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Establishment and Validation of Real-Time PCR
Assays for the Quantification of Different DNA-
Forms of Feline Immunodeficiency Virus
Master Thesis submitted to attain the degree of
Master of Science MSc.
Submitted by Matthias Hofer Bal<k.techn.
Supervisor: Ao.Univ.Prof.Dr. Dieter Klein Institute of Virology, Department of Pathobiology
University of Veterinary IVIedicine Vienna
Index
Index
ZUSAMMENFASSUNG -5-
ABSTRACT -6-
1. INTRODUCTION -7-
1.1. FIV-FELINE IMMUNODEFICIENCY VIRUS -7- 1.1.1. TAXONOMY -7- 1.1.2. STRUCTURAL CHARACTERIZATION -7- 1.1.3. GENOMIC AND MOLECULAR CHARACTERIZATION -8- 1.1.3.1. Cis acting elements -9- 1.1.3.2. Structural genes -11 • 1.1.3.3. Regulatory genes -12' 1.1.3.4. Accessory genes -13- 1.1.4. REPLICATION CYCLE -15- 1.1.4.1. Viral entry -15- 1.1.4.2. Reverse transcription -17 • 1.1.4.3. Nuclear entry -19- 1.1.4.4. Integration -20- 1.1.4.5. Transcription and translation - 21 • 1.1.4.6. Particle production, budding and maturation - 22 • 1.2. DIFFERENT RETROViRAL DNA FORMS -23- 1.2.1. INTEGRATED DNA/UNINTEGRATED LINEAR DNA -24- 1.2.2. 1-LTR CIRCLES -24- 1.2.3. 2-LTR CIRCLES -25-
1.2.4. AUTOINTEGRATION PRODUCTS - 25 • 1.2.5. ABUNDANCE AND STABILITY OF EPISOMAL VIRAL DNA -26- 1.2.6. GENE EXPRESSION AND FUNCTION OF EPISOMAL VIRAL DNA -27- 1.2.7. DETECTION AND QUANTIFICATION OF VIRAL DNA FORMS -29- 1.3. REAL-TIME PCR - 30 • 1.3.1. AMPLICON DETECTION - 31 • 1.3.1.1. DNA-binding fluorophores -31- 1.3.1.2. Hybridisation probes: light cycler probes - 31 • 1.3.1.3. Linear oligoprobes: 5' nuclease probes/ hydrolysis probes/ TaqMan probes - 32 • 1.3.1.4. Hairpin oligoprobes: molecular beacon probes - 32 • 1.3.1.5. Self fluorescing amplicon: sunrise primers - 32 • 1.3.1.6. Self fluorescing amplicon: scorpion primers - 33 • 1.3.2. QUANTIFICATION - 34 - 1.4. AIM OF THE STUDY -37-
2, MATERIALS & METHODS -38-
2.1. RESTRICTION ENZYMES AND DIGESTION - 38 • 2.2. AGAROSE GEL ELECTROPHORESIS - 38 • 2.2.1. PURIFICATION -39- 2.3. PLASMIDS AND CLONING - 40 • 2.3.1. IN siLico CLONING -40- 2.3.2. PLASMIDS -40- 2.3.2.1. pCTSefs -40-
-2-
Index
2.3.2.2. pCT5efsD66V - 41 - 2.3.2.3. pCT25egfpiresneo (pCT25ein) and pCT25einF - 41 - 2.3.2.4. pPetAenv - 41 - 2.3.2.5. pHCMV-G - 41 - 2.3.3. LIGATION - 42 -
2.3.4. BACTERIAL TRANSFORMATION - 42 - 2.3.4.1. Electroporation -43- 2.3.4.2. Heat shock -43- 2.3.5. BACTERIAL CULTURES - 44 - 2.3.5.1. Bacterial culturing on solid nfiedium - 44 - 2.3.5.2. Bacterial culturing in liquid medium - 44 - 2.3.5.3. Bacterial stock -44- 2.3.6. PLASMID PREPARATION - 45 - 2.3.6.1. Small scale plasmid preparation (Mini Prep) - 45 - 2.3.6.2. Large scale plasmid preparation (Maxi Prep) - 46 - 2.3.7. SEQUENCING -47- 2.4. MAMMALIAN CELL CULTURE - 47 - 2.4.1. CELL LINES -47- 2.4.2. CELL CULTURE MAINTENANCE - 48 - 2.4.3. PASSAGING / SPLITTING OF CELLS -49- 2.4.4. FREEZING/THAWING OF CELLS -49- 2.4.5. COUNTING OF CELLS -50- 2.4.6. TRANSIENT TRANSFECTiON -50- 2.4.6.1. Metafectene Pro -51- 2.4.6.2. Calcium phosphate - 51 - 2.4.7. INFECTION / TRANSDUCTION OF CELLS - 52 - 2.4.8. FACS ANALYSIS - 53 - 2.4.9. DNA-EXTRACTION - 53 - 2.4.10. MEASUREMENT OF DNA CONCENTRATION BY OPTICAL DENSITY (OD) - 54 - 2.5. POLYMERASE CHAIN REACTION (PCR) - 54 - 2.6. REAL-TIME PCR - 55 - 2.6.1. PRIMER DESIGN -56- 2.6.2. CELL NUMBER ASSAY -56- 2.6.3. TOTAL VIRAL DNA ASSAY -57- 2.6.4. 2-LTR CIRCLE ASSAY - 57 - 2.6.5. ALU-LTR BASED REAL-TIME NESTED PCR ASSAY FOR INTEGRATED DNA - 57 - 2.6.6. 1-LTR CIRCLE ASSAY -58-
2.6.7. ASSAY ANALYSIS - 59 - 2.6.8. ANALYSIS OF TIME COURSE INFECTION STUDY -59- 2.6.9. PRODUCT ENHANCED REVERSE TRANSCRIPTASE ASSAY (PERT-ASSAY) - 59 - 2.7. SOUTHERN BLOT - 61 - 2.7.1. SAMPLE PREPARATION BY ETHANOL PRECIPITATION - 62 - 2.7.2. PROBE LABELLING - 62 - 2.7.3. SAMPLE DIGESTION AND BLOTTING -63- 2.7.4. HYBRIDIZATION - 64 - 2.7.5. STRINGENCY WASHES AND ANTIBODY BINDING -64- 2.7.6. IMMUNOLOGICAL DETECTION - 65 -
3, RESULTS -67-
3.1. DEVELOPMENT OF REAL-TIME PCR ASSAYS - 67 - 3.1.1. TOTAL VIRAL DNA ASSAY -68- 3.1.2. INTEGRATED DNA ASSAY -69- 3.1.3. 2-LTR CIRCLE ASSAY -75- 3.1.4. 1-LTR CIRCLE ASSAY -76-
Index
3.2. EVALUATION OF REAL-TIME PCR SPECIFICITY - 81 3.2.1.1. Total viral DNA assay - 81 3.2.1.2. 2-LTR circle assay - 83 3.2.1.3. Integrated DNA assay - 83 3.3. VIRAL VECTOR PRODUCTION - 84 • 3.4. TIME COURSE INFECTION STUDY - 85 • 3.5. SOUTHERN BLOT - 91 •
4, DISCUSSION -93-
4.1. DEVELOPMENT OF REAL-TIME PCR ASSAYS - 93 • 4.2. EVALUATION OF REAL TIME PCR ASSAYS -96- 4.2.1. REALTIME PCR SPECIFICITY -96- 4.2.2. SOUTHERN BLOT - 97 • 4.3. VIRAL VECTOR PRODUCTION - 97 • 4.4. TIME COURSE INFECTION STUDY - 98 • 4.5. CONCLUSION -101- 4.6. OUTLOOK -102-
5, APPENDIX -103-
5.1. REFERENCES -103- 5.2. PUBLICATIONS -117- 5.3. ACKNOWLEDGEMENTS -119- 5.4. CURRICULUM VITAE -120-
Zusammenfassung
Zusammenfassung
Das Feline Immundefizienz-Virus (FIV) gehört zur Gattung der Lentiviren, einer Untergruppe
der Retroviren. FIV wird mit einer AIDS-ähnlichen Krankheit in Katzen assoziiert und spielt
eine Rolle als Modell für das humanen Immundefizienz- Virus (HIV). Ein wichtiger Schritt in
der lentiviralen Replikation ist die Reverse Transkription des viralen RNA-Genoms in DNA,
die dann in das Wirtsgenom integriert wird. Der Integrations-Prozess wird vom viralen Enzym
Integrase (IN) sichergestellt. Dabei dient das revers transkribierte Genom des Virus als
Substrat. Diese virale lineare DNA kann jedoch auch durch wirtseigene Prozesse zu
zirkulären DNA-Formen umgebildet werden: 1-LTR circles werden durch homologe
Rekombination der beiden long terminal repeats (LTRs) gebildet, die das virale Genom
flankieren. 2-LTR circles dagegen werden durch nicht homologe Ligation der Enden des
viralen Genoms gebildet. Beide zirkulären DNA-Formen dienen nicht als Substrat für die
Integration. Daher wurde die Bildung dieser Formen immer als „Sackgasse" der viralen
Replikation angesehen. Es wurde jedoch auch gezeigt, dass die episomalen DNA-Formen
stabil sind und deren Anzahl nur in Korrelation zum Zeil-Tod bzw. zur Zellteilung abnehmen.
Außerdem gibt es Hinweise, dass episomale DNA Formen eine Rolle im lentiviralen
Lebenszyklus spielen, da sie sowohl Gen-Transkription als auch Gen-Translation
ermöglichen. Daher kann die Untersuchung von Anzahl und Funktion der verschiedenen
viralen DNA- Formen neue Einblicke in die retrovirale Replikation ermöglichen.
Das Ziel dieser Studie war es verlässliche real-time PCR assays für verschiedene lentivirale
DNA-Formen zu entwickeln und zu evaluieren: gesamtvirale DNA, integrierte DNA, 1-LTR
circles und 2-LTR circles. Die Anzahl und die Stabilität dieser DNA-Formen konnten damit in
Infektionsstudien untersucht werden, in denen einerseits lentivirale Integration ermöglicht,
andererseits lentivirale Integration verhindert wurde. Wir konnten gesamtvirale DNA,
integrierte DNA und 2-LTR circles erfolgreich quantifizieren. Der integrationsfähige FIV-
Vektor zeigte die meiste DNA-Integration 48 Stunden nach Infektion, während die Anzahl der
2-LTR circles schon 24 Stunden nach Infektion ein Maximum zeigte. Über den Zeitraum der
Infektionsstudie wurde ein stabiler 2-LTR circle Anteil von 5 % festgestellt. Der
integrationsdefiziente FlV-Vektor dagegen zeigte nur marginale DNA Integration, dagegen
aber einen signifikant höheren 2-LTR circle Anteil. Im Gegensatz zu anderen Studien,
konnten wir 1-LTR circles mittels PCR-basierten Methoden nicht spezifisch quantifizieren.
Die hier gezeigten real-time PCR assays sind in der Lage die verschiedenen DNA-Formen
von FIV verlässlich zu quantifizieren.
Abstract
Abstract
Feline immunodeficiency virus (FIV) belongs to the genus of lentiviruses, a subgroup of
retroviruses. FIV is associated with an AIDS-like disease in cats and therefore has a great
potential as a model for human immunodeficiency virus (HIV) studies. An essential step in
lentiviral replication is reverse transcription of the viral RNA genome into double stranded
DNA, in order to integrate the viral DNA into the host genome. This integration process is
facilitated by the viral enzyme integrase (IN) that needs the linear double stranded viral DNA
as substrate. However, this linear viral DNA form can also be circularized by different host
mediated processes: 1-LTR circles are formed by homologous recombination of the two long
terminal repeats (LTRs) that flank the viral genome, while 2-LTR circles are formed by non-
homologous end to end ligation. These circular DNA forms are not substrates for integration,
stay episomal and thus were thought to be dead-end products. However, the viral episomal
DNA forms were shown to be stable and decrease only in correlation with cell death or cell
division. Furthermore, there is evidence that episomal DNA forms play a role in the lentiviral
replication cycle, because they can facilitate gene transcription and gene translation. Thus,
investigation of abundance and function of the different viral DNA forms can give new
insights into retroviral replication.
The aim of this study was to develop and evaluate reliable real-time PCR assays for the
different viral DNA forms occurring in FIV infected cells: total viral DNA, linear integrated
DNA, 1-LTR circles and 2-LTR circles. The abundance and stability of the viral DNA forms
were determined in time-course infection experiments in the context of either proficient or
impaired viral integration. As a result, 2-LTR circles, total viral DNA and integrated proviral
DNA could be successfully quantified in time-course infection experiments. The results
obtained with the integration proficient virus showed that most of the viral DNA is integrated
by 48 hours post infection, while the highest abundance of 2-LTR circles could be already
observed 24 hours after infection. Furthermore, a stable 2-LTR fraction of 5 % among all viral
DNA was observed during the complete time course. In contrast, when integration deficient
virus was used, minute amounts of integrated provirus and a significantly higher fraction of
episomal 2-LTR circles were detected. In contrast to reports by others, we were not able to
specifically quantify 1-LTR circles by PCR based methods. The herein reported real-time
PCR assays provide a valuable tool for the quantification of the different forms of FIV-DNA.
-6
Introduction
1. Introduction
1.1. FIV - Feline immunodeficiency virus
Feline Immunodeficiency Virus (FIV) was first isolated from a domestic cat by Pedersen and
co-workers in 1987 (Pedersen et al., 1987). The cat showed a chronic opportunistic infection
and neurological disease similar to the clinical picture of the human acquired
immunodeficiency syndrome.
1.1.1. Taxonomy
FIV is a member of the retroviridae family, subfamily orthoretrovirinae, genus lentivirus
(International Committee on the Taxonomy of Viruses, ICTV) (see Table 1.1). This genus
also includes human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV).
However, FIV is genetically closer related to ungulate lentiviruses (equine infectious anemia
virus or EIAV, visna virus, caprine arthritis encephalitis virus or CAEV) than to primate
lentiviruses. (Kanzaki and Looney, 2004).
Family Retroviridae
Subfamily Orthoretrovirinae Spumaretrovirinae
Genus
Alpharetrovirus
Betaretrovirus
Gammaretrovirus
Delta retrovirus
Epsilonretrovirus
Lentivirus
Spumavirus
Table 1.1. Classification of retroviruses: (International Committee of the Taxonomy of Viruses, ICTV)
1.1.2. Structural characterization
Mature FIV virions are enveloped, have a spherical to ellipsoid form and are 100 to 125 nm
in size. The outer envelope incorporates short projections or knobs representing the viral
glycoproteins. This envelope glycoprotein complex is assembled by the external,
-7
Introduction
glycosylated hydrophilic surface protein (SU) and the membrane-spanning transmembrane
protein (TIVI). Further Inside the virlon lies the internal protein core, whose shape and location
Is characteristic for different genera of the retrovlrus family. The FIV core Is cone-shaped and
Is enclosed by the capsid protein (CA). The matrix protein (MA) Is located In between
envelope and core. Inside the core, the nucleocapsid protein (NC) forms the
ribonucleoprotein complex together with the viral genomic RNA. Furthermore, the virlon
proteins Integrase (IN), reverse transcrlptase (RT), protease (PR) and dUTPase (DU) are
associated with this complex (Bendinelli et al., 1995; Coffin, 1997). An overview of the
particle composition Is given in Figure 1.1.
kilobases
MA CA NC PR RT DU IN SU TM
lipid membrane
RNA genome
Figure 1.1. Genomic organization and particie composition of FiV: (Inoshima et al., 1998)
Abbreviations are explained in the text.
1.1.3. Genomic and molecular characterization
The so called Petaluma strain was the first FIV Isolate (Pedersen et al., 1987). Furthermore,
It was the first strain that was characterized at the nucleotlde level (Olmstedt et al., 1989;
Talbottetal., 1989).
-8
Introduction
The genome of FIV is diploid with two copies of a positive sense RNA molecule; each copy is
9474 nucleotides in length. The RNA molecules show characteristic structural features of a
messenger RNA (mRNA) including a 5' cap and a 3' pot^-k tail. The genomic organization of
FIV with three main open reading frames (ORFs) can be found in all retroviral genomes. The
three ORFs code for the major structural genes gag, pol and env. In contrast to simple
retroviruses, lentiviruses - also defined as complex retroviruses - contain regulatory and
accessory genes. These genes are located up- and downstream of env and have various
supporting roles in regulation and coordination of viral gene expression. The coding region is
surrounded by the two unique sequences U5 at the 5' end and U3 at the 3' end, as well as by
a repetitional region (R) on both extremities of the genome. During the process of reverse
transcription, these unique and repetitional regions form 2 identical sequences, present on
both ends of the provirus. These sequences are called long terminal repeats (LTRs) and are
composed of U3, R, and U5 regions, respectively. The LTRs contain the c/s-acting elements
for promotion and termination of viral transcription (Olmsted et al, 1989.; Talbot et al., 1989;
Coffin, 1996). The LTR elements of different virus-families have common functional roles, but
nevertheless display little sequence relatedness (Thompson et al., 1994). A schematic
drawing of the genomic organization of FIV is given in Figure 1.2.
gag , pot RRE
5'LTR B E 3'LTR •^B MA CA NC vlf Ar|L SU •-• TM Ir^B "1 I I |PR RT DU IN| I 'iH \' -I r*^
'— ' i' I I I 'i—^ü—• Tin—tzr
-I 1 I 1 r 1 1 3 4 5 « 7 « 9 Kkp
Figure 1.2. Detailed genomic organization of a FIV provirus: (Bendinelli et al., 1995)
Abbreviations are explained in the text.
1.1.3.1. Cis acting elements
In general, c/5-acting elements act on the same molecule on which they are located. The FIV
genome contains several c/s-acting elements, which contribute to efficient reverse
transcription, integration, gene transcription and packaging. The role of the c/s-acting
elements in these processes shall now be explained in brief.
Introduction
Reverse transcription:
During reverse transcription, the viral RNA genome is transformed into DNA. The primer
binding site (PBS) is found near the 5'end of the genome, where the tRNA primer can
specifically bind and start synthesis of the minus-strand DNA. Another c/s-acting element is
the polypurine tract (3'PPT), near the 3' end of the genomic RNA, which acts as primer for
the synthesis of plus-strand DNA. Recently, a central polypurine tract (cPPT), similar to the
one in HIV-1, was described for FIV. This c/5-acting element is unique in lentiviruses and
primes a second plus-strand synthesis. The elongation of two plus strand fragments lead to
the production of a nucleotide overlap that is termed central DNA flap (Whitwam et al., 2001).
The central DNA flap is important for viral replication and is suggested to play a role in
nuclear import of the preintegration complex (PIC) (Zennou et al., 2000).
Integration:
Attachment sites determine the sites, where the viral DNA is attached to the host DNA.
Therefore they are important for viral integration. Attachment sites are CA/TG dinucleotide
pairs that are located at the internal edges of both LTRs. Within the reverse transcribed
linear precursor for integration, the attachment sites always reside two base pairs from each
end (Coffin, 1997).
