ell
s
wer
e tr
ansf
ecte
d w
ith e
xpre
ssio
n c
lone
conta
inin
g t
runca
ted a
nd f
ull
-length
hM
PV
N g
ene,
as
des
crib
ed i
n
sect
ion 2
.2.1
1.1
and i
mag
es f
rom
liv
e ce
lls
wer
e ta
ken
24 h
ours
po
st t
ransf
ecti
on a
s des
crib
ed i
n s
ecti
on
2.2
.11.2
. (B
) Q
uan
tita
tive
analy
sis
of
CL
SM
im
ag
ing i
s pre
sente
d a
s th
e ra
tio
bet
wee
n n
ucle
ar a
nd
cy
top
lasm
ic
conce
ntr
atio
n (
Fn/F
c) o
f G
FP
-tag
ged
hM
PV
N c
onst
ruct
s, w
hic
h w
ere
det
erm
ined
fro
m p
ixel densi
ty i
n t
he
imag
e and t
he
mea
n o
f th
e ra
tio
± S
EM
pre
sente
d. (C
) T
he
Fn/F
c ra
tio
valu
es o
f ea
ch h
MP
V N
co
nst
ruct
wer
e
com
par
ed a
gain
st G
FP
and f
ull
-length
hM
PV
N u
sing n
onp
aram
etri
c M
ann-W
hit
ney U
Tes
t (G
raphP
ad P
rism
,
La
Joll
a, U
SA
), (
*)
den
ote
s P
<0.5
; (*
*)
den
ote
s P
<0.0
05, (*
**)
den
ote
s P
<0.0
00
1.
115
5.6 Discussion
The N protein of RNA viruses that replicate in the cytoplasm has been shown to localise into
the nucleus during the course of infection, with the facilitation of NLS and NES. The
nuclear/nucleolar localisation of N protein has been well-characterised in the Coronaviridae
(SARS, borna disease virus, transmissible gastroenteritis virus, mouse hepatitis virus),
Arteriviridae (equine arteritis virus, porcine reproductive and respiratory viruses),
Bornaviridae (borna disease virus), and Paramyxoviridae (measles virus) families (Rowland
et al., 1999, Hiscox et al., 2001, Kobayashi et al., 2001, Wurm et al., 2001, Tijms et al., 2002,
Calvo et al., 2005, Surjit et al., 2005, You et al., 2005, Lee et al., 2006, Sato et al., 2006,
Takayama et al., 2012). The precise role of nuclear/nucleolar translocation of these N
proteins in virus replication has not been defined, but the transient localisation tends to
associate with processes that give advantage for virus replication. For example, the
Coronavirus N associates with nucleolar proteins including fibrillarin and nucleolin to hold
the cell cycle in interphase, during which the maximum translation of viral mRNA can occur
(Wurm et al., 2001, Chen et al., 2002). N association with fibrillarin has also been
documented in arteriviruses and inhibition of nuclear localisation of the N protein results in
attenuated virus replication (Yoo et al., 2003, Lee et al., 2006). In Paramyxoviridae (measles
virus), the nuclear localisation of N has been shown to block host interferon responses by
inhibiting the nuclear translocation of STATs (Takayama et al., 2012). These studies suggest
that disruption of the nucleocytoplasmic transport of N protein offers a way for attenuation.
Little is known about the nuclear localisation of hMPV N protein, but this study has
identified that there are putative NLS (185
RRANRVLSDALKRYPR200
) and NES
(1MSLQGIHLSDLSYKH
15) motifs in the N protein. To determine whether the motifs are
functional and can facilitate the nucleocytoplasmic localisation of hMPV N, GFP-tagged
hMPV N constructs were synthesised and the nucleocytoplasmic localisation observed in
transfected cells. Fusing a coding sequence to a fluorescent gene marker, followed by protein
expression in transfected cells, is a standard approach to characterise previously unknown
nucleocytoplasmic properties of a protein (Tijms et al., 2002, Rowland and Yoo, 2003, Surjit
et al., 2005, You et al., 2005, Sato et al., 2006).