Gene transcription:
Transcription of viral genes is mediated by the host-cellular RNA polymerase II. The viral
promoter is located in the 5' LTR, at the border between the U3 and the R region, where two
TATA boxes are located. Transcription is terminated within the 3' LTR, at the border between
the R and the U5 region, where finally also the polyadenylation takes place (Olmstedt et al.,
1989, Coffin, 1997). Furthermore, the R includes binding sites for regulatory elements. An
API site, an AP4 site, a C/EBP tandem repeat, and an ATF site were shown to be crucial for
the basal promoter activity of the LTR (Sparger et al., 1992; Thompson et al., 1994;
Kawaguchi et al., 1995). These sites are also thought to be potential binding sites for the FIV
Orf-A, speculated to act as transactivator (see 1.1.3.3) (de Parseval and Elder, 1999;
Chatterji et al., 2002).
Packaging:
The packaging signal (H^) or encapsidation sequence (E) is responsible for the efficient
packaging of viral genomic RNA into the virus. Genomic RNA with a packaging signal is
incorporated into new virions 20 - 200 times more often than genomic RNA lacking the
packaging signal (Coffin, 1997).
-10-
Introduction
/. 1.3.2. Structural genes
The gag gene encodes a polyprotein composed of matrix (MA), capsid (CA), and
nucleocapsid (NC) proteins. During or after the process of budding, this precursor protein is
cleaved by the viral protease (PR) (Elder et al., 1993).
The N-terminal domain of the precursor protein corresponds to the MA, which forms an outer
shell around the viral core. Furthermore, MA is responsible for the transport of the gag
polyprotein and its interaction with the plasma membrane. Finally, MA supports particle
formation and envelope glycoprotein incorporation into virions (Manrique et al., 2001).
The CA proteins are self-interacting and build up the protective shell around the viral
nucleus. A mature lentivirus is defined by a cone shaped capsid (Nath and Peterson, 2001).
The NC protein is essential for virus production by packaging viral genomic RNA into new
virions. It recognizes the packaging signal on the unspliced viral genomic RNA. (Manrique et
al., 2004). For retroviruses in general, it was shown that NC also plays a role during reverse
transcription (Allain et al., 1994; Tsuchihashi and Brown, 1994).
The HIV-1 gag precursor protein also contains the peptide P6, which is responsible for
efficient particle production. It was shown that a small C-terminal FIV Gag peptide is
functionally equivalent to HIV-1 P6 (Manrique et al., 2004).
The /DO/gene is expressed as a Gag-Pol polyprotein via ribosomal frameshifting (Morikawa
and Bishop, 1992). During the budding process, this precursor protein is cleaved into various
enzymes essential for the viral life cycle: protease (PR), reverse transcriptase (RT),
dUTPase (DU), and integrase (IN) proteins (Elder et al., 1993).
The role of the viral protease is to process the viral Gag and Gag-Pol precursor proteins into
their individual structural and enzymatic proteins. These essential proteolytic steps are
specific and occur during or after virus budding (Lin et al., 2000).
FIV reverse transcriptase is assembled of two polypeptides: a 66-kDa subunit (p66), and a
51-kDa subunit (p51). Both subunits are enzymatically active and present in equimolar
amounts; ratio of 1:1. The small subunit p51 enhances the rate and extent of the DNA
synthesis activity exerted by the large subunit p66. RT converts genomic single-stranded
RNA into double-stranded DNA by RNA- as well as DNA dependent DNA synthesis
(Amacker et al., 1995). HIV-1 RT and FIV-1 RT are very similar in terms of template
specificity and Mg^* requirements (North et al., 1990).
In contrast to primate lentiviruses, FIV and other nonprimate lentiviruses encode the enzyme
deoxyuridine-triphosphatase (DU or dUTPase). This enzyme prohibits misincorporation of
deoxy uridine into viral DNA during reverse transcription, which would result in a G to A
11
Introduction
transition. DU-defective FIV was shown to have a higher mutation frequency, which resulted
in a decreased viral burden (Lerner et al., 1995).
The integrase propagates the integration of the reverse transcribed viral cDNA into the host
genome. The HIV-1 integrase was also shown to play a role in the nuclear import of the
preintegration complex (PIC) (Gallay et al., 1997)
The envgene encodes a precursor protein that includes the glycoproteins within and on the
virus envelope. The polyprotein is produced on the rough endoplasmatic reticulum and
cleaved into the transmembrane protein (TM) and the surface protein (SU). The
transmembrane protein anchors the glycoprotein in the viral envelope and supports fusion of
virus- and cell membrane during viral cell entry, while the surface protein is responsible for
the interaction with cell receptors and thus determines cell tropism. The SU is also a major
target for the host's immune response (Stephens et al., 1991).
/. 1.3.3. Regulatory genes
Next to the main structural genes, which are present in all retroviruses, lentiviruses like FIV
additionally encode regulatory genes from multiply spliced mRNAs.
Rev has a RNA binding domain and forms a stable complex with viral mRNAs that contain
the Rev-responsive element (RRE). FIV RRE is a 243 bp fragment that forms a characteristic
stem loop structure, which is essential for interaction with Rev. Furthermore, RRE is located
at the 3' end of the envqeue, thus only full length or singly spliced mRNAs contain an RRE
and are bound by Rev (Cochrane et al., 1990; Phillips et al., 1992). Rev contains a nuclear
localization signal (NLS) and an export signal (NES) and can subsequently shuttle between
nucleus and cytoplasm. This enables Rev-dependent transport of single- or non-spliced
mRNAs into the cytoplasm, where Rev unloads the mRNA and moves back to the nucleus
(Henderson and Percipalle, 1997). This transport is essential for the viral life cycle, because
the host cellular mRNA export machinery only enables fully spliced mRNA to exit the
nucleus.
In conclusion. Rev determines whether an infection is latent or productive. High
concentrations of Rev results in the presence of full-length or singly spliced mRNAs in the
cytoplasm (Gag, Pol, Env), while low concentrations of Rev lead to small and multiply spliced
mRNA in the cytoplasm (Rev, Tat, Nef). Thus, high cellular concentrations of Rev result in
virus production, while low Rev concentrations maintain the virus in a more latent state
(Phillips et al., 1992).
12
Introduction
HIV-1 promotes viral transcription from tine LTR with the transactivator protein Tat. Until
today, no exact counterpart was found in FIV. However, Orf-A / Orf-2 shares certain
characteristics with HIV-1 Tat.
Orf-A deleted virus is still infectious and able to replicate. However, Orf-A deletion results in a
lower viral burden, milder reduction of CD4/CD8 ratios and a slower antibody development
(Inoshima et al., 1996). Furthermore Orf-A was shown to transactivate gene expression 14-
to 20-fold above the basal level by acting on potential binding sites within the LTR: the API
site, a C/EBP tandem repeat, and an ATF site are proposed (de Parseval and Elder, 1999).
However, a direct interaction between the LTR and Orf-A has not been proven. Similar to the
non-primate lentiviral Tax protein, but unsimilarto HIV-1 Tat, Orf-A requires additional factors
for sufficient transactivation (Chatterji et al., 2002). However, Orf-A only weakly promotes
viral RNA transcription (Sparger et al., 1992; Thompson et al., 1994; de Parseval and Elder,
1999; Chatterji et al., 2002), suggesting that it might have additional functions in FIV
replication and pathogenesis (Gemeniano et al., 2003). Effects on virus particle formation or
particle release as well as virus binding, entry or reverse transcription were proposed. Orf-A
localizes to the nucleus and induces cell cycle arrest. These properties, including a highly
conserved nuclear entry site (NES) site, can be related to the functions of HIV-1 Vpr.
(Gemeniano et al., 2003; Gemeniano et al., 2004) Furthermore, Orf-A probably down-
regulates ubiquitin conjugating enzymes and proteasome subunits as well as splicing factors,
in order to protect viral proteins or host proteins involved in viral replication (Sundstrom et al.,
2008).
/. /. 3.4. Accessory genes
FIV lacks lentiviral accessory genes like vpr, vpu, and net, which are not crucial but beneficial
for viral replication. Only vifoan also be found in FIV.
The FIV vif gene is located downstream of the 3' end of the pal gene (Tomonaga et al.,
1992). All lentiviruses, except the equine infectious anemia virus, encode the accessory
protein Vif (viral infectivity factor) (Oberste and Gonda, 1992). Vif is incorporated into the
virions (Liu et al., 1995). During infection, FIV Vif is located in the nucleus (Chatterji et al.,
2000). Vif is required for efficient viral replication in cats, CrFK cells as well as in primary
lymphoid and myeloid cell lines, termed "non-permissive" cells (Inoshima et al., 1996;
Lockridge et al., 1999; Paul et a!., 2007; Shen et al., 2007).
13
Introduction
HIV-1 Vif promotes viral infection of non-permissive cells by counteracting the cellular
deaminase AP0BEC3G. Cytidine deaminases of the apolipoprotein B mRNA-editing
enzyme-catalytic polypeptide-like (APOBEC) family are packaged into the virion and prevent
efficient infection by catalyzing the deamination of deoxycytidine to deoxyuridine on the DNA
minus strand during reverse transcription. Higher mutation rates, in particular G to A
transitions, are the consequence. HIV-1 Vif acts against this restriction by recruiting an E3-
ubiquitin ligase. As a result, APEBEC3G is ubiquitylated and degraded by the proteasome.
HIV-1 Vif additionally inhibits translation of AP0BEC3G mRNA (Paul et al., 2007).
For FIV it is reported that the level of G to A mutations in the proviral FIV-DNA is decreased
by Vif (Paul et al., 2007). Furthermore, interactions of Fe3, a feline cytidine deaminase
related to human AP0BEC3F, with the accessory protein Bet of feline spumavirus were
demonstrated (Löchelt et al., 2005). Recently, it was shown that feline analogues for
AP0BEC3G reduce the infectivity of Vif-deleted FIV (Münk et al., 2008). All these findings
confirm an interaction of FIV Vif with feline cytidine deaminases.
14
Introduction
1.1.4. Replication cycle
An overview of a typical lentiviral replication cycle is shown below in Figure 1.3. Most of the
following information about uncoating, the reverse transcription complex, the preintegration
complex, cellular trafficking, particle production and budding is deduced from HIV-1 and is
expected to be similar for FIV.
Oagand G«g-Pro-Pol proteins
Mature Virlon
singly spliced env mRNA
multiply spliced rev mRNA
incoming virion
Figure 1.3. Lentiviral Replication Cycle: (Saenz and Poeschia, 2004)
/. 1.4.1. Viral entry
In general, the retroviral glycoprotein SU must find and bind to a specific receptor molecule
on the target cell's surface to enter a target cell. This interaction activates the membrane
fusion-inducing potential of the TM protein and, as a consequence, the viral and the cell
membranes fuse. The host range and tissue tropism of a retrovirus is defined by this specific
virus-receptor interaction (Coffin, 1997).
FIV can infect a broad range of cells types, including CD4+ and CD8+ T lymphocytes, B
lymphocytes, and macrophages. Similar to HIV-1, FIV infections result in a loss of CD4+ T
cells and a subsequent immunodeficiency (Pedersen et al., 1987; Brown et al., 1991; English
et al., 1993). In contrast to HIV-1, FIV does not require the surface receptor CD4 for cell
15
Introduction
entry (Hosie et al., 1993; Willett et al., 1997), but uses CD134 (de Parseval et al., 2004b; de
Parseval et al., 2004a; Shimojima et al., 2004) as primary receptor and CXCR4 as co-
receptor. In acute FIV infections, the thymus is the major target, while the bone marrow
becomes a target and a reservoir later on. The chemokine receptor CXCR4 is needed as co-
receptor for FIV-infection. However, the existence of a CXCR4 independent infection process
was suggested (Troth et al., 2008). Furthermore, FIV also uses cell-surface heparans and
DC-SIGN as binding receptors to enhance infection (de Parseval and Elder, 2001; de
Parseval et al., 2004c).
CD134 expression is largely restricted to CD4+ T cells, explaining the massive CD4+ T cell
depletion during FIV infection without using CD4 for cell entry. However, CD134 expression
is promoted and enhanced due to CD4+ cell activation, which maintains cell-survival, cell-
proliferation and numerous immunological responses. By targeting CD134, FIV diminishes a
cell population integral to immunological interaction (Shimojima et al., 2004, de Parseval et
al, 2004a).
Comparing FIV and HIV-1, the cell entry procedure might be quite similar. Parallel to HIV-1 it
was suggested that the FIV SU domain of the viral envelope glycoprotein SU-TM binds to the
primary receptor CD134, resulting in a conformational change in SU-TM. Subsequently, SU-
TM can bind to the co-receptor CXCR4, thereby promoting cell entry (Elder et al., 2008) (see
Figure 1.4). This scenario was further confirmed by the finding that soluble CD134 can
promote FIV infection of CD134-/CXCR4* cells (de Parseval et al., 2005; de Parseval et al.,
2006). After receptor binding, SU dissociates from SU-TM, enabling TM to fuse with the cell
membrane (reviewed by Gallo et al., 2003).
Figure 1.4. FIV binding to receptor, which consequently enables viral entry: (Elder et al., 2008)
- 16
Introduction
Directly after release of the viral core into the cell cytoplasm the core undergoes structural
changes and forms the reverse transcription complex (RTC). This process is called
uncoating and involves the dissociation of most of the capsid, matrix and RT proteins, while
Vpr remains associated. However, a small amount of RT must remain associated to facilitate
reverse transcription (Fassati and Goff, 2001). The resulting RTC binds to the host cellular
cytoskeleton. Thus, reverse transcription occurs in association with the cytoskeleton and was
further suggested to depend on actin microfilaments (Bukrinskaya et al., 1998).
/. 1.4.2. Reverse transcription
An important step in the retroviral life cycle is reverse transcription (see Figure 1.5). The
transformation of single-stranded viral RNA into double-stranded DNA is driven by a
multifunctional viral enzyme, the reverse transcriptase (RT). The RT exerts a RNA-
dependent polymerase function, a DNA-dependent polymerase function and a ribonudease
H (RnaseH) function.
Reverse transcription starts at the primer binding site (PBS), which is located close to the 5'
end of the RNA genome. A host-encoded tRNALyss, complementary to the PBS, serves as
primer for the RT. While the RT elongates the primer till the 5' end of the genome, the
genomic RNA template is degraded at the same time by the RT RnaseH activity. The result
is a reaction intermediate called minus-strand strong-stop DNA (-ssDNA) (Figure 1.5, part 1).
For continuation of minus-strand synthesis, the -ssDNA is transferred to the 3' end of the
RNA genome. After successful binding of the R region of the -ssDNA to the complementary
R region on the 3' end of the genomic RNA, the -ssDNA fragment serves as primer and the
minus strand synthesis can be completed (Figure 1.5, part 2). Concurrently, the RNA-
template is degraded by the RT RnaseH activity. However, purine rich regions at the 3' end
and in the centre of the RNA genome are resistant to degradation and are referred to as
central polypurine tract (cPPT) and 3' polypurine tract (3'PPT), respectively. The polypurine
tracts prime the plus strand synthesis, which already starts during minus strand synthesis.
The central polypurine tract is unique to lentiviruses like FIV, while other retroviruses only
contain a 3' polypurine tract. As a consequence, lentiviruses start the plus strand synthesis at
two distinct sites producing an upstream and a downstream DNA fragment (Figure 1.5, part
3). The synthesis of the downstream fragment starts at the cPPT and is finished, when it
reaches the 3'PPT. The synthesis of the upstream fragment starts at the 3'PPT and
proceeds to the end of the plus-strand strong-stop (+ssDNA). The RNA polypurine tract
primer and the tRNA^Yss are degraded. Then, the second-strand transfer is facilitated by
formation of a circular intermediate due to the ligation of the plus-strand PBS with the
17
Introduction
homologous minus-strand PBS (Figure 1.5, part 4). As a result, the upstream fragment can
be further elongated until the central termination sequence (CTS) is reached, which is
located in the centre of the genome, about 100 nucleotides downstream of the 5' end of the
downstream fragment. Thus, the completed plus-strand synthesis leads to a displacement of
about 100 nucleotides from the cPPT to the CTS. The resulting DNA overlap is called central
DNA flap and represents a triple stranded intermediate (Figure 1.5, part 6). Finally, the RT is
released at the CTS and reverse transcription is completed (Götte et al., 1999).
1. I J RNasc H cuts
R . US . PBS 5-1 h
2. 7^ cPPT PPT, U3 . R ,„ H \ 13*
t-si^ cPPT
^ii t-5t~ t r • "Si"*" A S"
i D+ i U+
f-5Jr+ cPPT PPT. 113 , R , 15 , ^^ ^..,
cppt ppl u3 "• u5
^ (J
U+ PBS
ppl u3 >^ u5 pbs
cppt
D+
5.
D+
U3 R . 15 PBS
PPt u3 r
CTS cppt
:^ u5
pbs U+
6. U+
, 113 . R . 15 . PBS cPPT
u3 u5 pbs
D+
PPT. L'3 ^ R ^ US
CPP' CIS ppl u3 u5
Figure 1.5. Scheme of reverse transcription of lentivimses like HIV or FIV: (Götte et al., 1999)
- 18-
Introduction
/. /. 4.3. Nuclear entry
The preintegration complex (PIC) represents the viral integration machinery and is formed
after reverse transcription. While only sparse information about the FIV PIC is published, the
PIC of HIV-1 is composed of the reverse transcribed double stranded cDNA and the viral
proteins RT, MA, IN and Vpr. Furthermore, the three host cell co-factor proteins high mobility
group chromosomal protein Al (HMGA1) (Miller et al., 1997), lens epithelial derived growth
factor (LEDGF/p75) (Llano et al., 2004), and bamer to autointegration factor (BAF) (Lin and
Engelmann, 2003) are involved in the PIC. The cDNA forms a loop that is held together by a
protein bridge. It is likely, that IN associates with the cDNA ends, establishes the protein
bridge and protects the cDNA ends from exonucleases (Miller et al., 1997).
For integration of the viral genome into the host genome, the reverse transcribed cDNA in
the PIC must enter the nucleus. Retroviruses of the genus gamma-retrovirus depend on the
breakdown of the nuclear membrane duhng mitosis to translocate the PIC into the nucleus.
In contrast to that, infection with lentiviruses was shown to be independent from mitosis,
resulting in the ability to infect non-dividing cells (Lewis and Emerman, 1994). Therefore, the
PIC must traverse the lipid bilayer of the nuclear membrane.
Nuclear envelopes contain multiple nuclear pore complexes (NPC), which permit bi-
directional transport of macromolecules (Nigg et al., 1997). This transport system is
regulated by a class of proteins known as importins and exportins. In order to be translocated
into the nucleus, proteins must contain a nuclear localization signal (NLS), which is
selectively recognized by importin a that in turn binds to importin ß. The resulting protein
complex interacts with the NPC and the translocation of the protein complex into the nucleus
is facilitated under energy consumption (reviewed by Sherman and Greene, 2002).
Since the PIC can enter the nucleus before mitosis, it was suggested that the PIC contains
elements with NLS (Bukrinsky et al., 1992). In HIV-1, several NLS were found in the MA
protein (Bukrinsky et al., 1993a; von Schwedler et al., 1994), the IN protein (Gallay et al.,
1997; Bouyac-Bertoia et al., 2001) and the Vpr protein (Heinzinger et al., 1994; Jenkins et
al., 1998). However, the different NLSs were shown to be redundant, since HIV remained
infectious after deleting the NLSs (Fouchier et al., 1997; Reil et al., 1998; Petit et al., 2000;
Dvorin et al., 2002; Limon et al., 2002a; Yamashita and Emerman, 2005). Moreover, addition
of an NLS to gammaretroviruses did not facilitate infection of non-dividing cells (Deminie and
Emerman, 1994; Seamon et al., 2002; Caron and Caruso, 2005).