The results presented in this chapter indicates that GFP-tagged full length hMPV N
containing NES and NLS, GFP-tagged fragment A containing NES and a potential nuclear
116
localisation motif of NTD, and GFP-tagged fragment C containing the a potential nuclear
localisation motif of CTD localised in both the nucleus and cytoplasm at 24 hours after
transfection. In contrast, the GFP-tagged fragment B, containing intact NES and a potential
nuclear localisation motif of NTD, localised to the cytoplasm. Two indications can be
inferred based on this result. Firstly, the nuclear localisation motif of fragment B is functional
in facilitating nuclear import, considering that the nucleus presented a green fluorescence,
albeit dimmer than the cytoplasm (Figure 5-6 A). However, the rate of nuclear export
facilitated by the intact NES might be higher than the rate of nuclear import facilitated by the
disrupted nuclear localisation motif for hMPV N (1-199), such that it was more cytoplasmic
than nuclear. Secondly, hMPV N contains a functional NLS, based on the fact that full-length
hMPV N and fragment A constructs, having both the full-length putative NES (1-15) and
potential nuclear localisation motif of NTD (185-200) localised evenly in the cytoplasm and
the nucleus. This suggests that R200
has a determining role in facilitating the nuclear
localisation of hMPV N, and that the nuclear localisation activity might be contained in
residues 185 – 250. The highly cytoplasmic concentration of fragment B might be caused by
a dysfunctional nuclear localisation motif, most likely due to the lack of key residue R200
.
The size of the GFP-tagged constructs is an important factor that needs to be considered in
assessing the subcellular distribution of a construct in transfected cells. Proteins less than 50
kDa have been shown to passively diffuse across the NPC, causing even distribution in the
cytoplasm and in the nucleus; while proteins larger than 50 kDa require an active nuclear
transport system to facilitate the nucleocytoplasmic transport; causing cytoplasmic
localisation for inherently cytoplasmic proteins and nuclear concentration for the inherently
nuclear proteins (Terry et al., 2007). GFP has a molecular weight of 27 kDa; therefore it is
able to passively diffuse across the NPC, which results in equal distribution between the
cytoplasm and the nucleus (Chalfie et al., 1994, Macara, 2001). hMPV N is 43.5 kDa, which
may be small enough for a protein to passively diffuse but is most likely actively transported
across the NPC (Petraityte-Burneikiene et al., 2011). However, GFP-tagged hMPV N is 70.5
kDa, which is too big for passive diffusion and requires active transport to pass through the
NPC. Therefore, the equal distribution of hMPV N in the cytoplasm and in the nucleus is
most likely the result of active transport, which represents the ability of hMPV N to shuttle
between the nucleus and the cytoplasm.
117
GFP-tagged fragment A is approximately 57 kDa, while GFP-tagged fragment B and C are
approximately 51 kDa each. Therefore, the localisation of these hMPV N constructs is also
likely the result of nucleocytoplasmic transport.
This study suggests that there is an active NES in hMPV N coding sequence and a region
containing NLS motifs; and therefore confirmed that the hMPV N possesses inherent ability
for nucleocytoplasmic transport. The N protein is a structural protein that has various core
functions in viral replication, most notably in genome protection and packaging, ensuring that
only functional genome is inherited by infectious progeny (Narayanan et al., 2000). The
nucleocytoplasmic transport of hMPV N probably has a determining function in virus
replication, as already described in porcine reproductive and respiratory syndrome virus (Lee
et al., 2006). In that case, attempts to disrupt the movement can be useful for attenuation,
which is one known method to develop vaccines or antivirals. As such, by describing that
hMPV N possesses inherent nucleocytoplasmic transport ability; this study has opened a new
research avenue for future investigation on the development of vaccines/antivirals against
hMPV.
118
119
CHAPTER 6
GENERAL DISCUSSION
6 General Discussion
120
121
RSV and hMPV are the main cause of bronchiolitis and pneumonia in infants/ very young
children and the elderly. Being dispersed by aerosols, RSV and hMPV are easily transmitted.
This poses the risk of spreading the virus to a large proportion of the population in a short
time. Attempts to lessen the burden of RSV and hMPV are therefore required.
Options include the development of prophylaxis to ameliorate the severity of clinical
symptoms, antivirals to control infection, and vaccines to provide the human body with the
tools to fight viral infection such that the infection does not develop and virus spread can be
controlled. Of the three mechanisms, a vaccine is considered the best option for limiting the
risks of lower respiratory tract infections posed by RSV and hMPV because vaccination can
be applied to all members of the human population. This is important considering that,
although the main target of RSV and hMPV are the very young and elderly, people from
other age groups can become the reservoir of the virus once infected, spreading the virus to
the population in the high-risk age group.