Next to NLS in proteins, the cPPT element was suggested to support nuclear entry (Zennou
et al., 2000). Furthermore, Vpr was found to form nuclear hernations that disrupt the nuclear
19
Introduction
lamina. This may contribute to Vpr-mediated cell cycle arrest, but may also facilitate nuclear
uptake of the large PIC (de Noronha et al., 2001). Finally, also the uncoating after viral entry
was suggested to play a role in nuclear entry, since the CA protein of HIV-1 dissociates
easier from the viral nucleoprotein complex compared to the CA protein of the
gammaretrovirus MLV. As a consequence, HIV-1 uncoating seems to be more rapid and
could facilitate nuclear entry (Yamashita and Emerman, 2004).
Also the exact mechanism of FIV nuclear entry remains to be elucidated. In FIV, Vpr is
missing. As described before (see 1.1.3.3), FIV ORF-A shares properties with HIV-1 Vpr.
Orf-A is a nuclear protein, which includes a NLS (Gemeniano et al., 2004). Furthermore, FIV
IN has karyophilic properties, which depend on the conserved N-terminal zinc-binding
domain, while no canonical NLS can be found. The zinc-binding domain promotes protein-
protein interactions and may facilitate interaction with proteins of the nuclear import
machinery (Woodward et al., 2003).
/. 1.4.4. Integration
After the entry of the PIC into the nucleus, the viral double stranded cDNA genome can be
integrated into the host genome by action of the viral enzyme integrase (IN). The FIV IN
functions as a multimer and has a 3' end processing function, a 3' end joining function and a
disintegration function (Shibagaki et al., 1997). First, two nucleotides from the 3' end of each
viral DNA strand are cleaved by IN to produce a free 3'OH group. Then, IN breaks the host
DNA and joins it with the viral 3' ends in one transesterification reaction. The same
procedures are then repeated at the 5' end of each viral DNA and the 3' end of the host
DNA. Finally, host DNA repair enzymes remove resulting nucleotide overhangs and fill
resulting nucleotide gaps (reviewed in Ciuffi and Bushman, 2006). The integrated viral
genome is termed provirus.
The integration target site selection differs among retroviruses; it is not sequence specific,
but also not random (reviewed in Bushman et al., 2005). Integration target sites for FIV were
determined based on infections with an FIV vector in a human cell line. FIV prefers to
integrate into transcriptionally active regions of chromatin: 79% of all integrations occurred in
genes; only 4% of these integrated into exons. Furthermore, FIV integrates over the entire
length of transcriptional units. Interestingly, 21% of all integrations were shown to occur in
genes regulated by the transcriptional coactivator LEDGF/p75 (Kang et al., 2006).
-20
Introduction
For HIV-1, host integration co-factors have been extensively described (reviewed by van
Maele et al. 2006). The lens epithelial derived growth factor/p75 (LEDGF/p75) is suggested
to be part of the PIC, and was shown to interact with lentiviral but not MLV integrase. It is a
transcriptional activator of stress-related or anti-apoptotic proteins. LEDGF/p75 contains a
classical NLS and therefore has a possible role in nuclear import. However, nuclear
localization as well as viral replication does not depend on LEDGF/p75. Furthermore,
LEDGF/p75 acts as a tethering factor for HIV-1 integrase to chromosomes and thus probably
targets the PIC to actively transcribed regions.
The barrier-to-autointegration factor (BAF) is another component of the PIC and prevents
suicidal autointegration.
The high mobility group chromosomal protein AI (HMGA1) is a non-histone DNA-binding
protein and also part of the PIC. It has DNA-protein and protein-protein binding capacities
and can thus modulate transcriptional regulation and chromatin structure. HMGA1 can bind
within the HIV-1 5'LTR and may play a role in viral transcription.
/. /. 4.5. Transcription and transiation
Once integrated, transcription of the provirus is mediated by the host cellular RNA
polymerase II. Transcription regulatory elements are located within the proviral LTR. In FIV,
the following binding sites for nuclear transcription factors and enhancers were found to be
responsible for the basal promoter activity of the LTR: AP-1, AP-4, C/EBP and ATF
(Thompson et al., 1994; Kawaguchi et al., 1995; Ikeda et al., 1996). Furthermore, a NFkB
site is found in the LTR, whose role in LTR transactivation must still be elucidated (Olmsted
et al., 1989; Talbott et al., 1989).
Next to the c/s-acting regulatory elements, the integration site within the heterogeneous
chromatin determines transcriptional activity (Jordan et al, 2001). Furthermore,
transcriptional activity was shown to be negatively influenced by DNA methylation in HIV-1
(Bednarik et al., 1987) and FIV (Ikeda et al., 1996).
The FIV provirus contains 3 splice donors and 5 splice acceptors. Thus, transcription can
result in a non-spliced genomic full length RNA and in at least 5 other spliced RNA species
(Tomonaga et al., 1993). The multiply spliced RNAs that do not contain any introns can leave
the nucleus towards the cytoplasm like all cellular mRNAs. They encode the early gene
products Vif, Rev and ORF-A. ORF-A may act as transactivator equal to the HIV-1 Tat, a role
that still has to be fully elucidated (see 1.1.3.3). The singly spliced and non spliced RNAs
-21
Introduction
contain introns and therefore have to be shuttled to the cytoplasm for virus production. This
shuttling function is maintained by the early expressed Rev protein (see 1.1.3.3) that
consequently enables expression of the late gene products including the structural proteins
and the transcribed viral RNA genome.
The Rev activity connected with the expression of the differentially spliced RNAs mainly
regulates latency of FIV-infection. Thus, peripheral blood mononuclear cells (PBMCs) of
asymptomatic cats with a latent FIV-infection predominantly express multiply spliced RNA
over non- or singly spliced RNAs. After stimulation, expression of non- or singly spliced RNA
is markedly increased in comparison to multiply spliced RNAs (Tomonaga et al., 1995).
Another transcript is found to be conserved in FIV. It is an antisense transcript
complementary to the envgene and therefore may play a role in translational regulation. The
gene product would be a highly hydrophobic and 103 amino acids long protein, but there is
no evidence for expression (Bhquet et al., 2001).
/. /. 4.6. Particle production, budding and maturation
The unspliced genomic RNA is translated into the precursor polyproteins Gag and via
ribosomal frameshifting also into GagPol. The N-terminus of both precursors is myristylated,
which is essential for its transport to the cell membrane (Göttlinger et al., 1989; Manrique et
al., 2001). Thus, the polyproteins traffic to the plasma membrane, where the virus assembly
takes place (Jouvenet et al., 2006). Meanwhile, the Env-polyprotein, translated at the rough
endoplasmatic reticulum, is cleaved into the TM and SU proteins and is incorporated into the
plasma membrane (Stephens et al., 1991). At the assembly site, Gag proteins associate and
multimerize. Furthermore, the NC domain of the Gag-precursor protein binds and packages
the viral RNA genome via RNA binding zinc-finger motifs and a high content of basic
residues (Manrique et al., 2004). Packaging of full-length unspliced genomic RNA is
facilitated due to 2 major packaging signals within the 5' untranslated region and the first
nucleotides of gag. However, there is evidence that next to these major encapsidation
signals additional weaker encapsidation determinants are spread out in the 5'LTR as well as
in the 3'LTR (Mustafa et a!., 2005; Ghazawi et al., 2006).
The viral particles are released by viral budding, which results in an enveloped spherical
immature virion. Finally, maturation occurs during and after particle release. Maturation is
performed by the viral enzyme PR and involves cleaving of the Gag precursors into the
functional and mature structural proteins and cleaving of the Gag-Pol precursor into the
different viral enzymes (Luttge et al., 2008).
22
Introduction
1.2. Different retroviral DNA forms
Several aspects about the different retroviral DNA forms will be discussed in this chapter,
with a focus on the episomal DNA forms. Studies on the topic of episomal DNA forms have
been done with different retroviral models, mainly with the lentivirus HIV-1 and the gamma-
retrovirus MLV. However, since episomal DNA forms are conserved within the retroviridae
family, all studies are combined here to provide a uniform picture about the different DNA
forms in retroviruses. These results can also give information about the different FIV DNA
forms, since there are few publications dealing directly with FIV (Saenz et al., 2004).
After nuclear entry, integration of the reverse transcribed double stranded viral DNA genome
is a crucial step for completion of the retroviral replication cycle. This step enables
transcription and translation of viral proteins in order to produce new virions. Nevertheless,
next to the provirus also unintegrated DNA forms can be found in the nucleus. There is linear
unintegrated DNA as precursor for integration. Furthermore, two different circular episomal
forms exist, containing the whole viral genome next to one or two copies of the LTR. These
circular forms arise from the linear form. Finally, the retroviral genome can also integrate into
itself and as a consequence form circular autointegration products that also stay
extrachromosomal. An overview of the different viral DNA forms during infection is given in
Figure 1.6.
The circular episomal DNA forms were first considered to be precursor molecules for
integration (Panganiban and Temin, 1984). It is now accepted that the unintegrated linear
DNA molecule is the direct precursor for integration, while the circular episomal DNA forms
don't play a role as templates for integration Thus, circular unintegrated DNA forms are
considered to be dead-end products of viral replication (Brown et al., 1989, Lobel et al.,
1989).
Episomal DNA forms are generated after nuclear entry and are thus found in the nucleus.
However, there is a report that 2-LTR circles of MLV are built directly after reverse
transcription and can be found already in the cytoplasm and not exclusively in the nucleus
(Serhan et al., 2004). Within the nucleus, episomal DNA is localized between the
chromosomes and aggregates into clusters. Furthermore, episomal DNA accumulates before
first viral transcripts are produced (Bell et al., 2001). In contrast to the linear episomal cDNA,
23
Introduction
2-LTR circles are not associated with proteins of the PIC like IN, but show binding to a not
identified nucleoprotein (Bukrinsky et al., 1993b).
RNA C RT
I A gs DNA ^
\Ji\ ——^.H A -H A US
unintegrated
/vy^Tj^ ^"'^"'"^ |7]^ nucleus
cytoplasm
Figure 1.6. All retrovlral DNA forms In a cell during infection: DNA resulting from autointegration is not shown.
1.2.1. Integrated DNA / unintegrated linear DNA
As described above, the unintegrated linear DNA is generated by reverse transcription within
the host cell and is the precursor for integration (Brown et al., 1989, Lobel et al., 1989). All
aspects about the integration and about the integrated provirus are described in chapter
1.1.4, since the integrated DNA mainly facilitates the viral replication cycle.
1.2.2. 1-LTR circles
1-LTR circles represent the whole reverse transcribed retrovlral genome, but contain only
one LTR. Different pathways might lead to the formation of 1-LTR circles. One possibility is
that 1-LTR circles arise from reverse transcription intermediates that do not finish the reverse
transcription process (Lee and Coffin, 1990; Miller et al., 1995). Alternatively, 1-LTR circles
might be formed after complete reverse transcription by homologous recombination between
the two homologous LTRs (Farnet and Haseltine, 1991). Homologous recombination is a
conserved process facilitating repair of DNA double strand breaks. The nuclear host cell
complex of Rad50/Mre11/NBS1 is involved in natural recombination processes, checkpoint
control, telomere maintenance and meiosis (Symington et al., 2002; Tauchi et al., 2002) and
-24-
Introduction
furthermore is also shown to be related to 1-LTR circle formation. Therefore,
Rad50/Mre11/NBS1 might create single stranded LTRs, which can subsequently anneal
again and so create circular DNA with only one LTR. Mutations in the exonuclease Mre11,
which has a central role in the complex formation, subsequently inhibit 1-LTR circle formation
(Kilzer et al., 2003).
1.2.3. 2-LTR circles
2-LTR circles represent a complete and circularized reverse transcribed retroviral genome,
and thus contain both of the two LTRs. 2-LTR circles are formed by the host cell
nonhomologous DNA end joining (NHEJ) pathway. This is the major double-strand break
repair pathway during GO and G1 phase and early S-phase (Grawunder et al., 1998). NHEJ
pathway is facilitated by the DNA-dependent protein kinase catalytic subunit (DNA-PKcs),
which binds Ku with its two subunits Ku70 and Ku86/80 to free double stranded DNA ends.
After this association, the DNA ligase IV and its co-factor XRCC4 are recruited to ligate the
free DNA ends. The result of viral reverse transcription is a linear double stranded DNA
molecule with two free ends. Ku was shown to be associated with the cDNA ends of the PIC.
Ku and the XRCC4-DNA ligase IV complex probably ligate the free ends and form a 2-LTR
circle (Li et al., 2001). However, there is evidence that the PKcs of the NHEJ pathway is not
essential for 2-LTR circle formation (Kilzer et al., 2003). Recently, RAD52 was shown to
inhibit 2-LTR circle formation. RAD52 is a protein of the homologous recombination process
and is suggested to compete with and replace Ku from the cDNA ends. Additionally, RAD52
is associated with the cDNA and can block other proteins involved in integration. In
conclusion, RAD52 prohibits integration as well as 2-LTR formation (Lau et al., 2004).
1.2.4. Autointegration products
Circular episomal viral DNA forms also arise due to autointegration. During this
intramolecular integration process, the linear cDNA integrates into itself and not into
heterologous chromosomal DNA. Autointegration is facilitated by the same enzymatic
pathways as "conventional" integration. The resulting circular autointegration products
contain one or two LTRs, need not to represent the whole retroviral genome and can contain
inversions. Higher concentrations of heterologous target DNA, as present in the nucleus, are
suggested to inhibit episomal circle formation by autointegration, but not 1-LTR and 2-LTR
formation by host-cell factors (Shoemaker et al., 1980; Lee and Coffin, 1990; Farnet and
-25-
Introduction
Haseltine, 1991). Furthermore, the host cellular protein barrier to autointegration factor (BAF)
was shown to inhibit autointegration. In this context it was suggested, that the viral DNA
genome is prevented from autointegration through intramolecular bridging by the DNA
binding properties of BAF (Lee and Craigie, 1998).
1.2.5. Abundance and stability of episomal viral DNA
Retroviral cDNA accumulates quickly after infection and reaches a maximum after 12 to 24
hours, while integrated DNA forms or episomal forms like 2-LTR circles accumulate slower
and reach a maximum after 24h. This is in line with the hypothesis that the linear double
stranded cDNA serves as a template for integrated as well as unintegrated forms. Thus, only
a fraction of all cDNA copies in cells are integrated and can maintain the virus replication
cycle. After 24 to 48 hours, all linear cDNA should be either integrated or transformed to a
circular episomal form (Butler et al., 2001). In HIV-1 infected resting and activated CD4*
cells, unintegrated DNA is the most abundant form of DNA (Chun et al., 1997). Also in brain
tissue of HIV-1 infected patients, up to 80-fold more unintegrated DNA than integrated DNA
was found during HIV-1 infection (Pang et al., 1990). These large amounts were correlated
with the pathogenesis of AIDS in brain tissues (Pang et al., 1990; Pauza et al., 1990; Teo et
al., 1997; Panther et al., 1998).
Concerning FIV, 2 LTR circles of FIV vectors were shown to be stable and competent for
gene expression (Saenz et al., 2004). However, the stability of episomal DNA forms has
been mainly investigated based on 2-LTR circles during HIV-1 infection. Several studies
show short half-lifes of 2-LTR circles in dividing cells (Pauza et al., 1994; Sharkey et al.,
2000; Sharkey et al., 2005). In contrast to that, another study reveals that 2-LTR circles are
stable and only decrease by a rate that is equal to the rate of cell division (Pierson et al.,
2002). This is supported by the observation that 2-LTR circles, once formed, persist when
the cell cycle and further cell division are stopped (Butler et al., 2002). Consistent with this
view, 2-LTR circles are stable for up to 30 days in non-dividing cells like macrophages
(Gillim-Ross et al., 2005a; Kelly et al., 2008). Recently, a report compared 2-LTR circles of
patients before and after highly active antiretroviral therapy (HAART) therapy. As a result,
they found that in contrast to the integrated provirus, 2-LTR circles harboured mutations. This
indicated a steady 2-LTR circle turnover and thus could be another in vivo evidence for 2-
LTR instability (Chavez et al., 2007). Further studies will have to elucidate the stability of
episomal DNA forms during infection.
26
Introduction
Several studies detected persistence of 2-LTR circles in HIV-1 infected individuals, who have
no detectable plasma viraemia due to HAART therapy (Sharkey et al., 2000; Cara et al.,
2002; Brüssel et al., 2003). The hypothesis of instability of 2-LTR circles led to the
suggestion to use these episomal DNA form as marker for active viral replication (Pauza et
al., 1994; Sharkey et al., 2000). However, the 2-LTR persistence during an antiretroviral
therapy can have two reasons in tenns of episomal circle stability. If 2-LTR circles are
Instable, their persistence could be only explained by viral replication and would confirm the
use of episomal DNA forms as markers for ongoing viral replication. If 2-LTR circles are
stable, they would first increase in number before antiretroviral therapy and could then stably
persist in non-dividing cells (Butler et al., 2002). However, a longitudinal study reports the
clearing of 2-LTR circles 8 years after the onset of antiretroviral treatment. The reason for
this could likely be the suppression of replication, while a clearing of a reservoir also cannot
be excluded (McDermott et al., 2005).
Finally, a new aspect in 2-LTR circle biology was shown by Delelis and co-workers. They first
found that the spumaretroviral IN cleaves the unique LTR-LTR junction of 2-LTR circles.
Furthermore, they could support this observation by demonstrating that also HIV-1 IN
specifically cleaves 2-LTR circles (Delelis et al., 2007). This would be consistent with the
observed accumulation of 2-LTR circles in cells infected with IN-defective viruses as well as
with the finding, that both circular episomal forms are processed in a different manner.
However, these recent results make it necessary to reconsider the functional role of 2-LTR
circles in the retroviral life cycle (Delelis et al., 2005).
1.2.6. Gene expression and function of episomal viral DNA
Non-integrated viral DNA forms cannot produce fully infectious virions (Stevenson et al.,
1990; Engelman et al., 1995; Wiskerchen and Muesing, 1995). However it was demonstrated
that episomal DNA forms are transcriptionally and translationally active.
Concerning FIV, it was demonstrated that integrase defective FIV-vectors produce stable
amounts of unintegrated DNA forms under certain conditions. Furthermore, these IN" vectors
showed a high-level transgene expression, equivalent to WT vectors (Saenz et al., 2004).
Concerning HIV-1, it was demonstrated that episomal HIV-1 DNA expressed Tat when the
integrase was mutated (Engelman et al., 1995; Wiskerchen and Muesing, 1995).
Furthermore, multiply spliced mRNAs like nef, env and tat are transcribed and translated
27
Introduction
from episomal HIV-1 DNA. There is evidence that transcription of unintegrated DNA is a
normal early step in HIV-1 replication and that simultaneously also the singly spliced and
unspliced mRNAs are produced, but not translated due to a lack of Rev-function in the
absence of integration (Wu and Marsh 2001; Wu and Marsh 2003). Moreover, also non-
dividing macrophages with reported stability of unintegrated viral DNA forms show a stable
transcription of the early genes nef, tat rev and vif. However, at the protein level, again only
Nef-expression could be identified (Kelly et al., 2008). Nevertheless, transcription and
translation of all viral proteins from HIV-1 DNA was already observed by DNA molecules,
mimicking the extrachromosomal DNA forms (Cara et al., 1996). However, the gene
transcription and expression of naturally occurring unintegrated DNA forms still has to be
analyzed. Furthermore, it is still not clear, which unintegrated viral DNA form is responsible
for protein expression.