To develop an effective vaccine, it is important to understand the molecular biology of viral
replication because disrupting the biological processes of viral replication can facilitate virus
attenuation for the purpose of vaccine development. Viruses that replicate in the cytoplasm
have been shown to have structural proteins transiently translocating into the nucleus to play
a non-structural function that optimise viral replication; therefore, can be used as a target of
attenuation.
To attain attenuated virus by disrupting the nucleocytoplasmic transport of the structural
proteins involves four major steps. Firstly, the nucleocytoplasmic shuttling of a structural
protein in infected and transfected cells needs to be identified. Secondly, the nuclear transport
motifs need to be defined by expressing the protein in cell culture. Thirdly, to gather evidence
on whether the nucleocytoplasmic transport of the structural protein has a significant function
in virus replication, the key residues of the nuclear transport motifs need to be mutated and
the impact of the mutation on virus production analysed in vitro and in vivo. The final stage is
to apply the knowledge for the production of live-attenuated virus vaccine or antivirals.
6.1 Respiratory syncytial virus
Attempts to develop a vaccine for RSV have been hindered by problems such as the
difficulties in culturing the virus in cell lines (Collins and Crowe Jr., 2007), to allow further
122
study, and the partial immunity developed against the formalin-inactivated virus that turned
out to cause deadly clinical outcomes in infants (Dudas and Karron, 1998). After years of
study, live-attenuated virus has been shown to be the best option for an RSV vaccine because
of the capability to induce immune responses similar to the natural infection (Collins, 2011).
However, attenuating RSV for vaccine purpose is a challenge itself, particularly in terms of
balancing the immunogenicity and the safety (not causing severe disease). Methods to
attenuate RSV include multiple passaging to obtain mutants that are temperature sensitive (ts)
and host specific. However, multiple passaging is a costly and time-consuming method to
generate attenuated RSV. The phenotypes of the mutants are not predictable, and subsequent
in vitro and in vivo characterisation might result in phenotypes that are not suitable for
vaccine production (Karron et al., 1997b). To overcome the obstacles in the development of
RSV vaccine, attempts of attenuation are directed towards disrupting biological processes in
virus replication (Collins et al., 1995), with the nucleocytoplasmic transport of RSV M being
one potential target of attenuation.
The nucleocytoplasmic transport mechanism of RSV M is well characterised. The RSV M
translocates to the nucleus early in infection and localises in the cytoplasm later (Ghildyal et
al., 2002, Ghildyal et al., 2003, Ghildyal et al., 2005b). The nuclear localisation of RSV M is
associated with binding to the host DNA and the inhibition of host transcription; therefore it
is thought that RSV M nuclear localisation aims to hold host transcriptional activity to allow
the viral replication machinery to function optimally in the cytoplasm (Ghildyal et al., 2003,
Latiff et al., 2004). The cytoplasmic localisation of RSV M in the late stage of infection aims
to facilitate the formation of infectious progeny during assembly and budding processes (Li et
al., 2008). Disrupting nuclear localisation has been shown to decrease the production of
infectious progeny, while disrupting cytoplasmic localisation (nuclear export) has been
shown to cease the infection as no infectious progeny are produced due to the failure of
assembly/budding (Ghildyal et al., 2009). The RSV M nucleocytoplasmic transport has been
shown to be regulated by CK2 phosphorylation, which is thought to promote nuclear export
(Alvisi et al., 2008). RSV M205 (T) was proposed as the possible candidate of CK2
phosphorylation site. Therefore, the site was mutated into alanine (A) to identify whether this
key residue has a role in viral replication.
This study characterises the ability of a recombinant mutant having RSV M205 (A) to
replicate and to induce pro-inflammatory response in vitro. The impact of RSV M205 for
123
virus replication was studied in single cycle replication and multiple cycle replication assays.
The single cycle replication assay aimed to observe the capability of RSV to infect, while the
multiple cycle replication assay aimed to observe the capability of RSV to transmit the
infection.
In contrast to the starting hypothesis regarding the role of M205 (T) as a putative CK2
phosphorylation site that promotes nuclear export, this study, indirectly, identified that
M(205) T is not the regulatory CK2 phosphorylation site and that the RSV M nuclear export
was not affected by the substitution. This was evidenced by the ability of rRSV A2
M(T205A) to produce a relatively similar titre of infectious particle at the end of single cycle
replication assay (Figure 3-1 A). However, this site does appear to be required to facilitate
viral egress to reinitiate infection in uninfected cells, as shown by the results of the multiple
cycle replication assay (Figure 3-2 A). In doing so, results obtained in this study present a
new finding that the M205 (T) is an important site for the ability of RSV to transmit the
infection in vitro. The M(T205A) substitution restricted viral spread (Figure 3-2 A), leading
to reduced amount of infectious progeny; and therefore, attenuating the virus.
The ability of rRSV A2 M(T205A) to replicate further affected the pro-inflammatory
responses that were produced upon RSV infection. The expression of pro-inflammatory
mediators, IL-8 and RANTES, requires the production of infectious particles (Fiedler et al.,
1996, Saito et al., 1997). As the M(T205A) substitution limited the growth of RSV, the
induction of IL-8 and RANTES production (Chapter 4) was further suppressed.
In addition, this study also suggested that the M protein possesses an important role for
suppressing the antiviral IFN-α/β activity that can restrict viral spread, and that the M205 (T)
might be a key residue in regulating this role. The failure to suppress the antiviral activity of
rRSV A2 M(T205A) was shown by the reduced titres in A549 cells, compared to the rRSV
A2 (Figure 3-1 B). The titre of rRSV A2 M(T205A), however, was relatively similar to the
rRSV A2 in the IFN-α/β-defective Vero E6 cells (Figure 3-1 A); suggesting that M may have
a role in regulation of IFN response in RSV infected cells. This is the first evidence that
suggests the role of RSV M in the induction of the IFN-α/β activation.
The attenuation of RSV presented in this study provides an early characterisation of an
attenuated RSV strain. It is important to repeat the characterisation in a whole organism (in
vivo) to ensure whether the attenuated phenotype of rRSV A2 M(T205A) displayed in cell
124
culture (in vitro) is stably expressed in vivo, considering that cell culture is an environment
with limited immune capabilities, while an organism has a complex immune network.
Attenuation by generating recombinant virus poses a good potency of a safe and useful
vaccine or antiviral. For example, many forms of recombinant RSV has been generated via
reverse genetics, including rRSV A2 M(T205A) that was studied in this thesis. Questions
about the length of protection it may give cannot be assessed based on the experimental
results presented in this thesis. However, to infer from another experience using recombinant
mutant RSV as a vaccine candidate, a recombinant mutant RSV has been shown to provide
protection up to 147 days against challenge of natural RSV infection (Widjojoatmodjo et al.,
2010). Based on this experience, the potential of rRSV A2 M(T205A) to provide protection
against natural RSV infection is possible to last more than two months (approximately 4.5
months). Given that RSV infections mostly peaks during late spring-winter (4 – months), a
single dose of vaccination could be expected to protect against RSV during the RSV season.
Even if the protection is induced for only one season, RSV vaccination should lead to
significant reduction in morbidity, mortality and socio-economic costs given that more than
10,000 elderly patients die each year from RSV disease.
6.2 Human metapneumovirus
Obstacles of developing an hMPV vaccine include, like RSV, difficulties in growing the
virus in cell culture, to enable further study of the biology of the virus (van-den-Hoogen et
al., 2001, Kuiken et al., 2004). This results in limited availability of knowledge regarding the
development of host immune responses and viral factors of infection that can be targeted as a
basis of vaccine development. As hMPV is from the same family as RSV, i.e
Paramyxoviridae, knowledge of the development of immune response and clinical symptoms
for hMPV can be inferred from knowledge assessed in RSV, including the development of a
live-attenuated vaccine.
In the case of hMPV, the N protein has been shown to localise in the nucleus in the very late
stage of infection (day 5 post infection) (Ghildyal R and Gahan, M; unpublished). The
mechanism of nuclear localisation has never been described; therefore, this study attempts to
gather evidence of the inherent ability of the hMPV N for nucleocytoplasmic transport. As
the first step in characterising the nucleocytoplasmic transport ability, the presence of nuclear
125
transport motifs in hMPV N was identified using the expression of full-length and truncated
hMPV N constructs in transfected cells.