Protein expression from unintegrated DNA also seems to be regulated. HIV-1 Vpr was found
to increase LTR-driven protein expression from unintegrated DNA templates (Poon and
Chen, 2003; Poon et al., 2007).
Considering these results, unintegrated DNA forms may have a function in protein
expression during the retroviral life cycle. HIV-1 Nef is an early protein that was shown to be
produced by unintegrated DNA in active T-cells, in resting T-cells and in macrophages. After
expression, Nef downregulates the CD4 expression in activated T-cells and facilitates viral
replication and T-cell activation in resting T-cells (Wu and Marsh 2001; Wu and Marsh 2003;
Gillim-Ross et al., 2005b; Kelly et al., 2008). Thus, Nef is important for preventing
superinfection and viral budding. The production of Nef from episomal DNA is also regulated
by Vpr as mentioned before (Poon et al., 2007).
DNA-methylation was suggested to be another function of unintegrated HIV-1 DNA. Thereby,
episomal DNA forms might increase the expression of eukaryotic DNA methyltransferases
(DNMTs) maybe due to production of the transactivator Tat. As a result, the overexpressed
DNMTs increase methylation and subsequent silencing of cellular genes. This could
contribute to HIV-1 pathogenesis (Fang et al., 2001).
Finally, unintegrated retroviral DNA also seems to play a direct role in pathogenesis. There is
a correlation between accumulation of episomal DNA and cell killing in different retroviruses:
spleen necrosis virus (Keshet and Tenim, 1979), avian leukosis virus (Weiler et al., 1980),
feline leukemia virus (Mullins et al., 1986), and equine infectious anemia virus (Rice et al.,
1989). High copy numbers of HIV-1 episomal DNA are correlated with high levels of plasma
-28-
Introduction
HIV-1 RNA, rapid decline in CD4''"-cell count, and clinical progression of AIDS (Panther et al.,
1998). Furthermore, circular episomal HIV-1 DNA forms can be associated with dementia,
cerebral atrophy and multinuclear giant cell in the brains of AIDS patients (Pang et al., 1990;
Pauza et al., 1990; Teo et al., 1997).
1.2.7. Detection and quantification of viral DNA forms
The Southern blot method was used as a first tool in order to get insights into the abundance
of the different viral DNA forms (Kim et al., 1989) and was also used as independent method
to evaluate real-time PCR results (Butler et al., 2001). Additionally, also polymerase chain
reaction (PCR) could improve detection and quantification of viral DNA (Pang et al., 1990;
Benkirane et al., 1993; Courcoul et al., 1995, Frey et al., 2001). With the emergence and
improvement of real-time PCR, this method provides the most sensitive and precise tool for
detection and quantification of various DNA forms in HIV-1 (Butler et al., 2001; Butler et al.,
2002; Pierson et al., 2002; Brüssel and Sonigo, 2003) and FIV (Saenz et al., 2004; Savarino
et al., 2007).
-29
Introduction
1.3. Real-time PCR
Real time PCR is based on the PCR method, developed in the 1980s by Kary Mullis and co-
workers. PCR allows amplification of specific DNA fragments more than a billion fold (Mullis
et al., 1986). The advancement of PCR in the form of real-time PCR was developed by
Higuchi and co-workers in the 1990s. They used the intercalating fluorescent dye ethidium
bromide to monitor the PCR-reaction under UV-light and could thus simultaneously amplify
and detect specific DNA sequences (Higuchi et al., 1992, Higuchi et al., 1993). In 1996, the
first real-time PCR instrument was commercially available from Applied Biosystems. Several
companies followed and added further machines to the market.
PCR uses a pair of oligonucleotides or primers that specifically hybridize to DNA sites, which
flank the region to be amplified. The primers are substrates for the DNA polymerase. Due to
its thermo-resistance and stability Taq-polymerase (derived from Thermus aquaticus) is
mainly used today. With its 5' to 3' polymerase activity, it creates complementary strands via
adding deoxynucleotides. One cycle of amplification consists of three steps: (1) separation of
the dsDNA at >90°C, (2) primer annealing at 50°C to 75°C, (3) primer extension at 72°C -
78°C. A typical PCR-reaction consists of 30 - 50 cycles (Powledge et al., 2004).
The disadvantage of PCR often lies in the post-PCR handling steps that are laborious and
prone to cross-contamination. Post-PCR steps include the agarose-gel electrophoresis with
ethidium bromide detection. Southern blot or PCR-ELISA, respectively. Real-time PCR
eliminates post-PCR steps by simultaneous amplification and detection without opening the
tube and therefore is referred to be a closed or homogenous system. Real-time PCR is more
precise than conventional PCR, minimizes the possibility of cross-contamination between
samples due to the closed system and enables fast and continuous data collection. However,
amplicon size determination still requires the opening of the tubes. Furthermore, higher start-
up costs and expenses are needed compared to conventional PCR (outlined in Mackay et
al., 2002 and Houghton et al., 2006).
30
Introduction
1.3.1. Amplicon detection
During real-time PCR, amplification is monitored by recording changes in samples
fluorescence. Both, specific and unspecific detection methods are used. All establish a link
between amplification and increase in fluorescence (see Figure 1.7).
1.3.1.1. DNA-binding fluorophores
This is the earliest and simplest mechanism, but still in use. DNA-binding fluorogenic
molecules including Ethidium bromide, BEBO, YOYO-1 and SYBR-green can intercalate into
double stranded DNA and then fluoresce under exposure to light with a suitable wavelength.
SYBR green is the most commonly used intercalator. The bound SYBR green exhibits 1000-
fold more fluorescence than free dye and the binding affinity is 100-times higher than that of
ethidium bromide. In general, intercalation happens independent from the DNA sequence,
thus this type of detection is unspecific (Figure 1.7a). That is why unwanted amplification
products like primer-dimers are also detected. Therefore, melting curve analysis of the
amplicon should be considered. If it shows two or more peaks, there is evidence that not only
one amplicon was generated. However, DNA-binding fluorophores have the advantage that
no specific probe has to be designed. That reduces time and costs. Furthermore, amplicon
size is irrelevant for detection (Wilhelm and Pingoud, 2003).
/. 3.1.2. Hybridisation probes: ligtit cycler probes
This method uses fluorescence resonance energy transfer (FRET) for amplicon detection.
FRET is the transfer of excitation energy from one fluorophore to the other - from dipole to
dipole - that share overlapping emission and excitation spectra.
Two fluorophore-labelled probes are used in one assay. The donor probe has a 3' label,
while the acceptor probe is labelled on the 5' end. In case that both probes are not bound,
only the donor-fluorophore is exited. During the annealing step of the real-time PCR, both
probes specifically bind onto the template in close proximity. As a result, the 5' donor
fluorophore transfers its energy on the 5' acceptor fluorophore (Figure 1.7b). The emerging
fluorescence of the acceptor is detected and correlates with amplification. A disadvantage
can be the use of Taq-polymerase that can partly hydrolyze the probes through its
endonucleolytic activity. The resulting higher signal-to-noise ratio can be prevented by the
use of other polymerases (Wilhelm and Pingoud, 2003).
31
Introduction
/. 3.1.3. Linear oligoprobes: 5' nuclease probes/ hydrolysis probes/ TaqMan probes
This method to detect amplification was described first in 1991 for the use of radiolabelled
probes (Holland et al., 1991). In 1993, the method was improved by the use of a dual-
fluorophore labelled probe (Lee et al., 1993).
This amplicon-detection method uses the 5' - 3' exonuclease function of the Taq-polymerase
and a single sequence specific oligonucleotide probe, which binds to the template between
the primers, before phmer-annealing. The probes are labelled with a quencher fluorescent
dye and a reporter fluorescent dye, respectively. When both fluorophores are bound on the
probe, they are in close proximity and the quencher subsequently absorbs the reporter's
energy by FRET. During amplification, the probe is cleaved by the Taq polymerase and the
reporter is separated from the quencher, resulting in liberated reporter fluorescence (Figure
1.7c). This fluorescence is measured and is proportional to amplification. 5' nuclease probes
should be 20-40 nucleotides in length, should have a GC content of 40-60%, should not
include single nucleotide runs and should not have repeated sequence motifs or overlapping
regions with the primers. Common quenchers include fluorescing and non-fluorescing
quenchers (NFQ). Additionally, all obtained results can be normalized to the passive internal
reference fluorophore ROX, in order to adjust for non-PCR related fluctuations in
fluorescence (Mackay et al., 2002). In conclusion, the advantage of TaqMan probes is the
specificity of amplicon detection. However, TaqMan probes are more expensive and probe
design can be time consuming and challenging (Wilhelm and Pingoud, 2003).
/. 3.1.4. tiairpin oligoprobes: molecular beacon probes
Molecular beacon probes are a variation of dual labelled oligoprobes. They also have a
reporter and a quencher fluorophore at the probe's ends that can inhibit reporter
fluorescence due to FRET. The probe is designed in such a manner, that only a part in the
middle of the probe is homologous to the template, while the terminal 10 to 15 nucleotides
are self-complementary. Thus, the free probe builds a stem-loop and facilitates FRET due to
close proximity of the fluorophores (Figure 1.7d). In order to bind to the template, the probe
must open the stem-loop structure and thus releases reporter fluorescence (Tyagi and
Kramer, 1996).
/. 3.1.5. Self fluorescing amplicon: sunrise primers
The sunrise primers work similar to molecular beacon probes, except that the fluorescent
label is incorporated into the PCR product. At its 5' end, the dual-fluorophore labelled sunrise
-32-
Introduction
primer forms a hairpin structure, which maintains close proximity of reporter and quencher,
inducing FRET. The sunrise primer acts as forward primer and is elongated during real-time
PCR. This single stranded DNA amplicon serves as template for the reverse primer. During
elongation, the hairpin structure is opened and reporter fluorescence is released
(Figure1.7e). The amplicon detection method is not highly specific, because also primer
dimers lead to opening of the stem-loop (Nazarenko et al., 1997).
/. 3.1.6. Self fluorescing amplicon: scorpion primers
The dual fluorophore labelled scorpion primer is structurally similar to the molecular beacon
probe or the sunrise primer. Like the molecular beacon probe, but unlike the sunrise primer,
the hairpin region is complementary to a part of the amplicon. The 3' end of the scorpion
primer acts as fonward primer and a single stranded amplicon is elongated, which can then
serve as template for the reverse primer. However, the stem loop region is complementary to
a target sequence further downstream, subsequently binds to it and thus separates the
fluorophores and induces the fluorescence signal of the reporter (Whitcombe et al., 1999)
(Figure 1.7f).
33
Introduction
Figure 1.7. Different types of amplicon detection in a real-time PCR assay: (Wilhelm and Pingoud, 2003)
(D) donor, (A) acceptor, (R) reporter, (Q) quencher
(a) DNA binding fluorophores, (b) hybridization probes (c) 5' nuclease probes, (d) molecular beacon probes, (e)
sunrise primers, (f) scorpion primers
1.3.2. Quantification
Real-time PCR offers a wide dynamic range of quantification of 7-8 logarithmic decades with
a high precision (<2 % standard deviation) and a high technical sensitivity (< 5 copies).
However, these advantages also increase the risk of false negative results. Also, variation
was showed to increase in correlafion with the cycle number (Klein, 2002).
Quantitative analysis is based on the evaluation of the amplification curves, showing the
increase in fluorescence obtained during the cycles of the real-time PCR (Figure 1.8). Three
phases are visible in an amplification curve: (1) an initial lag phase, (2) an exponential phase
and (3) a plateau phase. A direct correlation between product accumulation and signal
increase is only given during the exponential phase. Thus, accurate quantificafion can only
be performed in the exponential phase. The amount of template is estimated from the
-34-
Introduction
number of cycles needed for the signal to reach a threshold. The threshold must cross the
signal curve in its exponential phase. Thus, the crossing point represents the number of
cycles to reach the threshold and is defined as threshold cycle (CT). The CT is inversely
proportional to the amount of template. For quantification, reference samples are needed:
defined standards as well as reference genes can be used. However, all quantifications by
real-time PCR are relative to the applied reference. Nevertheless, an externally defined
standard results in an absolute quantification of template copy number, while comparison to
an internal standard only results in relative quantification (Klein, 2002; Wilhelm and Pingoud,
2003).
t Fl
RFU
35
30H
25
20
15
10 H
5
Log template
molecules
0-K 0
Threshold
IS 20 25 30 35 40 45
cycle number —^ Figure 1.8. Amplification curves in an amplification plot:: (Wilhelm and Pingoud, 2003)
Fluorescent curves result from a dilution series with template copy numbers ranging from 100 to 1000 000.
(NTC) no template control/negative control
For absolute quantification an external standard is needed. Therefore, a sample with known
copy number (RNA, cDNA, plasmid DNA, genomic DNA) is diluted in order to create a
standard dilution series for a standard curve. The CTs of unknown samples and of the
external standard dilution series are compared and can be used to estimate the copy number
of the unknown samples. Thus, absolute numbers of quantification largely depend on the
accuracy of the external standard. Furthermore, the amplification efficiency of samples and
the amplification efficiency of standards must be identical. Finally, the same reaction
conditions should be applied to standards as well as unknown samples. Therefore, the CT-
values of an external standard are always detennined together with those of the samples in
each assay. To compare results of different laboratories differences in standards, reagents,
35
Introduction
instrumentation and data analysis must be taken into account and should be standardized as
well (Klein, 2002; Valasek and Repa, 2005).
For relative quantification, an internal standard like a housekeeping gene can be used. A
relative quantification records changes in the steady state of unknowns relative to an
invariant internal standard like a control gene. For relative quantification of gene expression,
housekeeping genes are chosen that should always show the same level of expression.
Before usage in a relative quantification, this constant expression must be demonstrated in
order to obtain reliable data (Klein, 2002; Valasek and Repa, 2005).
36
Introduction
1.4. Aim of the study
During lentiviral infection, different viral DNA forms can be found in infected cells after
reverse transcription of the viral RNA genome into DNA. The most important DNA form for
virus replication seems to be the integrated DNA, which enables protein expression and
virion production. However, other episomal DNA forms are also formed during the infection
process. Next to autointegration products that evolve due to the viral integration process,
other circular episomal DNA forms are generated by host-cellular processes: 1-LTR circles
are formed by homologous recombination, while 2-LTR circles are formed by non-
homologous end-joining. The abundance of these episomal forms within a cell and the
stability after formation are important characteristics and might be associated with the
function of these forms. Information about circular episomal forms at the first sight does not
seem to be important for our understanding of viral replication, since these forms were
thought to be dead-end products of the virus. However, there is evidence that viral
unintegrated DNA also enables gene expression, mainly of the early genes. These findings,
together with observations showing a regulation of episomal gene-expression, would suggest
a function of these DNA forms in the viral replication cycle. Contributing to that, the
abundance and stability of lentiviral episomal DNA forms has been extensively investigated
for HIV-1, with the help of molecular methods like real-time PCR and Southern blot. The aim
of this study is to develop and evaluate reliable real-time PCR assays for the different viral
DNA forms occurring in FIV infected cells in order to get insight into the abundance and
stability of episomal DNA forms during an FIV infection. Considering that FIV is an accepted
HIV and AIDS model, the results could contribute to our knowledge about the different viral
DNA forms in lentiviral replication.
Real-time PCR assays shall be developed for the different episomal FIV- DNA forms that
occur during FIV infection: total viral DNA, linear integrated DNA, 1-LTR circles and 2-LTR
circles. Similar assays have already been described for HIV-1 and will now be adapted for
FIV. By using the developed assays, abundance and stability of the viral DNA forms will be
determined in time-course infection experiments in the context of either proficient or impaired
viral integration. This will be facilitated by the use of an integration proficient FIV vector and
an integration deficient FIV-vector, the latter containing an amino acid substitution in the viral
enzyme integrase.
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Materials & Methods
2. Materials & Methods
2.1. Restriction enzymes and digestion
Restriction enzymes or restriction endonucleases were originally found in bacteria as a
defence mechanism against foreign double stranded DNA, for example of bacteriophages.
They are widely used for cutting double stranded DNA specifically at restriction sites.
Restriction sites are distinct and often palindromic DNA sequences, which are recognized by
endonucleases and subsequently cut. Depending on the restriction enzyme blunt ends or
sticky ends can occur on the cut DNA fragment.
Restriction enzymes were used for qualitative analysis of plasmids after plasmid preparation
or as a preparative step during the creation of new plasmids.
Restriction enzymes were purchased from New England Biolabs or Promega, together with
suitable buffers. Restriction digests were usually prepared in a total volume of 20 |jl with a
DNA amount of 0.5 |jg to 1 pg and an enzyme amount of 0.2 to 1 pi (2-10 Unit). In order to
get the best digestion-result the recommended 10 x buffer were used and 10 x BSA (see
below) was added. The reaction mix was then filled up with dH20 to 20 pi and incubated for 1
hour at the suitable incubation temperature for the enzyme; in general at 37°C.
Bovine serum albumine (BSA): 0.1 pg/pl
2.2. Agarose gel electrophoresis
Agarose Gel electrophoresis is a method for separation of DNA molecules according to size,
while the DNA moves trough a gel consisting of an agarose matrix, with variable agarose
concentration. Migration is accomplished by an electric field, in which the negatively charged
DNA moves to the positive pole. Smaller molecules migrate faster through the matrix-pores
than larger ones and can therefore move longer distances under the same conditions and in
the same time. As a result, DNA molecules with different sizes are separated within the gel.
Agarose Gel Electrophoresis was used for identification of DNA molecules according to their
banding patterns after restriction digests. Additionally, it was used as a preliminary step for
creating new plasmids/vectors by separation of DNA fragments for further ligation.
-38-
Materials & Methods
Depending on the expected fragment sizes, agarose gels contained 0.8 % to 1 % agarose
dissolved in 1 x TAE buffer. DNA-samples were mixed with 5 x loading buffer and loaded
onto the gel along with a 1 kb marker for fragment size determination. Gels were run in 1 x
TAE buffer at 90 - 130 V. After electrophoresis gels were stained for up to 30 minutes in an
ethidium bromide solution. Ethidium bromide intercalates into the DNA and fluoresces under
UV-light exposure. The DNA-bands were visualized via an UV-transilluminator and
subsequently photographed for documentation.
50 x TAE buffer:
5 X DNA loading buffer:
Ethidium bromide solution:
1-kb Marker:
UV-transilluminator:
2 M Tris
0.05 M EDTA pH 8.0
57.1 ml acetic acid (100 %)
dH20 to 1 L
0.25 % bromphenol blue
0.25 % xylene cyanol
0.25 % orange G
30 % glycerol
10nM EDTA
in dHaO
0.5 |jg ethidium bromide / ml 1 x TAE
from Invitrogen
UV Vilber-Lourmat gel
documentation system
2.2.1. Purification
After a restriction digest or after a PCR, the DNA was first loaded onto an agarose gel for
identification and separation. In order to obtain specific DNA fragments, the DNA had to be
purified from the gel.
The gel was inspected under an UV-handlamp, to avoid major DNA-damages under the
intense UV-light of a transilluminator. The specific bands were cut out with a sterile scapel
and DNA fragments were subsequently purified by the Wizard® SV Gel and PCR Clean-Up
System (Promega) following the operators instructions.
After DNA purification, a second gel, so called check gel, was prepared in order to verify the
obtained DNA fragment and to estimate the DNA amount: 4 |jl of purified DNA were loaded
onto the gel together with 10 pi of the 1 kb DNA-ladder in an adjacent lane. The DNA amount
39
Materials & Methods
was approximated by comparison of the sample's band-intensity with the band intensities of
the 1636 bp band and the 506/517 bp band of the marker, representing a DNA amount of 50
ng and 30 ng.
2.3. Plasmids and cloning
A cloning procedure is used to create new plasmids and to propagate the plasmids by
bacterial cloning.
Therefore existing plasmids were cut by restriction enzymes to gain backbone-DNA and
insert-DNA, which together should form the new plasmid. The cut DNA-fragments were then
isolated by agarose gel electrophoresis and subsequently purified from the gel. A following
ligation of backbone and insert resulted in a new plasmid. The plasmid was transformed into
bacteria and first propagated only in small scale bacterial cultures just for identification of
positive bacterial clones that have acquired the correct plasmid. The identified bacteria were
then grown in large scale bacterial cultures for further propagation of the plasmids. These
plasmids were extracted by large scale plasmid preparation and finally tested by restriction
digest.
2.3.1. In silico cloning
Prior to realization in the laboratory the software Sei Ed Central (Clone Manager 6.0, Align
Plus 4.1 Primer Designer 4.2) was used to create plasmid maps and to plan further
experiments. The software provides all tools for planning a cloning experiment in silico:
restriction digests with various restriction enzymes, ligations, primer designing and DNA-
alignment.
2.3.2. Plasmids
2.3.2.1. pCTSefs
CT5efs ("efs" represents "envelope frameshift") is a proviral FIV-plasmid that contains an env
frameshifting 29 bp insertion at nucleotide 7146 of the full-length proviral construct CT5. CT5
expresses the FIV 34TF10 molecular clone in human cells from a fusion of the
-40-
Materials & Methods
cytomegalovirus immediate-early promoter to the viral R repeat (Poeschia et al., 1998;
Whitwam et al., 2001). In this project, pCTSefs was used in transient transfection to produce
viral vector CTSefs for the time-course infection study. Plasmid was kindly provided by Dr.
Eric Poeschia.
2.3.2.2. pCT5efsD66V
PCT5efsD66V is based on pCTSefs, but is integrase deficient due to an amino acid
substitution from aspartic acid to valine on position 66 of the protein integrase (Saenz et al.,
2004). In this project, pCT5efsD66V was used in transient transfection to produce viral vector
CT5efsD66V for the time-course infection study. Plasmid was kindly provided by Dr. Eric
Poeschia.
2.3.2.3. pCT25egfpiresneo (pCT25ein) and pCT25einF
pCT25ein is based on the proviral FIV-plasmid pCT25 that contains the 5' hybrid LTR of
pCTSefs, the first 311 bp of the gag open reading frame (ORF), the Rev responsive element,
an internal cytomegalovirus (CMV)-lacZ cassette (Loewen et al., 2001). The pCt25ein
additionally was cloned from the pCT25, exchanging the /acZ gene with an egfp gene and
adding and a neomycin resistance gene under the control of an internal ribosomal entry site
(Steinrigl, unpublished). The pCT25einF is based on pCT25ein, but additionally contains a
central polypurine tract (cPPT) and a central terminal site (CTS) (Steinrigl, unpublished). In
this project, pCT25ein was used as template for PCR during the development of the 1-LTR
circle assay. pCT25einF was used to produce CT25einF vector by transient transfection for
the creation of the HeLa^ integration standard.
2.3.2.4. pPetAenv
pPetAenv is a proviral FIV plasmid that is based on the Petaluma-14 strain. It has no
functional e/7i/gene. In this project it was used for a dilution series as standard for the total
viral DNA assay.
2.3.2.5. pHCMV-G
pHCMV-G is a plasmid that expresses the envelope glycoprotein G from vesicular stomatitis
virus (VSV-G) (Burns et al., 1993). In this project, pHCMV-G was used in transient
-41 -
Materials & Methods
transfection together with pCTSefs or pCT5efsD66V, respectively, in order to pseudotype the
vectors, which leads to a change in vectors cell-type tropism. Plasmid was kindly provided by
Dr. Eric Poeschla.
2.3.3. Ligation
Ligases are enzymes that form covalent phosphodiester-bonds between 3' hydroxyl ends
and 5' phosphate ends of nucelotides under ATP usage. Ligation was used to create new
plasmids by joining backbone- and insert- DNA. These DNA fragments originated from
restriction digests of existing plasmids (see 2.1), further gel electrophoresis (see 2.2) and
purification (see 2.2.1).
T4-Ligase and the supplied 5 x T4-ligase buffer were used for all ligation-reactions. A single
ligation reaction was performed in a total volume ranging from 20 pi to 50 pi containing 1 pi
T4 ligase and the 5 x T4 ligase buffer. A total DNA-amount of 100 ng was provided,
representing a molar backbone to insert ratio of 1:3. Additionally, a negative control-reaction
without any insert was performed to test for self ligation of the backbone. Ligation mixes were
incubated over night at 14°C.
Ligase: T4-ligase (Invitrogen)
lOU/pl
Ligase buffer: 5 x T4-ligase buffer (Invitrogen)
2.3.4. Bacterial transformation
Transformation is the uptake of foreign genetic material into bacterial cells. The result is a
genetic alteration leading to recombinant bacteria.
Two methods were used to accomplish bacterial uptake of plasmid DNA: electroporation and
heat shock. These methods were selected for every individual cloning.
42-
Materials & Methods
2.3.4.1. Electroporation
The method enables DNA-uptake due to a short-time permeability of the bacterial cell
membrane, caused by an electric pulse.
The success of elctroporation strongly depends on the purity of the plasmid DNA. Therefore,
butanol precipitation was performed to purify plasmid solutions from salts prior to
electroporation. The plasmid solution (100 ng DNA from ligation mix, see 2.3.3) was filled up
to 50 pi with dH20. 500 pi butanol were added in order to precipitate the DNA. The pelleted
DNA was air dried and resuspended in 30 pi dHaO.
A 25 pi aliquot of electrocompetent E.coli, strain DH10B, stored on -80°C, was thawed on
ice. 15 pi of the purified plasmid DNA were added to the bacterial suspension and
subsequently incubated on ice for 5 minutes. The Gene Power II was switched on and set to
1.8 kV for bacteria. A cooled, sterile and dry cuvette was filled with the transformation mix
and placed into the Gene Power II, followed by pulsing the bacteria. After electroporation, the
transformation mixture was immediately mixed with 1 ml SOC medium and incubated by
shaking for 1 hour at 37°C in an eppendorf tube. Finally, 200 pi of the cells were spread onto
agar plates.
Bacteria:
Electroporation:
SOC-medium:
(Invitrogen)
Electromax• DH10B• Cells (Invitrogen, Life Technologies)
Cuvette (Biozym)
Biorad Gene Pulser II system at 1.8 kV
2 % Bactotrypton
0.5 % yeast extract
lOmMNaCI
2.5 mM KCI
10 mM MgS04
20 mM D-Glucose
dissolved in dH20
2.3.4.2. Heat shock
A 50 pi aliquot of MAX Efficiency DH5a competent Cells, stored at -80°C, was thawed on ice
and mixed with 2 pi of plasmid suspension (100 ng DNA from ligation mix, see 2.3.3). Cells
were heat shocked for 45 seconds at 42°C and subsequently placed on ice for 2 minutes.
0.95 ml SOC medium were added and the bacterial cells were incubated by shaking for one
hour at 37°C. Finally 100 pi of the transformed bacteria were spread onto agar plates.
43
Materials & Methods
Bacteria: MAX Efficiency® DH5a• competent cells (Invitrogen)
SOC-medium: See electroporation
(Invitrogen)
2.3.5. Bacterial cultures
2.3.5.1. Bacterial culturing on solid medium
For selection of positive recombinant bacterial colonies directly after transformation, bacteria
were cultured on a solid agar-medium. Transformed plasmids contained an antibiotic
resistance gene, which only enabled recombinant bacteria to grow on the plates. Positive
recombinant colonies can be referred to one bacterial cell with a successful plasmid-uptake.
In general, 100-200 |jl of bacterial suspensions were plated onto one agar plate containing a
suitable selection antibiotic, usually Ampicillin. The plates were incubated over night at 37°C.
2.3.5.2. Bacterial culturing in liquid medium
Bacterial colonies, grown on solid medium after bacterial transformation, were picked with a
sterile pipette-tip and transferred into a glass-tube with 3 ml LB-medium (selective with
antibiotic). These small scale bacterial cultures were incubated by shaking over night at
37°C. Positive bacterial clones containing the correct plasmid were further identified by a
restriction analysis (see 2.1) of the plasmid DNA recovered from a small scale plasmid
preparation. Small scale bacterial cultures containing positive clones were finally transferred
into a large scale liquid culture for obtaining large amounts of highly pure plasmid DNA.
2.3.5.3. Bacterial stock
0.5 ml of a large scale bacterial culture (grown over night by shaking at 37°C) was
transferred in a cryo-tube and mixed with 0.5 ml glycerol in order to protect cells during
freezing and unfreezing. The bacterial stock was stored at -80°C. A large scale bacterial
culture could be restored by adding 10-30 pi of the bacterial stock to 200 ml LB medium and
subsequent incubation at 37°C over night by shaking.
44-
Materials & Methods
Luria Bertani- (LB) medium: 1 % NaCL
I % select peptone 140 (Invitrogen)
0.5 % yeast extract
pH7.5 10MNaOH
Agar plates: 15 g bacterial grade agar
II LB-medium
10 cm bacterial culture plates
Antibiotic selection medium: final concentration of 100 pg/ml antibiotic in LB-medium
2.3.6. Plasmid preparation
2.3.6.1. Small scale plasmid preparation (Mini Prep)
Small scale bacterial cultures of 3 ml, representing individual bacterial clones, were analyzed
for correct plasmid-uptake: plasmids were extracted by small scale plasmid preparation and
further screened by restriction analyses (see 2.1).
1 ml of the small scale bacterial suspension was pelleted by centrifugation for 5 minutes at
6000 rpm. The pellet was resuspended in 300 pi buffer PI. Resuspended cells were lysed in
alkaline conditions by adding 300 pi of buffer P2 and subsequent incubation for 5 minutes at
room temperature. Lysis was stopped by adding 300 pi of neutralization buffer P3, leading to
precipitation of proteins, RNA and genomic DNA. A following centrifugation at 13000 rpm for
30 minutes excluded the precipitated bacterial elements. The plasmid DNA containing
supernatant was transferred into a new tube for DNA precipitation by adding 650 pi
isopropanol. The precipitated DNA was pelleted by another centrifugation for 30 minutes at
4°C. An additional DNA washing step with 500 pi of 70% ethanol was performed. The
resulting DNA pellet was air-dried or in a vacuum centrifuge. Finally, the DNA pellet was
resuspended in 50 pi TE buffer. This DNA solution was then further analyzed by restriction
analysis.
Resuspension buffer P1: 50 mM Tris pH 8.0
lOmMEDTA
100 pg RNAse A/ml
pH 8.0, stored at 4°C
Lysis buffer P2: 200 mM NaOH
45
Materials & Methods
1 % SDS
Neutralization buffer P3: 3 M KCI
pH5.5
TE buffer: 10mMTris-CI
1 mM EDTA
pH8.0
2.3.6.2. Large scale plasmid preparation (Maxi Prep)
Identified positive bacterial clones, containing correct plasmids, were grown in large scale
bacterial cultures for subsequent propagation of the plasmid. The plasmids were then
extracted by large scale plasmid preparation and were further used for transfection of
mammalian cells (see 2.4.6), or further cloning experiments.
The large scale plasmid preparation was performed with the QIAGEN Plasmid Maxi Kit
following the manufacturer's instructions.
A large scale bacterial culture of 200 ml was pelletd by centrifugation. The principle of
alkaline cell-lysis by usage of buffer P1, P2 and P3 was similar to the Mini Prep procedure.
After alkaline lysis plasmid DNA was separated from RNA, proteins and genomic DNA by
centrifugation. The supernatant containing plasmid DNA was directly transferred onto a
Qiagen tip, prior equilibrated with 10 ml equibrilation buffer. The resin-bound plasmid DNA
was purified by two washing steps with each 30 ml washing buffer. The purified plasmid DNA
could be eluted from the resin with 15 ml elution buffer. Finally the purified plasmid DNA was
precipitated with isopropanol and pelleted by centrifugation for 30 minutes at 20000 rpm. The
DNA pellet was further washed with 5 ml 70 % ethanol. The resulting pure plasmid DNA
pellet was dried in a vacuum centrifuge and finally resuspended in 500 pi TE buffer.
Resuspension was carried out at 4°C, under shaking for at least one hour.
Large scale plasmid preparation kit: QIAGEN Plasmid Midi and Maxi Kit
Buffer PI,P2,P3,TE: See Mini Prep
Equilibration buffer QBT: 750 mM NaCI
50 mM MOPS pH 7.0
15 % isopropanol
0.15% Triton X-100
Wash buffer QC: 1 M NaCI
50 mM MOPS pH 7.0
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Materials & Methods
Elution buffer QF:
Centrifuge:
15 % isopropanol
1.25MNaCI
50 mM Tris-Cl pH 8.5
15 % isopropanol
Beckmann Avanti J-25
2.3.7. Sequencing
Sequencing was performed in order to control the nucleotide sequence of a certain DNA-
fragment. In this project, plasmids after cloning and amplicons during the 1-LTR circle assay
development were sequenced for identification.
0.8 fjg target DNA and 20 pmol of appropriate primer were mixed with H2O in a total volume
of 10|jl. This mixture was sent to Microsynth (Balgach, Switzerland), where sequencing was
performed, after the method developed by Sänger. The results of sequencing were analyzed
in chromatograms with the help of the software Chromas 1.45. Finally, the DNA sequences
were analyzed for alignment against known plasmid maps with the software Sei Ed Central
(Clone Manager 6.0, Align Plus 4.1 Primer Designer 4.2).
2.4. Mammalian cell culture
2.4.1. Cell lines
HeLa cells (ATCC® Number: CCL-2•)
Organism: Homo sapiens (human)
Growth properties: adherent
Source:
Organ: cervix
Cell type: epithelial
Disease: adenocarcinoma
Used for transduction
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Materials & Methods
293T cells (ATCC® Number: CRL-11268•)
Organism: Homo sapiens (human)
Growth properties: adherent
Source:
Organ: kidney
293T/17 is derivative of the 293T cell line. 293T is a highly transfectable derivative of the
293 cell line into which the temperature sensitive gene for SV40 T-antigen was inserted.
293T cells were cloned and the clones tested with the pBND and pZAP vectors to obtain a
line capable of producing high titers of infectious retrovirus, 293T/17
Used for transient transfection
NIH/3T3 cells (ATCC® Number: CRL-1658•)
Organism: Mus musculus (mouse)
Growth properties: adherent
Source:
Organ: embryo
Cell type: fibroblast; fibroblast
Used for PALSG-standard
2.4.2. Cell culture maintenance
Cell culture manipulations were done in a safety-level-3 laboratory under aseptic conditions
within a laminar air flow bench. Before and after usage the laminar air flow was disinfected
and irradiated with UV-light.
Dulbecco's modified Eagles's minimal medium (DMEM; Invitrogen) with the supplement of 10
% fetal calf serum (FCS) was used as cell culture-medium. FCS was heated to 56°C for half
an hour in a water bath before first use. Before adding to the medium, FCS was additionally
filtered through a 0.45 pm filter.
All cell lines were maintained in monolayer cultures within T75 and T175 flasks at 37°C in 5%
CO2 incubators with >80% humidity.
Cell culture medium: Dulbecco's modified Eagles's minimal medium (DMEM)
(Invitrogen) + 10 % Fetal Calf Serum (FCS) (Invitrogen)
Phosphate buffered saline (PBS): 1.04 mM KH2PO4
155.17 mM NaCI
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2.96 mM Na2HP04 x 7 H2O
pH 7.4; autoclaved or sterile filtered
Trypsin: Trypsin-EDTA solution (Invitrogen)
2.4.3. Passaging / splitting of cells
Splitting and passaging of cells was required at as soon as cells were grown to confluence
within the culture flask. The medium was removed and the monolayer culture was washed
once with PBS. Cells were further detached from the flasks surface by the endopeptidase
trypsine, which unspecifically cleaves extracellular proteins. Therefore 2 ml (for a T75 flask)
or 3 ml (for a T175 flask) trypsine were added to the cells. Until all cells were detached from
the flask-surface, the flask was incubated on the 37°C incubator. At least the triple volume of
DMEM was added to neutralize the trypsine activity. The resulting cell-suspension was
pipetted up and down for a few times to singularize cells. An aliquot of the cell-suspension
was put back into the same or into a new cell culture flask, while the rest of the cell-
suspension was discarded. The ideal cell culture volume is re-established by adding fresh
medium.
In general, cells were splitted 1:8 and culture flasks were exchanged after 5 passages.
2.4.4. Freezing / thawing of cells
Cell stocks were created as backups in case of contamination and for maintenance of cell-
lines at lower passage numbers. Therefore cells were grown to confluence in one T175-flask.
The cells were washed and trypzinized as described before and transferred into a 15 ml
falcon tube. Cells were pelleted by centrifugation for 5 minutes at 1000 rpm. After discarding
the supernatant, the cells were resuspended in PBS and centrifuged again. The resulting
washed pellet was resuspended in 3 ml freezing medium. The cell suspension was aliquoted
in 3 freezing vials. First, freezing vials were stored at ice for one hour, then they were stored
over night in a polystyrene box in the -80°C freezer. Final storage of the freezing vials was in
cardboard boxes at -80°C.
Freezing medium: 90 % FCS (Invitrogen) and 10 % DMEM (Invitrogen)
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Materials & Methods
For reculturing the backup-cells, the frozen vials were quickly thawed in the 37°C incubator.
The cells were transferred into a 15 ml falcon tube with 10 ml DMEM, in order to dilute the
freezing medium. Cells were subsequently pelleted by centrifugation for 5 minutes at 1000
rpm. After discarding the supernatant, the cells were washed by resuspension in 10 ml PBS
and followed centrifugation at 1000 rpm for 5 minutes. The washed cells were resuspended
in DMEM and transferred in a new cell culture dish, where they were cultured at general
growth conditions described before (see 2.4.2).
2.4.5. Counting of cells
For transfections and transductions, certain numbers of cells were needed. Therefore cell
numbers had to be estimated with the help of a 16 square disposable haemocytometer
(FastReadTM 102, Megumed Diagnostics). Cell were washed with PBS, trypsinized and
resuspended (as described 2.4.3). Before splitting and passaging of cells, an aliquot of the
cell suspension was transferred into the haemocytometer for further counting of cells by eye
under a microscope. Cell number was estimated in 4 squares and averaged. The average
cell number multiplied by 10"* is equal to the cell number in 1 ml of cell suspension.