This study identified the presence of components for active nucleocytoplasmic transport in
hMPV N protein. The NES motif identified in EXPASY (hMPV N 1 – 15:
MSLQGIHLSDLSYKH) was indicated to be functional by the cytoplasmic localisation of
fragment B that contains this motif and lacks the complete sequence of the potential nuclear
localisation motif (Figure 5-6). A region containing the nuclear localisation motif was
manually identified in amino acid 185 – 200: RRANRVLSDALKRYPR. This study
confirmed that an active NLS is contained within this sequence; with the key residues being
the amino acid residue R200
and other important residues might reside in amino acids 185 –
250. This finding was based on the ability of fragment C (hMPV N(192-394)) and fragment
A (hMPV N (1-250)) to be equally distributed in both the nucleus and cytoplasm, while
fragment B (hMPV N (1 – 199)) was more cytoplasmic (Figure 5-6).
The ability of active nucleocytoplasmic transport in hMPV N was confirmed by the size of
GFP-tagged hMPV N constructs, either full-length or truncated, which were larger than the
size threshold of proteins that can diffuse passively across the nuclear pore complex (50 kDa)
(Terry et al., 2007). In addition, the biology of cells used as the transfection host has been
well characterised; therefore, ensuring that the expression of a transfected gene truly
represents the phenotype of a protein without interference from other components (Kingston,
2003). Therefore, it can be concluded that the hMPV N possesses inherent nucleocytoplasmic
transport ability.
Nevertheless, the precise NLS motif, as well as the mechanism of localisation, cannot be
determined based on the result obtained in this study. This prompts further study to define the
precise NLS motif in transfected cells and investigate the subnuclear localisation of hMPV N
in infected cells, as a basis to determine its nuclear functions and suitability as an attenuation
target.
6.3 Conclusion
Study of the nucleocytoplasmic transport of viral structural proteins contributes to the
knowledge of attenuation targets for the purpose of developing a safe live-attenuated vaccine,
which is needed to control the spread of RSV and hMPV worldwide. The nuclear transport of
126
RSV M protein has been well characterised and recombinant mutants were generated by
targeting this mechanism. This study characterised a recombinant RSV with M(T205A)
substitution in vitro. The results showed that RSV M205 (T) is a site required to transmit
infection, and that RSV M has a role in the suppression of IFN-α/β. On the other hand, the
subcellular localisation of hMPV N has never been assessed and this study identified for the
first time that the protein has inherent nucleocytoplasmic transport ability, therefore
providing a possible means of attenuation. To sum up, the disruption of the
nucleocytoplasmic transport of a structural protein provides a new method of attenuation, and
enables biological processes of a virus to be studied and characterised.
127
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APPENDIX
8 Appendix
148
1
Appendix: Confirmation of hMPV N constructs ligated in the entry and expression clones by DNA sequencing
10 20 30 40 50 60 70 80 90 100
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
N_Can97-83 ATGTCTCTTCAAGGGATTCACCTGAGTGATCTATCATACAAGCATGCTATATTAAAAGAGTCTCAGTATACAATAAAGAGAGATGTAGGCACAACAACAG
pDONR_hMPVN TCA.................................................................C...............................
pDONR_hMPVN-frA TCA.................................................................C...............................
pDONR_hMPVN-frB TCA.................................................................C...............................
pDONR_hMPVN-frC ----------------------------------------------------------------------------------------------------
pEPI_hMPVN TCA.................................................................C...............................
pEPI_hMPVN-frA TCA.................................................................C...............................
pEPI_hMPVN-frB TCA.................................................................C...............................
pEPI_hMPVN-frC ----------------------------------------------------------------------------------------------------
110 120 130 140 150 160 170 180 190 200
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
N_Can97-83 CAGTGACACCCTCATCATTGCAACAAGAAATAACACTATTGTGTGGAGAAATTCTATATGCTAAGCATGCTGATTACAAATATGCTGCAGAAATAGGAAT
pDONR_hMPVN .....................................G..............................A...............................
pDONR_hMPVN-frA .....................................G..............................A...............................
pDONR_hMPVN-frB .....................................G..............................A...............................
pDONR_hMPVN-frC ----------------------------------------------------------------------------------------------------
pEPI_hMPVN .....................................G..............................A...............................
pEPI_hMPVN-frA .....................................G..............................A...............................
pEPI_hMPVN-frB .....................................G..............................A...............................
pEPI_hMPVN-frC ----------------------------------------------------------------------------------------------------
210 220 230 240 250 260 270 280 290 300
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
N_Can97-83 ACAATATATTAGCACAGCTCTAGGATCAGAGAGAGTACAGCAGATTCTAAGAAACTCAGGCAGTGAAGTCCAAGTGGTTTTAACCAGAACGTACTCCTTG
pDONR_hMPVN ........................G................................................AC.....................T...
pDONR_hMPVN-frA ........................G................................................AC.....................T...
pDONR_hMPVN-frB ........................G................................................AC.....................T...
pDONR_hMPVN-frC ----------------------------------------------------------------------------------------------------
pEPI_hMPVN ........................G................................................AC.....................T...
pEPI_hMPVN-frA ........................G................................................AC.....................T...
pEPI_hMPVN-frB ........................G................................................AC.....................T...
pEPI_hMPVN-frC ----------------------------------------------------------------------------------------------------
49
150
310 320 330 340 350 360 370 380 390 400
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
N_Can97-83 GGGAAAGTTAAAAACAACAAAGGAGAAGATTTACAGATGTTAGACATACACGGAGTAGAGAAAAGCTGGGTGGAAGAGATAGACAAAGAAGCAAGAAAAA
pDONR_hMPVN .................T.........................................A........................................
pDONR_hMPVN-frA .................T.........................................A........................................
pDONR_hMPVN-frB .................T.........................................A........................................
pDONR_hMPVN-frC ----------------------------------------------------------------------------------------------------
pEPI_hMPVN .................T.........................................A........................................
pEPI_hMPVN-frA .................T.........................................A........................................
pEPI_hMPVN-frB .................T.........................................A........................................
pEPI_hMPVN-frC ----------------------------------------------------------------------------------------------------
410 420 430 440 450 460 470 480 490 500
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
N_Can97-83 CAATGGCAACTTTGCTTAAAGAATCATCAGGCAATATTCCACAAAATCAGAGGCCTTCAGCACCAGACACACCTATAATCTTATTATGTGTAGGTGCCTT
pDONR_hMPVN .............A......................................................................................
pDONR_hMPVN-frA .............A................................................................................C.....
pDONR_hMPVN-frB .............A......................................................................................
pDONR_hMPVN-frC ----------------------------------------------------------------------------------------------------
pEPI_hMPVN .............A......................................................................................
pEPI_hMPVN-frA .............A................................................................................C.....
pEPI_hMPVN-frB .............A......................................................................................
pEPI_hMPVN-frC ----------------------------------------------------------------------------------------------------
510 520 530 540 550 560 570 580 590 600
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
N_Can97-83 AATATTTACCAAACTAGCATCAACTATAGAAGTGGGATTAGAGACCACAGTCAGAAGAGCTAACCGTGTACTAAGTGATGCACTCAAAAGATACCCTAGG
pDONR_hMPVN ...................................................................................................A
pDONR_hMPVN-frA ...................................................................................................A
pDONR_hMPVN-frB ................................................................................................----
pDONR_hMPVN-frC ----------------------------------------------------------------------------------------------------
pEPI_hMPVN ...................................................................................................A
pEPI_hMPVN-frA ...................................................................................................A
pEPI_hMPVN-frB ................................................................................................----
pEPI_hMPVN-frC ----------------------------------------------------------------------------.......................A
151
610 620 630 640 650 660 670 680 690 700
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
N_Can97-83 ATGGACATACCAAAAATCGCTAGATCTTTCTATGATTTATTTGAACAAAAAGTGTATTACAGAAGTTTGTTCATTGAGTATGGCAAAGCATTAGGCTCAT
pDONR_hMPVN .................T........C........C................................................................
pDONR_hMPVN-frA .................T........C........C................................................................
pDONR_hMPVN-frB ----------------------------------------------------------------------------------------------------
pDONR_hMPVN-frC .................T........C........C................................................................
pEPI_hMPVN .................T........C........C................................................................
pEPI_hMPVN-frA .................T........C........C................................................................
pEPI_hMPVN-frB ----------------------------------------------------------------------------------------------------
pEPI_hMPVN-frC .................T........C........C................................................................
710 720 730 740 750 760 770 780 790 800
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
N_Can97-83 CCTCTACAGGCAGCAAAGCAGAAAGTTTATTCGTTAATATATTCATGCAAGCTTACGGTGCTGGTCAAACAATGCTGAGGTGGGGAGTCATTGCCAGGTC
pDONR_hMPVN .......................................................T.............................G.....C........
pDONR_hMPVN-frA .......................................................T...............-----------------------------
pDONR_hMPVN-frB ----------------------------------------------------------------------------------------------------
pDONR_hMPVN-frC .......................................................T.............................G.....C........
pEPI_hMPVN .......................................................T.............................G.....C........
pEPI_hMPVN-frA .......................................................T...............-----------------------------
pEPI_hMPVN-frB ----------------------------------------------------------------------------------------------------
pEPI_hMPVN-frC .......................................................T.............................G.....C........