2.4.6. Transient transfection
Transient transfection was used for production of infectious viral vectors. Transient
transfection is the uptake of foreign plasmid DNA into mammalian cells, similar to
transformation in bacterial cells. The transfection is transient, because the plasmid DMA does
not stably integrate into the genome and subsequently disappears after a few cell divisions.
Transient transfection accomplishes a short term expression of all virus components,
resulting in the release of infectious viral particles into the medium.
To produce infectious viral particles, all components of a virus must be produced by the
transfected plasmids. Therefore structural vectors containing most of the structural genes are
mixed with plasmids containing the env and/or transfer vectors containing egfp or antibiotic
resistance.
The used FIV-plasmids (described in 3.2) lacked a functional env. Therefore, they were co-
transfected with pHCMV-G, which expresses the VSV-G envelope protein. As an additional
advantage, the resulting VSV-G pseudotyped viruses can infect a broader range of
mammalian cells than the natural FIV Env-proteins.
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Materials & Methods
Two transient-transfection methods were used in this project.
2.4.6.1. Metafectene Pro
Transient transfection with Metafectene Pro was performed in 10 cm dishes with 8 ml
DMEM. In general, 3.5*10^ 293T cells were seeded per dish 24h prior transfection.
Two eppendorf tubes per transfection were prepared - one for transfection-solution A and
one for transfection-solution B. First, 700 pi of solution A were prepared by mixing 1.5 pg of
pHCMV-G and 13 pg of the viral plasmid with PBS. Then also 700 pi of solution B were
prepared by mixing 658 pi PBS with 42 pi Metafectene pro. Solution A was pipetted to
solution B. This transfection-mixture was incubated at room temperature for 15-20 minutes
before it was finally added to the cells in the 10 cm dish.
Metafectene: Metafectene Pro (Biontex)
2.4.6.2. Calcium phosphate
Transient transfection with calcium phosphate was performed in 10 cm dishes with 8 ml
DMEM. In general, 3.5*10^ 293T cells were seeded per dish 24h prior transfection.
13 pg of the viral plasmid were mixed with 1.5 pg of pHCMV-G in a total volume of 240 pi
dH20. Then 240 pi of transfection buffer A were added to the DNA suspension. After 10
minutes of incubation at room temperature 480 pi of transfection buffer B were added and
the mixture was again incubated for 15 minutes at room temperature. Finally, the transfection
mixture was distributed to the seeded cells in the 10 cm dish.
Calcium phosphate kit: Cellphect® (Pharmacia)
Transfection buffer A: 0.5 M CaCb
0.1 M HEPES
pH7.0
Transfection buffer B: 0.28 M NaCI
0.75 mM NaH2P04
0.75 mM Na2HP04
0.05 M HEPES
pH7.0
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Materials & Methods
In general, 24h after both transfection methods the medium, containing the transfection-
mixture, was replaced by fresh medium. 32 h, 48 h and 72 h after transient transfection, the 8
ml supernatant, containing infectious virus, were collected in a 50 ml falcon tube without
disturbing the attached producer cells. 8 ml fresh DMEM were subsequently added at all time
points. After 72 h the cells were discarded. The infectious supernatant was centrifuged for 5
minutes at 1000 rpm to remove producer cells. The supernatant was decanted into a 50 ml
syringe and filtered through a 0.45 pm filter, in order to purify the virus supernatant. Finally
the purified viral supernatant was aliquoted and either used directly for further infection or
was stored at -80°C.
2.4.7. Infection / transduction of cells
Infection was usually performed in 6-well plates with 2 ml DMEM per well. In general, 2*10^
HeLa cells/well were seeded 24h prior to infection.
The infectious supernatant was used in different concentrations, resulting in a virus titration.
Therefore, the viral supernatant was diluted in a serial dilution 1:1, 1:10 and 1:100.
For infection the former medium was withdrawn and the cells were infected with 1 ml of the
diluted infectious agent. Additionally 10 pi polybrene (0.8 pg/ml) were added to each well for
enhancement of infection. Six hours after the infection, 2 ml of DMEM were added to each
well. Cells were further cultured for 72 hours at usual culture conditions (see 2.4.2).
To analyze the infected cells by real-time PCR or FACS analysis, the infectious medium was
discarded and the infected cells were washed with PBS. Then, cells were detached by 300 pi
trypsine in the 37°C incubator. When all cells were detached, the trypsine was neutralized
with 700 pi DMEM. The cells were transferred into an eppendorf-tube and pelleted by
centrifugation at full speed for 15 seconds. The supernatant was discarded and 1 ml of PBS
was added to wash the cells. The pellet was detached from the tube wall by vortexing and
again centrifuged at full speed for 15 seconds. Finally the supernatant was withdrawn.
For DNA-extraction and further real-time PCR assays, the cell pellet was stored at -20°C in a
small residual volume of PBS.
For FACS-analysis 1 ml of PBS was added to the cell pellet, which was again detached by
vortexing. The tube was finally placed on ice prior to FACS analysis.
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Materials & Methods
2.4.8. FACS analysis
FACS analysis is a flow-cytometric technique to count, examine and sort cells. Cells are
singularized in a fluid stream and pass a light source, for example laser. Cells are examined,
in real time, according to light scattering and fluorescent properties. Light scattering allows
conclusions regarding morphology parameters like cell-size and cell-granularity, while
fluorescence properties can give conclusions about specific gene expression or specific cell
surface proteins.
In this project we used the FACScalibur• system (Becton Dickinson) to evaluate
transduction-efficiency of viral vectors containing enhanced green fluorescent protein
(eGFP). eGFP, originally found in a jelly fish, fluoresces green when exposed to blue light
(395 nm and 475 nm). If viral vectors carry an egfp gene, infected cells will express eGFP.
Subsequently, infected cells can be identified from non-infected cells by FACS upon their
green fluorescence.
Cells from transduction experiments were collected and resuspended in 1 ml PBS. The cell
suspension was stored on ice and filtered trough a nylon mesh (48 pm) into a FACS tube
before analysis. In general 10.00 cells were analyzed. The eGFP emission, activated by a
laser at 488 nm, was measured at 530 nm +- 30 nm.
The most important results of a FACS analysis were the absolute and relative number of
transduced cells and the MFI (mean fluorescence intensity). The MFI represents the average
eGFP- fluorescence in all transduced cells. Finally the transduction efficiency was calculated:
Percentage of eGFP positive cells x number of cells at time of transduction x dilution of the
supernatant /100 = infectious particles / ml supernatant.
FACS-machine: FACScalibur• system (Becton Dickinson)
2.4.9. DNA-extraction
DNA extraction from mammalian cells was performed in a separate room to ensure
protection against contamination. DNA extraction was performed with the DNeasy Blood and
Tissue Kit (QIAGEN) following the manufacturer's instructions. The cells were lysed in lysis
buffer plus proteinase K. The suspension was then transferred into the DNeasy mini spin
column, where DNA could specifically bind to a silica membrane during centrifugation.
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Materials & Methods
Contaminants were removed in two following washing steps with washing buffers. Finally, the
purified DNA was dissolved and eluted in a provided buffer. The eluted DNA was further
stored at -4°C.
DNA-extraction kit: DNeasy Blood and Tissue Kit (QIAGEN)
2.4.10. Measurement of DNA concentration by optical density (OD)
Double-stranded DNA has its UV absorbance maximum at 260 nm. Optical density is the
absorbance of a DNA suspension under 260 nm UV-light irradiation. There is a correlation
between the DNA concentration and the optical density of a DNA suspension - reflected in
the following formula:
C of DNA (|jg/ml) = OD260 X dilution x 50 pg/ml
In general, DNA suspensions were diluted 1:40 with dH20. 80 pi of this dilution were
transferred into a clean quarz cuvette and measured with the UV spectrophometer. The
background absorbance of pure dH20 was additionally measured and subsequently
subtracted.
The absorbance of a sample should be between 0.1 and 1, in order to get reliable results.
Additionally, the absorbance ratio OD260/OD280 (potentially contaminating proteins have their
UV-absorbance maximum at 280nm) was evaluated, in order to get information about
sample purity. The OD260/OD280 ratio should be between 1.7 and 2. Lower ratio would be
indicative of an impure DNA solution containing contaminating proteins after DNA extraction.
UV spectrophometer: Gene Quant II ( Pharmacia Biotech)
2.5. Polymerase chain reaction (PCR)
PCR is a molecular biological method for specific DNA amplification by the heat-stable Taq-
DNA-polymerase. Specificity of amplification is accomplished by 2 primers, which selectively
bind to distinct DNA sites, surrounding the desired target DNA fragment. To obtain an
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Materials & Methods
exponential DNA increase, 3 main important steps are repeated several times: 1-
Denaturation: The double stranded DNA is separated; 2- Annealing: the specific primers
anneal on to the target DNA sites; 3- Elongation: the Taq polymerase binds and starts
amplification of the template;
These 3 steps represent one cycle. One cycle is controlled by 3 different optimum
temperatures and lead -if the conditions are optimal- to a doubling of the template DNA. The
cycle is repeated several times resulting in an exponential increase of template DNA. All
temperature changes are planned and arranged by a thermocycler.
PCR reactions were performed in a total volume of 25 pi. In general, a standardized PCR
mixture was used for all assays. It consisted of 5 pi DNA template, 200 pM dNTPs, 2.5 pi 10
X PCR buffer, 3 mM MgCb, 300 nM of each primer, 200 nM probe and 1.25 U of Taq-
polymerase. The mastermix was prepared in a separate room within a laminar air flow
bench.
No standardized PCR-temperature profile was used. The temperature profile for the PCR
consisted of an initial denaturation step for 10 minutes at 95° C, followed by varying cycle
numbers of denaturation (15 sec/ 95°C), different annealing temperatures (depending on the
primers) and different extension times at 72°C (depending on the amplicon length: in general
1 minute for 1000 bp).
The amplified DNA can be analyzed by agarose gel electrophoresis. For further analyses the
DNA can be purified from the agarose gel (see 3.2, 3.2.1)
In this project standard PCR was used as a first amplification step for detection and
quantification of integrated proviral DNA with the nested-Alu-real-time PCR assay. Further it
was used during the establishment phase of the 1-LTR circle assay. Finally, the detection of
plasmids was performed by PCR.
2.6. Real-time PCR
The real time PCR method is based on the PCR method. Real time PCR allows tracing of
amplification in real-time without additional handling of the tube (further description see 1.3).
All real-time PCR assays used in this project used TaqMan probes. Only the 1-LTR circle
assay was designed to use SYBR green for amplicon-detection. The real-time PCR assays
were performed with the 7500 Real-Time PCR System of Applied Biosystems. Real-time
PCR reactions were performed in a total volume of 25 pi. In general, a standardized PCR
mixture was used for all assays. It consisted of 5 pi DNA template, 200 pM dNTPs, 2.5 pi 10
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Materials & Methods
X PCR buffer, 3 mM MgCb, 300 nM of each primer, 200 nM probe, 50 nM ROX (as passive
fluorescence reference) and 1.25 U of Taq-polymerase. The mastermix was prepared in a
separate room within a laminar air flow bench. Samples and standards were measured in
duplicate.
A standardized PCR-temperature profile for all assays was used. The temperature profile for
the real-time PCR consisted of an initial denaturation step for 2 minutes at 95° C, followed by
45 cycles of denaturation (15 sec/ 95°C) and annealing and extension (1 min/ 60°C).
2.6.1. Primer design
Primers were designed with the help of the software Sei Ed Central (Clone Manager 6.0,
Align Plus 4.1 Primer Designer 4.2) and the internet application primer 3.0 (Rozen and
Skaletsky, 2000). Probes were designed with the software primer express (version 3.0) by
Applied Biosystems. Primers and probe were purchased at Mycrosynth (Balgach,
Switzerland).
2.6.2. Cell number assay
Cell number was determined by real-time PCR quantification of a reference gene. Either
18S-rDNA or human apoB were used as reference. For absolute quantification the HeLa
integration standard was used (see 3.1.2)
rRNA343F: 5'-CCA TCG AAC GTC TGC CCT A-3'
rRNA409R: 5'-TCA CCC GTG GTC ACC ATC-3'
rRNA370P: 5'-FAM-CGA TGG TGG TCG CCG TGC CTA-TAMRA-3'
hApoB F: 5'-TTC TTA CCA CAC ATC TCT TGA TTC TCT T-3'
hApoB R: 5'-GGA CTT CAC TGG ACA AGG TCA TAC T-3'
hApoB P: 5'-CAC TCG TCC AGG TGC GAA GCA GAC T-3'
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Materials & Methods
2.6.3. Total viral DNA assay
Total viral DNA was quantified with specific primers and probes that amplify a fragment that
is present in all viral DNA forms: between a left part of the LTR and a part downstream of the
gag. A 1:10 dilution series of the FIV plasmid pPetAenv was used as standard for this assay.
viral DNA_F: 5'-CCT AAC CGC AAA ACC ACA TC-3'
viral DNA_R: 5'-GGA GTT CTG CTT AAC AGC TTT CT-3'
viral DMA P: 5'-FAM-AAC CAG TGC TTT GTG AAA CTT CGA GGA-TAMRA-3'
2.6.4. 2-LTR circle assay
2-LTR circles were quantified with primers and probe, which specifically bind at the U3-U5
junction, unique to 2-LTR circles. A 1:10 dilution series of plasmid p2LTR sense was used as
standard for this assay.
q2LTR_F: 5'-AAT CCG GGC CGA GAA CTT C-3'
q2LTR_R: 5'-TAA ACA GTC CCT AGT CCA TAA GCA TTC-3'
q2LRT_P: 5'-FAM-AGT ATT GGA ACC CTG AAG AA-NFQ-3'
2.6.5. Alu-LTR based real-time nested PCR assay for integrated DNA
This Alu-LTR based real-time PCR assay is a nested PCR with 2 round s(Brussel and
Sonigo, 2003). In a first round PCR, a primer specific for Alu-sites and a primer specific for
LTR, amplifies the DNA sequence between the LTR and its nearest Alu-site, which are
regular and abundant in primates genomes. The forward primer, which binds in the LTR, is
additionally extended by a A-phage heel sequence. To exclude genomic amplicons between
the Alu primers that do not represent integrated provirus, a second nested real-time PCR is
performed that exclusively amplifies a fragment within the LTR by a A-heel specific forward
primer and a LTR specific reverse primer. In the first round, single stranded amplicons of all
different viral DNA forms can be produced by the forward primer, since all contain the binding
site within the LTR. These amplicons will be again amplified in the second PCR step and
thus influence the result of the integrated-DNA real-time PCR assay. To exclude this false-
positive detection, the assay is performed twice: once without Alu primer to detect and
quantify the false-positive background amplification; and once with Alu primer, to detect and
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Materials & Methods
quantify integrated DNA as well as all background amplification. Finally, the -Alu setup is
subtracted from the +Alu setup to obtain the abundance of integrated DNA.
A 1:4 dilution series of DNA-extracts from HeLa cells infected with FIV-vector CT25einF was
produced called HeLa integration standard. The HeLa integration standard was improved by
diluting the DNA extracts with uninfected HeLa DNA, in order to maintain a stable amount of
DNA in all dilution steps. This termed HeLa^ integration standard reflects the natural situation
in an unknown sample and therefore improves the reliability of the standard.
The mastermix for the 1^' "conventional" PCR contained 200 |JM dNTPs, 2.5 |jl10 x PCR
buffer, 3 mM MgCb, 200 nM of primer IN_1rd_F, 100 nM of Alu reverse primer or of Alu
forward primer and 1.25 U of Taq-polymerase. The PCR was performed in a total volume 25
|jl with 20 pi of mastermix and 5 pi of template DNA. The temperature profile for the PCR
consisted of an initial denaturation step for 10 minutes at 94° C, 20 cycles of 10 seconds at
94° C for denaturation, 10 seconds at 60° C for annealing, and 2:50 minutes at 72° C for
extension. Finally, the PCR-reaction was cooled down to 4° C and kept at this temperature
until further analysis. The 2""^ nested real time PCR is performed under the general
conditions described above.
Alu fon/vard: 5'-TCC CAG CTA CTG GGG SGG CTG AGG-3'
Alu reverse: 5'-GCCTCCCAAAGTGCTGGGATTACAG-3'
IN_1 rd_F: 5'-ATG CCA CGT AAG CGA AAC TAC GGG AGA CAG CAC AGT AGA-3'
5'-ATG CCA CGT AAG CGA AAC T-3'
5'-GTA AAC AGT CCC TAG TCC ATA AGC ATT-3'
5'-FAM-AGT ATT GGA ACC CTG AAG AA-NFQ-3'
LambdaT
IN 2rd R
q2LRT_P
2.6.6. 1-LTR circle assay
1-LTR circle assay was believed to quantify 1-LTR circles with primers that anneal in the gag
and the env to amplify over the single LTR. Therefore 2 primer pairs were designed and
tested (F1/R1 and F2/R2). Plasmid detection should have been accomplished by the
intercalator SYBR green. Standards were not designed, since the assay failed to uniquely
detect 1-LTR circles.
1-LTR _F1: 5'-CGACTTCTACAACGGGAGACAGC-3'
1-LTR_R1: 5'-CACTCTCAATCAAGTCCCTGTTCG-3'
1-LTR_F2: 5'-CCTCCTGAGTTGTGGACAAG-3'
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Materials & Methods
1-LTR R2: 5'-ACCAGGTTCTCGTCCTGTAG-3'
2.6.7. Assay analysis
The assays were tested for usability with the standards that were then also used for absolute
quantification: the standard curves were analyzed in context of slope, correlation and
efficiency. The slope and correlation could be obtained by the standard curve, while the
efficiency was calculated with the following formula:
E = 10-i's.i
E = PCR efficiency
S = slope
In general, a perfect real-time PCR reaction is characterized by 100 % efficiency and a slope
of -3.3, when the template is duplicated in every cycle. However, optimal efficiency should
range between 90 % and 100 %. The correlation should be higher than 0.99. Furthermore
the standard curve also showed the linear range of quantification, in which linearity for
quantification is given. Template concentrations in this range of log decades can be
quantified by the assay (Mocellin et al, 2003; Nolan, 2004).
2.6.8. Analysis of time course infection study
The DNA extracts from the time-course infection study (see 2.4.9) were analyzed twice and
in duplicate by every real-time PCR assay. Results from the total viral DNA assay, integrated
DNA assay and of the 2-LTR circle assay were normalized to the cell number, obtained from
the cell-number assay. The mean of the resulting values and the connected standard
deviation were calculated with Microsoft Excel. Statistical analysis was performed with
Windows Excel: 2-sided paired T-test. The statistical cut-off value was defined by p^O.05.
2.6.9. Product Enhanced Reverse Transcriptase Assay (PERT-Assay)
PERT assay (Lovatt et al., 1999) was performed to evaluate the amount of viral particles
produced by transient transfection. PERT assay measures the activity of reverse
transcriptase, present in viral particles.
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Materials & Methods
The principle of the method is that reverse transcriptase, extracted from the infectious
supernatant, converts an exogenous RNA from brome mosaic virus (BMV) into cDNA. The
resulting cDNA is further quantified by specific BMV primers and probe in a real-time PCR
assay. The amount of converted cDNA allows conclusions about the activity of reverse
transcriptase in the supernatant, and therefore indirectly measures the amount of retroviral
particles.
Aliquots of the purified and filtered infectious supernatant (see 2.4.6) were mixed with the
same volume of disruption buffer and incubated at room temperature for 2 minutes. Thus, the
viral particles were lysed and the enzyme reverse transcriptase was released into the
supernatant. The lysates were further stored on ice and diluted in disruption buffer 1:10,
1:100, 1:1000 and sometimes 1:10000. 5 \i\ of these dilutions were used as templates in a
real time PCR assay. Samples and standards were measured in duplicate. Dilutions of MLV-
Reverse Transcriptase were used as standards.
The PERT assay was performed with the 7500 Real-Time PCR System (Applied
Biosystems). Real-time PCR reactions were performed in a total volume of 25 |jl. A
standardized PCR mixture was used for all assays. It consisted of 5 |jl DNA template, 200
pM dNTPs, 2.5 pi 10 x PCR buffer, 3 mM MgCb, 300 nM of each primer, 200 nM probe, 50
nM ROX (as passive fluorescence reference) and 1.25 U of Taq-polymerase.
As a difference to conventional PCR profiles a step, in which reverse transcription of the
BMV-RNA occurs, is added prior to the normal real-time PCR reaction. The temperature
profile for the PERT assay consisted of an initial reverse transcription step for 45 minutes at
37°C, followed by a denaturation step for 2 minutes at 95° C, followed by 45 cycles of
denaturation (15 sec/ 95°C) and annealing and extension (1 min/ 60°C).
BMVF: 5'-FAM-TGA AGG AAT TTG TGC GTT ATT GTA A-TAMRA-3'
BMVR: 5'-FAM-GAT GCT CTT ATT TGC GAT CTC ACT T-TAMRA-3'
BMV probe: 5'-FAM-TCT TCG TCA CCT ATG GGA CAT TTC CGG-TAMRA-3'
Disruption buffer: 40 mM Tris-HCL
50 mM KCI
20 mM DTT
0.2 % NP-40
0.2% Triton X-100
pH8.1
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2.7. Southern blot
Southern blot is a method to transfer DNA from an agarose gel to a nitrocellulose membrane.
There, specific transferred DNA fragments can be detected using labelled nucleic acid
probes.
Southern blot was performed to evaluate the results of the real-time PCR assays. A Southern
blot setup was chosen that detects all different viral DNA forms in one approach and the total
viral DNA in a second approach, respectively, by using only one probe for all setups.
Therefore, three restriction enzymes were selected to cut the viral DNA forms into specific
DNA fragments that can all be detected by one probe. Three probes were designed and
tested (Table 2.1). For detection of the total viral DNA, the DNA extracts were digested with
the restriction enzyme Hindi 11 for all three different probes. For detection of the different viral
DNA forms, the DNA extracts were digested with the restriction enzymes EcoRI and Spel in
case of probe 1 and probe 2, and with Ncol and Spel in case of probe 3 (for cutting pattern
see Table 2.2).
Probe 1 Probe 2 Probes
Origin pPetAenv pCTSefs
Restriction enzyme Hind III Spel/Ndel —
production Random primed DNA-labelling PCR-labelling
Site In provlrus CTSefs 5'nR-gag
(148-1242 nt)
env
(8316-8934 nt)
gag-pol
(1608-1952 nt)
Length [bp] 1094 619 345
Table 2.1.: Overview of all three designed and produced probes
In all experiments a fluorescence detection system was used. Therefore, the probe was
labelled with the protein digoxigenin (DIG). The labelled probe hybridizes to a specific DNA
target on the membrane. A a-DIG-antibody could be used to selectively bind to the probe.
The anti-DIG-AP antibody is linked to alkaline phosphatase (AP), which enables the use of
chemoluminescent CSPD as substrate for detection. CSPD was added to the membrane,
where the AP, linked to the antibody, could dephosphorylate it. As a consequence, light at a
maximum wavelength of 477 nm was emitted and could be detected by X-Ray film.
Probe 1 Probe 2 Probes
Total viral DNA Hindlll Hindlll Hindlll
Total viral DNA [bp] 1094 3591 2739
Different DNA forms EcoRI/Spel EcoRI/Spel Ncol/Spel
61
Materials & Methods
Unintegrated DNA [bp] 1872 1187 2499
Integrated DNA [bp] >1872 >1187 >2499
1-LTR circle [bp] 2704 2705 3331
2-LTR circle [bp] 3058 3059 3686
Table 2.2.: Overview of Southern blot setup for the three different probes: restriction enzymes and fragment
sizes of different detected DNA-forms of the different approaches
2.7.1. Sample preparation by ethanol precipitation
In order to concentrate the DNA extracts from the different time-points of the infection study
(see 2.4.9), ethanol precipitation was performed. 1/10 volume of 3 M sodium acetate was
added to the DNA solution. After vortexing, another 2 - 2.5 volumes of ice cold 100 % ethanol
were added and the mixture was placed at -20°C for at least 30 minutes. Then the mixture
was centrifuged at maximum speed and the supernatant was decanted. One ml of 70 %
ethanol at room temperature was added followed by another centrifugation at maximum
speed for 5 minutes. Finally the supernatant was withdrawn again and the DNA pellet was
air-dried and afterwards dissolved in H2O. The DNA concentrations of the resulting extracts
were measured by OD (see 2.4.10).
2.7.2. Probe labelling
The probes were produced either with the DIG High Prime DNA Labelling and Detection
Starter Kit II (Roche), or the PCR DIG Probe Synthesis Kit (Roche).
Probe 1 and probe 2 were developed with the DIG High Prime DNA Labelling and Detection
Starter Kit II. For that, 14 pg of the plasmid pPetAenv were digested with Hindlll to produce
probe 1. Probe 2 was produced from 14 pg of pPetAenv by digestion with Spel/Ndel (location
and length of probe see Table 2.1). The restriction digestion mix containing the probe-
fragment was loaded onto an agarose gel and the probe-band was identified by size, purified
from the gel and DNA concentration was determined with a check gel (see 2.2.1). Then, the
purified DNA-fragment was DIG labelled as described in the manual of the DIG High Prime
DNA Labelling and Detection Starter Kit II. 16 pl of the DNA-fragment (200 ng) was
denatured in a boiling water bath for 10 minutes. After chilling on ice, 4 pl of the kit's probe-
production-mixture were added and the whole reaction mixture was incubated for 20 hours at
37°C. The reaction was stopped by heating for 10 minutes at 65°C. After this incubation step,
the primary 200 ng of unlabeled probe should be transformed into 1700 ng of DIG labelled
62
Materials & Methods
probe. This labelling efficiency was checked by a blotting test. For that, serial dilutions of the
probe and a comparable reference probe were applied onto a positively charged nylon
membrane and immunologically detected (see below). The resulting intensities of the DNA
spots of probe and reference were compared, thus estimating the labelling efficiency of the
probe.
Probe 3 was developed with the PCR DIG Probe Synthesis Kit. 1 ng of plasmid pCTSefs was
used as template for PCR. During this PCR reaction, the probe is labelled by random
incorporation of digoxigenin-11-dUTP. The PCR was performed with 300 ng of forward
primer, 300 ng of reverse primer and reaction ingredients provided by the kit including dig-
11-dUTPs. The temperature profile of the PCR started with an initial denaturation step of 2
minutes, followed by 30 cycles of denaturation (95°C/ 30sec), annealing (60°C/ 30sec) and
elongation (72°C/ 40sec) and finished with an elongation step of 4 minutes on 72°C. A
control PCR was performed without the digoxigenin-11-dUTP. DIG-labelled ad -unlabelled
reactions were analyzed by agarose gel electrophoresis in a 1.5 % agarose gel. The
digoxigenin labelled probe moves slower than its unlabelled counterpart and therefore an
upshift of the labelled probe was visible. If the probe labelling was successful, the probe was
purified from the gel and the DNA-concentration was evaluated by a check gel (see 2.2.1).
The hybridization temperature for all probes was obtained with the help of the melting
temperature (Tm) and was calculated with the following formula:
Tm = 49.82 + 0.41 (% G + C) - (600/1) [I = length of hybrid in base pairs]
Hybridization temperature = Tm - 20 to 25°C
2.7.3. Sample digestion and blotting
5 pg DNA extract from each sample from the infection study was used for digestion. The
digestion was performed in a total volume of 20 pi and was incubated at 37°C over night (for
an overview of the restriction enzymes see Table 3.2). These digested samples and one
control-sample with 1 pg of plasmid pCTSefs were loaded onto a 0.8 % agarose gel. The
agarose gel electrophoresis was performed in 1 x TAE buffer at 70 V. The gel was stained
with ethidium bromide and photographed under UV-light for documentation (see 2.2). Then,
the gel was washed in H2O for 15 minutes for elimination of the ethidium bromide.
Furthermore, the gel was washed twice for 15 minutes with the denaturation buffer in order to
separate the two DNA strands. Finally, this reaction was stopped by washing the gel twice for
15 minutes with the neutralization buffer. After these washing steps, the gel was ready for
blotting. For that, a Whatman paper, soaked with 20 x SSC, was put on an elevated layer
-63-
Materials & Methods
within an electrophoresis chamber. The ends of the Whatman paper reached the bottom of
the chamber filled with 20 x SSC in order to provide the capillary transfer medium. The gel
was put upside-down onto this Whatman paper and was overlaid with a positively charged
nylon membrane (Hybond N+, Amersham) in the exact size of the gel. The next layers were
2 Whatman papers, also in the exact size of the gel and the membrane. These Whatman
papers were additionally overlaid with a 10 cm stock of paper tissue. Finally, a plexiglas plate
was put onto the whole assembly and functioned as basis for a weight of about 200 - 500 g.
The capillary transfer of 20 x SSC through the whole Southern blot assembly transferred the
DNA out of the gel onto the nylon membrane. After blotting over-night blotting, the blotting
assembly was removed and the membrane was carefully turned so that the DNA side was
upside. The membrane, placed on a Whatman paper soaked with 2 x SSC, was put into an
UV-crosslinker (Amersham) in order to fix the DNA to the membrane. The agarose gel was
stained again with ethidium bromide in order to check if the DNA transfer was successful.
2.7.4. Hybridization
The membrane was soaked with 2 x SSC and was placed inside a hybridization flask. The
gel side of the membrane had to face inwards in order to be exposed to the following
solutions of the hybridization procedure: first, the membrane was exposed to 10 ml
prewarmed DIG Easy Hyb, for 30 minutes at the calculated hybridization temperature.
Meanwhile, 3 |jl of probe 1 or probe 2 or respectively 20 pi of probe 3 were added to 50 |jl of
H2O in order to be denatured for 5 minutes at 97°C. After an immediate shock on ice, the
probe-solution was added to 10 ml of DIG Easy Hyb. This hybridization solution replaced the
DIG Easy Hyb solution inside the hybridization flask. The hybridization flask with the
membrane and the hybridization solution was incubated over night at the calculated
hybridization temperature.
2.7.5. Stringency washes and antibody binding
After hybridization, the membrane was washed twice for 5 minutes with low stringency buffer
by shaking at room temperature. Then, the membrane was washed twice for 15 minutes with
high stringency buffer at 68°C. After this removal of unspecific bound probe, the membrane
was washed for 5 minutes in washing buffer. Next, unspecific binding sites on the membrane
were blocked in a washing step with 1 % blocking solution for at least 30 minutes.
Meanwhile, the antibody solution was prepared by adding 2.5 pi of anti-DIG-AP antibody to
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Materials & Methods
25 ml of 1 % blocking solution. In order to obtain pure anti-DIG-AP, the anti-DIG-AP solution
was centrifuged for 10000 rpm for 5 minutes and the anti-DIG-AP was obtained from the top
of the solution. Finally, the membrane was incubated in the prepared antibody solution for 30
minutes under shaking at room temperature.
2.7.6. Immunological detection
In order to prepare the membrane for immunological detection, the antibody solution was
discarded and the membrane was washed twice in washing buffer for 15 minutes under
shaking, followed by membrane equilibration in TBS for a few minutes. Then, a development
folder was prepared, in order to place the equilibrilated membrane inside. CSPD working
solution was dropped over the membrane and the development folder was closed
immediately. The membrane was incubated for 5 minutes at room temperature, followed by
another incubation step at 37°C for 10 minutes. Finally, an X-ray film was placed onto the
development folder just over the membrane. Thus, the X-ray film was exposed to the
chemiluminescent signal originating from the sites, where the probe hybridized. Finally, the
X-ray film was developed with the Agfa Curix 60 film processor for visualization of the bands.
Probe labelling:
Denaturation buffer:
Neutralization buffer:
20 X SSC:
Positively charged nylon membrane:
Low stringency buffer:
High stringency buffer:
Washing buffer:
DIG High Prime DNA Labelling and Detection
Starter Kit II (Roche)
PCR DIG Probe Synthesis Kit (Roche)
1.5 M NaCI
0.5 M NaOH
1.5 M NaCI
0.5 M Tris-CI
pH7.0
3 M NaCI
300 mM NaCitrat
pH7.0
Hybond N+ (Amersham)
2xSSC
0.1 % SDS
0.1 xSSC
0.1 % SDS
1 X Maleic acid buffer
0.3 % Tween 20
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Materials & Methods
1 X blocking solution: 10 x blocking solution (Roche) diluted 1:10 in 1 x
maleic acid buffer
Antibody: Anti-digoxigenin-AP antibody (Roche)
TBS: 0.9 % NaCI
30 mM Tris-CI
pH9.5
CSPD: "CSPD ready-to-use" substrate (Roche)
X-ray film: Hyperfilm MP (Amersham)
Developer: Agfa Curix 60 film processor
-66
Results
3. Results
3.1. Development of real-time PCR assays
The first part of this project was to develop real-time PCR assays that specifically quantify
the different FIV DNA forms, present in a cell during FIV infection. Therefore, primer and
probes had to be designed and tested. The PCR primers were designed based upon the
maps of viral plasmids, viral vectors and viral DNA forms that were constructed in silico
(Figure 3.1) with the software Sei Ed Central (Clone Manager 6.0, Align Plus 4.1 Primer
Designer 4.2). The plasmid maps and maps of the DNA forms are presented in Figure 3.1;
maps for pCTSefs and pCT5efsD66V are identical with the exception of a D-V mutation in
codon No. 66 of the FIV integrase, present in pCT5efsD66V.
a)
b)
5' LTR' gag pol
C)
Figure 3.1. Map of viral plasmid pCTSefs and maps of
different DNA forms resulting from the viral vector
CTSefs during infection: maps were constructed in
silico with tlie software Sei Ed Central (Clone Manager
6.0, Align Plus 4.1 Primer Designer 4.2)
a) plasmid pCTSefs, b) CTSefs provirus/ linear
unintegrated, c) CTSefs 1-LTR circle, d) CTSefs 2-LTR
circle
efs
^ ^=t= mm y LTR'
IN
d)
eft
-67
Results
3.1.1. Total viral DNA assay
This assay should detect all viral DNA forms, thus a fragment should be amplified that can be
found in all viral DNA forms. Therefore, primers and probe were designed to amplify a part of
the common LTR region extending into the 5' untranslated region. The real-time PCR assay
for quantification of total viral DNA had already been created at the institute (Figure 3.2)
(Steinrigl, unpublished). Thus, the probe as well as the primers had already been designed
and tested for usability.
I r
U3 R US gag'
f p r
Figure 3.2. Total viral DNA assay: The total viral DNA assay Is shown with primers (f and r) and probe (p) on a
viral DNA fragment containing the 5'LTR and part of the gag
The standard for this assay was created with the plasmid pPetAenv. The plasmid PetAenv
represents a FIV provirus based on the FIV-14 molecular clone of the Petaluma strain. For
standard creation, pPetAenv was propagated in a large scale bacterial culture. After
evaluation of the plasmid DNA concentration by optical densitometry, the plasmid was diluted
1:10 in a dilution series ranging from 1 copy to 10^° copies per |jl. The dilution series was
tested by real-time PCR with the total viral DNA assay. The standard curve shows a good
correlation with 0.9975 and a slope of -3.3 thus can be used for quantification (Figure 3.3).
The standard shows a linear range of quantification over 7 logarithmic decades.
-68-
Results
«»o -
40 r2: 0.9975 slope: -3.331
35 P
i
a E: 0.9962
b
num
ber
to
<j3 m
!• • Ö o 20
^^ •
rhre
shol
d
10
5
0 1 r 1 1 r 1 1 r 1
1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E+05 1.E+06
log copy number
1.E+07 1.E+08 1.E+09 1.E+10
Figure 3.3. Standard curve of pPetAenv standard for quantification of total viral DNA: The standard curve is
generated from the (threshold cycle) CT values of the dilution series of pPetAenv. The log copy number of the
template input is shown on the x-axis and the CT values on the y-axis. The mean and the standard deviation of
the duplicates are indicated. The correlation coefficient (r^) of the standard curve, the slope of the standard curve
and the real-time PCR efficiency (E) are listed in the box.
3.1.2. Integrated DNA assay
The principle behind the quantification of integrated DNA with the help of Alu- sites within the
genome was described before (see 2.6.4). The primers and probe needed to perform this
nested PCR assay are shown in Figure 3.4.
-69-
Results
r«' round
Alu site LTR ^ -
Iambda-f1 Alu-r
1 Alu-f
2"'' round
Alu site LTR
amplicon 1 St round
f2 p2r2
amplicon 2nd round
Figure 3.4. Integrated DNA assay: Both rounds of the assay are shown on a fragment containing a part of the
provirus, integrated into host DNA containing an Alu repetitive site. In the 1st round, the forward primer (Iambda-f1),
tagged with an lambda phage heel sequence, and the reverse Alu primer (Alu-r) or forward Alu primer (Alu-f) amplify a
fragment spanning from the integrated provirus to the first adjacent Alu-site. The 2nd round specifically amplifies a
proviral DNA fragment by using a lambda-heel specific forward primer (f2), next to the reverse primer (r2) and the
probe (p2).
The standard for this assay was obtained from HeLa cells stably infected with an egfp-
harbouring FIV-vector (pCT25einF). HeLa-DNA was extracted and the cell provirus copy
number was quantified with the PALSG-standard by real-time PCR. The PALSG standard
contains genomic DNA obtained from NIH/3T3 cells infected with an MLV-vector also
harbouring an egfp-gene. Thus, this PALSG standard could be used as a standard for
provirus copy number discrimination. After quantification, a 1:4 dilution series with known
provirus copy number was created with the DNA extract. The standard dilution was tested
with a real-time PCR for human apoB gene prior to usage in order to check the linearity of
the standard. The standard curve showed a good correlation of 0.998 and a slope of -3.43
(Figure 3.5).
-70
Results
HO
40 r2: 0.9979
35 ^..^^ slope: -3.426 P ü
• i
8 -
E: 0.9583
num
ber
Ol
a
--I
S" 20
»
thre
shol
d
5-
0- 1 , , 1 i
1.E+00 1.E+01 1.E+02 1.E+03
log copy number
1.E+04 1.E+05
Figure 3.5. Standard curve of HeLa integration standard In human ApoB real-time PCR assay: The standard
curve is generated from the (threshold cycle) CT values of the dilution series of HeLa integration standard. The
log copy number of the template input is shown on the x-axis and the CT values on the y-axis. The mean and the
standard deviation of the duplicates are indicated. The correlation coefficient (r^) of the standard curve, the slope
of the standard curve and the real-time PCR efficiency (E) are listed in the box
Although this HeLa integration standard could already be used for quantification, it also has
the disadvantage that with every dilution step the complete DNA amount is further diluted.