810 820 830 840 850 860 870 880 890 900
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
N_Can97-83 ATCTAACAATATAATGTTAGGACATGTATCTGTCCAAGCTGAGTTAAAACAAGTCACAGAAGTCTATGACCTGGTGCGAGAAATGGGCCCTGAATCTGGG
pDONR_hMPVN .................................G.....C.....G.....G....................A........G..................
pDONR_hMPVN-frA ----------------------------------------------------------------------------------------------------
pDONR_hMPVN-frB ----------------------------------------------------------------------------------------------------
pDONR_hMPVN-frC .................................G.....C.....G.....G....................A........G..................
pEPI_hMPVN .................................G.....C.....G.....G....................A........G..................
pEPI_hMPVN-frA ----------------------------------------------------------------------------------------------------
pEPI_hMPVN-frB ----------------------------------------------------------------------------------------------------
pEPI_hMPVN-frC .................................G.....C.....G.....G....................A........G..................
152
910 920 930 940 950 960 970 980 990 1000
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
N_Can97-83 CTCCTACATTTAAGGCAAAGCCCAAAAGCTGGACTGTTATCACTAGCCAATTGTCCCAACTTTGCAAGTGTTGTTCTCGGCAATGCCTCAGGCTTAGGCA
pDONR_hMPVN ....................T....................CT......................................G..................
pDONR_hMPVN-frA ----------------------------------------------------------------------------------------------------
pDONR_hMPVN-frB ----------------------------------------------------------------------------------------------------
pDONR_hMPVN-frC ....................T....................CT......................................G..................
pEPI_hMPVN ....................T....................CT......................................G..................
pEPI_hMPVN-frA ----------------------------------------------------------------------------------------------------
pEPI_hMPVN-frB ----------------------------------------------------------------------------------------------------
pEPI_hMPVN-frC ....................T....................CT......................................G..................
1010 1020 1030 1040 1050 1060 1070 1080 1090 1100
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
N_Can97-83 TAATAGGTATGTATCGCGGGAGAGTGCCAAACACAGAACTATTTTCAGCAGCAGAAAGCTATGCCAAGAGTTTGAAAGAAAGCAATAAAATTAACTTTTC
pDONR_hMPVN ................A...................................C.............................T........C........
pDONR_hMPVN-frA ----------------------------------------------------------------------------------------------------
pDONR_hMPVN-frB ----------------------------------------------------------------------------------------------------
pDONR_hMPVN-frC ................A...................................C.............................T........C........
pEPI_hMPVN ................A...................................C.............................T........C........
pEPI_hMPVN-frA ----------------------------------------------------------------------------------------------------
pEPI_hMPVN-frB ----------------------------------------------------------------------------------------------------
pEPI_hMPVN-frC ................A...................................C.............................T........C........
1110 1120 1130 1140 1150 1160 1170 1180
....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|....|
N_Can97-83 TTCATTAGGACTCACAGATGAAGAAAAAGAGGCTGCAGAACACTTTCTAAATGTGAGTGACGACAGTCAAAATGATTATGAGTAA
pDONR_hMPVN .............................................C.......................................
pDONR_hMPVN-frA -------------------------------------------------------------------------------------
pDONR_hMPVN-frB -------------------------------------------------------------------------------------
pDONR_hMPVN-frC .............................................C.......................................
pEPI_hMPVN .............................................C.......................................
pEPI_hMPVN-frA -------------------------------------------------------------------------------------
pEPI_hMPVN-frB -------------------------------------------------------------------------------------
pEPI_hMPVN-frC .............................................C.......................................
Legends: Full-length and truncated hMPV N constructs were cloned into entry (pDONR207) and expression (pEPI-DESTC) vectors and sequenced as described in section 2.2.7.2. Sequences resulted in this study were compared to the reference N gene of hMPV Can97-83 (GenBank accession number AY145278). Dots (…) = residues similar to reference
Dash (---) = regions that cannot be sequenced using primers that were used in this study
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