Thus, the standard does not reflect the natural situation in a cell extract, represented by a
relatively stable amount of DNA. To achieve a more natural situation for primers and probe,
the HeLa integration standard was re-made by dilution in DNA obtained from uninfected
HeLa cells in order to create the HeLa^ standard. Thus, every standard dilution step of this
HeLa2 standard contains the same amount of HeLa DNA, but at the same time has a
decreasing and known fraction of proviral DNA for assay quantification.
The usability of the two standards for the integration assay was compared using the
conditions of the integrated DNA real-time PCR assay. Both standards had nearly the same
correlation. A correlation of 0.986 of the HeLa integration standard was comparable to a
correlation of 0.972 of the HeLa^ integration standard (Figure 3.6). However, the efficiency of
the real-time PCR with the HeLa^ integration standard of 104 % is not perfect but more
appropriate for quantification than the 86 % of the HeLa integration standard. Furthermore,
71
Results
the HeLa2 integration standard enabled quantification of smaller DNA sample concentrations
(Figure 3.6b).
Concluding, the HeLa^ integration standard could be used for quantification and represented
the natural conditions better than the conventional HeLa integration standard. As a result, the
HeLa2 integration standard was used for further quantification of the integrated DNA.
a) HeLa integration standard 45
40
35
r^: 0.9862 slope: -3.712 E: 0.8595
p 30
20
1 15
10
5
0
•
--•
1 E+02 1 E+03
log copy number
b) HeLa^ integration standard 45
40
35
p 30 Ü t>
I" u
O 20
i e 15
10
S'
r2: 0.9723 slope: -3.229 E: 1.0402
Figure 3.6. Comparison of
standard curves of l-leLa^ - and
IHeLa integration standard with
complete integrated DNA real-
time PCR assay: 1=' step of
nested PCR was performed with
lambda forward primer and AluF
primer. The standard curves are
generated from the (threshold
cycle) CT values of the dilution
series of (a) HeLa and (b) HeLa2
standard. The log copy number of
the template input is shown on the
X-axis and the CT values on the y-
axis. The mean and the standard
deviation of the duplicates are
indicated. The correlation
coefficient (r^) of the standard
curve, the slope of the standard
curve and the real-time PCR
efficiency (E) are listed in the box.
1 E*02 1 E+03
log copy number
After deciding for the HeLa^ standard, the assay setup had to be tested and improved. There
was the question, which primers to use in the first round PCR. Former tests (with the HeLa
integration standard) showed that using a fon/vard and a reverse Alu primer - as described in
the literature - resulted in a worse standard curve and an impaired detection of integrated
DNA, compared to a setup with only one Alu primer (data not shown). Therefore, we tested 4
possible setups for the first round PCR of the nested real-time PCR assay for integrated DNA
with the HeLa^ integration standard (Figure 3.7): a setup with both Alu primers (+Alu) (Figure
3.7a), a setup without Alu primers (-Alu) (Figure 3.7b), a setup only with the Alu forward
-72
Results
primer (AluF) (Figure 3.7c) and a setup with only the Alu reverse primer (AluR) (Figure 3.7d).
The AluR setup showed the best standard curve of all approaches with a correlation of 0.996
and a slope of -3.46. The efficiency of the real-time PCR reaction was 95 % (Figure 3.7d).
a) +Alu setup
i 30 E
1,5
r^: 0.9808 slope: -4.011 E: 0.7755
1.E+02
log copy number
b) -Alu setup
40
35 -
-___^^ r^: 0.9930 slope: -3.372 E: 0.9795
1- i .25-
& 2 20
10
s
0
a
'•
1 ,E+02
log copy number
c) Alu-F setup
40 r^: 0.9298 slope: -3.642
36 E: 0.8818
«30 —~~^~^* F • "•——.
a 25 ~—*-—-^
•D 20 ^^~~"~~*'~~—.^ ^ ' •"—-^-._^
S 16
••
10'
s
0
Figure 3.7. Comparison of
standard curves of l-ieLa^
standard in different setups of the
integrated DNA real-time PCR
assay: The standard curves of (a)
the assay with both AluF and
AluR primer, (b) the assay with no
Alu primer, (c) the assay with the
AluF primer, (d) and the assay
with the AluR primer are
generated from the (threshold
cycle) CT values of the dilution
series of HeLa2 standard. The log
copy number of the template input
is shown on the x-axis and the CT
values on the y-axis. The mean
and the standard deviation of the
duplicates are indicated. The
correlation coefficient (r^) of the
standard curve, the slope of the
standard curve and the real-time
PCR efficiency (E) are listed in
the box
1.E+02
log copy number
73
Results
d) Alu-R setup
•to ~
r2: 0.9960 40
slope: -3.461
35 E: 0.9450
w
^ 30 E 3 • C « 25
••» u >> s u "^~—-. «
•O 20 - o « £ — W » £ 15 £ +rf
10 -
5 -
0 _ 1.E+00 1.E+01 1 .E+02
log copy number
1.E+03 1.E+04
Figure 3.7 continued: description In first part of ttie figure.
As described earlier, the principle of the assay additionally depends on the subtraction of the
-Alu approach from one of the +Alu approaches in order to subtract background amplification
from linearly amplified unintegrated DNA. This is crucial, because the forward primer in the
first step of the nested integrated DMA assay can produce single stranded DNA from all viral
DNA forms. These single stranded fragments are detected in the second step of the
integrated DNA assay. This false positive detection is measured by a -Alu approach and
consequently is subtracted from the +Alu approach in order to quantify only integrated DNA.
Concluding, all the different +Alu test setups were also analyzed for usability concerning
subtraction from the -Alu test setup. The +Alu setup showed higher CTs than the CTs of the
AluR- and the Alu-F-setup respectively, which implied that the setup with both Alu primers
detected less integrated DNA (Figure 3.8). Concluding, again the AluR setup leaded to the
best result, because the subtraction showed a constant CT-differences of about 9 cycles,
while the subtraction of the AluF or the +Alu setup led to more variable CT-differences
(Figure 3.8).
Concluding, the AluR setup in combination with the HeLa^ standard seemed to be best suited
to quantify integrated DNA. The assay shows a linear range of quantification over 3
logarithmic decades. Compared to the other assays, the range of quantification is smaller.
74
Results
which is explained by the use of an 1:4 dilution series of the standard compared to 1:10
dilution series by the other assays.
CT difference from -Alu setup
-+Alu-«-AluF ---AluR
O
•o
12 3 4 5 6
Standard dilutions
Figure 3.8. Comparison of the different +Alu setups of the integrated DNA assay: CT-difference of standard
dilutions: The +Alu setups (+Alu, AluF and AluR) and the -Alu setup of the real-time nested PCR for integrated
DNA were performed as described in the text. The 6 standard dilutions of the HeLa2 standard resulted in
different CTs depending on the setup. The CTs of each +Alu setup have to be subtracted from those of the -Alu
setup as described in the text. The 6 standard dilutions are plotted on the x-axis. The subtracted ACT values
(between the different +Alu setups and the -Alu setup) are plotted on the y-axis. The AluR setup shows the
highest and most constant ACT, which makes this setup superior to the other tested setups.
3.1.3. 2-LTR circle assay
The real-time PCR assay for quantification of 2-LTR circles uses primers and probe for
amplification of the U5-U3 junction that is unique in 2-LTR circles (Figure 3.9). The assay has
already been created at the institute (Figure 3.9.) (Steinrigl, unpublished). Thus, the probe as
well as the primers had already been designed and tested for usability.
U3 ' ' I I I h-
R U5 U3 R U5
f p r
Figure 3.9. 2-LTR circle assay: The 2-LTR circle assay is shown with primers (f and r) and probe (p) on a 2-LTR
circle fragment with both LTRs
75-
Results
The standard for this assay was a 1:10 dilution series of plasmid p2LTRsense that contains a
FIV 2-LTR-junction. The standard was created and tested for usability with the primers and
probe for this assay (Figure 3.10). The standard could be used for quantification, because it
had a good correlation of 0.99 and a slope of -3.6. The standard shows a linear range of
amplification over 7 logarithmic decades.
45
40
35
30 E 3 C « 25 o >• o
o 20
K 15
10
5 -
r2: 0.9903
slope: -3.596
E: 0.8971
1.E+00 1.E+01 1.E+02 1.E+03 1.E+04 1.E+05
log copy number
1.E+06 1.E+07 1.E+08 1.E+09
Figure 3.10. Standard curve from 2-LTR circle standard for quantification of 2-LTR circles: The standard curve is
generated from the (threshold cycle) CT values of the dilution series of p2LTRsense. The log copy number of the
template input is shown on the x-axIs and the CT values on the y-axIs. The mean and the standard deviation of
the duplicates are indicated. The correlation coefficient (r^) of the standard curve, the slope of the standard curve
and the real-time PCR efficiency (E) are listed in the box.
3.1.4. 1-LTR circle assay
1-LTR circles have no unique sequences that could be used to place primers and probe. As
a consequence, primers that bind in the area surrounding the LTR must be used: the reverse
primer in the gag and the forward primer in the e/7i/(Figure 3.11). These primers specifically
amplify circular episomal DNA, while the 1-LTR circles can be distinguished from the 2-LTR
circles by amplicon size (Figure 3.11).
First, the primers were designed and tested for usability before designing the complete assay
including a probe. Furthermore, once the assay with the primer is established, there would
also be the possibility to use SYBR green for real-time PCR detection.
76
Results
Figure 3.11. 1-LTR circle assay: Tlie two different primer pairs that were designed to detect 1-LTR circles are
stiown together in this figure. Both primer pairs can amplify parts of 1-LTR circles - as shown in the figure - but
can also amplify 2-LTR circles with a 355 bp longer amplicon as described in the text. f1 and r1 are fonward and
reverse primer that bind adjacent to the LTR and produce an amplicon of 459 bp from a 1-LTR circle and an 814
bp amplicon from a 2-LTR circle. f2 and r2 are forward and reverse primers that produce a larger amplicon of
1510 bp from a 1-LTR circle and an 1865 bp amplicon from a 2-LTR circle.
A first primer pair was designed with primers binding just outside of the LTR (Figure 3.11:
fl/rl): a 1-LTR circle amplicon would be 459 bp long, while a possible amplicon from a 2-
LTR circle would have a size of 814 bp. The primers were tested in a conventional PCR on
the following templates: DNA extracts of CrFK cells infected with CT25ein, plasmids
pCT25ein and pCT5efs and a negative control. The PCR on the infected DNA extracts
showed the correct band at 459 bp. However, also the plasmid controls showed a band of
the same size (Figure 3.13a). Theoretically, plasmids should lead to larger amplicons
because they contain two LTRs and various plasmid-backbone elements like ampicilin
resistance genes between both primer binding sites (Figure 3.13a).
Thus, the next step was to identify the binding sites of the 1-LTR primers on one of the tested
plasmids. Therefore, the plasmid pCT5efs was sequenced with both 1-LTR primers (F1/R1)
and the resulting sequences were aligned with the sequence of pCTSefs. The result
indicated that the primers bound to the expected sites: the reverse primer bound to the
region between gag and CMV-LTR hybrid and elongated a fragment over the complete CMV-
LTR hybrid sequence that ended upstream of the CMV-LTR hybrid and downstream of the
ampicilin resistance gene. The forward primer bound upstream the 3'LTR and elongated a
fragment over the total 3'LTR that ended in the ColEI origin (Figure 3.12d: F/R). Thus, we
concluded that primers annealed at the correct sites.
For identification of the unexpected PCR-amplicons, obtained after using pCT5efs or
pCT25ein as templates, the pCT5efs-amplicon was purified out of the agarose gel and
sequenced with both 1-LTR primers. The results are shown in Figure 12e. The reverse
primer produced an amplicon spanning over the complete LTR and ending upstream of the
3'LTR (Figure 3.12e: R2). The fonA^ard primer unfortunately produced only a 61 bp long
fragment that represents a piece of the LTR (Figure 3.12e: F2). Upon repetition of
sequencing, the fon^/ard primer again only yielded a 206 bp long fragment of the amplicon,
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Results
which again only aligns with the LTR. The reverse primer again sequenced an amplicon
including the LTR and a part of the sequence upstream of the LTR (figure 3.12: F3/R3).
Concluding, the results sufficiently showed that the PCR-amplicon, obtained from pCTSefs,
contains one complete LTR, equal to the expected amplicon that should be obtained from a
1-LTR circle. However, generation of such an amplicon should not be possible, according to
the maps of either pCTSefs or pCT5efsD66V. However, the sequencing results also showed
that the primers bind correctly so that the reason for that unexpected amplicon is probably
not due to misbinding of the primer.
a) b)
'•nv 3' LTR' C0IEI origin Amplcillin res. CMV-LTR gag'
F primer R primer
C)
3' LTR' C0IEI origin'
F primer
d)
n—I-
•nv 3' LTR' C0IEI origin Ampicillin res. CMV-LTR gag'
F primer F R R primer
e)
••nv 3' LTR' C0IEI origin"
F primer F2
R2
"•nv 3" LTR' Col El origin'
F primer F3
R3
Figure 3.12. Sequencing of plasmids and 1-LTR circle amplicons: (a) to (c) shows different views and fragments of
the plasmid pCTSefs that was used for sequencing and alignment: (a) whole plasmid (b) LTR regions and
backbone elements of pCTSefs (c) 3'LTR plus adjacent sequences (d) The plasmid was sequenced with the 1-
LTR circle primers: alignment between pCTSefs and the F primer amplicon (F) and the R primer amplicon (R).
78
Results
(e) The PCR product of the 1-LTR circle assay was sequenced with the 1-LTR circle primers: alignment between
pCTSefs and the F primer amplicon (F2) and the R primer amplicon (R2) (f) The PCR product of the 1-LTR circle
assay was sequenced with the 1-LTR circle primers: alignment between pCTSefs and the F primer amplicon (F3)
an the R primer amplicon (R3).
Since similar 1-LTR circle assays have been described in the literature, another attempt for
unique 1-LTR quantification/detection was made by adopting the amplification conditions
from reported work (Jacque and Stevenson, 2006). For that, a new primer-pair, amplifying a
larger amplicon of 1510 bp, was designed (Figure 3.11: f2/r2). This time, primers were tested
on infection extracts of HeLa cells infected with CTSefs and on different plasmid
concentrations. The PCR on the FIV infected DNA extracts showed the correct band at 1510
bp. Unfortunately, the plasmid controls again showed a band of the same size.
Consequently, conditions of the PCR reaction, in particular the annealing temperature, were
altered for further testing. The result showed little influence of the annealing temperature on
the PCR reaction, because pCTSefs leads to a 1510 bp amplicon at all annealing
temperatures. Lower concentrations of pCT5efs lead to amplification only at 65°C (Figure
3.13b).
Figure 3.13. Conventional PCR of 1-LTR circle assay: (a) A 1-LTR circle PCR was performed with different templates
and the first designed primer pair that produces a 459 bp amplicon from 1-LTR circles (indicated with an arrow).
Templates: lane 1: infection CT25ein IN+ 8h p.i., lane 2: infection CT25ein IN+ 144h p.i., lane 3: plasmid pCT25ein,
lane 4: plasmid pCTSefs, lane 4: negative control; (b) Four different 1-LTR circle PCR were performed with the
79-
Results
second designed primer pair that produces a 1510 bp amplicon from 1-LTR circles (indicated with an arrow). Different
annealing temperatures were used (shown in the boxes below the lanes). Templates: lane 1: infection CT5efs IN+ 8h
p.i., lane 2: infection CTSefs IN+ 144h p.i., lane 3: negative control, lane 4: plasmid pCTSefs 1 ng lane 5: plasmid
pCTSefs 1 pg;
We then argued that the observed amplicon might also be due to recombination in the
bacteria used for plasmid propagation. In order to test this possibility a new primer pair was
designed that amplifies a similar 1500 bp fragment from the e/7/gene. A SYBR green real-
time PCR with a dilution series of the plasmid pCTSefs as template was performed including
one approach with the newly designed e/7i<-primers and one approach with the 1-LTR circle
primers. The two real-time PCR reactions were comparable due to the same PCR conditions
and the same amplicon lengths. Thus, a comparison of the PCR amplification would show if
the 1-LTR circle primer amplifies as well as the e/7i/primer. This result would help to reveal if
the plasmid itself or contaminations in the plasmid-solution serve as template for creating a
1-LTR circle amplicon. However, no correct amplification could be obtained, although the
real-time PCR was performed as described in literature (Jaque and Stevenson, 2006). There
was amplification, but there was not a kind of linearity corresponding to a pCTSefs dilution
series. It has to be mentioned that surprisingly the 1-LTR circle primers (Figure 3.14b)
showed better linearity than the reference primer in the e/7i/gene (data not shown). However,
the melting curve indicated that with both primer setups, there is no unique and pure
amplification (Figure 3.14a).
As a consequence of all these results, we decided not to use the 1-LTR circle assay,
because we cannot specifically detect, amplify and quantify 1-LTR circles.
a)
i^ fmiiA rw WÄ iXl e—^'-" - V" ...^^^^^^
// V
Figure 3.14. Melting curve
analysis and amplification plot of
the 1-LTR circle assay: The assay
was performed with the 1-LTR
circle primer that produces a 1500
bp amplicon, as described in the
text. A SYBR green real-time
PCR was conducted with a 1:10
dilution series of pCTSefs (Ipg -
100 ng). (a) The melting curve
plots the change in fluorescence
with temperature on the y-axis
against the temperature on the x-
axis. The melting curve does not
-80
Results
b) Delia RnvsCycl«
^^1^^^ •Ip^^S 41 i 1
1 1 1 Xi i i / M
^L ""fiPn 1 Ur ' 'ß j|!' 4 MHIH
ötffMl 1 1 1 i L/! i 1 ' >/ • /
1 2 3 4 5 6 7 8 9 1Ü 11 1213 141516 17 18 192021 22232425262728 M3031 3233 3435 363738 39404t 12431445
Cycle Numbtf
show a specific peak at the only
melting temperature of the
amplicon, indicating detection of
different templates, (b) The
amplification plot shows the cycle
numbers on the x-axis and the
fluorescence intensity on the y-
axis. A kind of linearity concerning
the dilution series can be
observed. However, the melting
curve indicates not a pure
amplification and the amplification
with the reference primer showed
no linearity (data not shown).
3.2. Evaluation of real-time PCR specificity
Reliability of the real-time PCR results is important in order to draw correct conclusions. One
important aspect, concerning reliability of the created real-time PCR assays, is the
simultaneous detection of plasmids. Due to the fact that the virions were produced by
transient transfection with FIV vector plasmids, plasmid carryover could be possible and
could therefore influence results.
3.2.1.1. Total viral DNA assay
The primers that are used for detection of total viral DNA produce a 316 bp amplicon in all
reverse transcribed DNA forms. There should not be amplification from the plasmids
pCTSefs or pCT5efsD66V, because the forward primer binding site in the U3-region of the 5'
LTR is not present on the plasmid due to the use of a CMV-hybrid 5'LTR in the plasmid
(Poeschia et al, 1998). Therefore, only a large amplicon would be produced if the forward
primer would bind in the US-region of the 3'LTR. However, this should be prevented because
of the limited elongation time of the PCR.
A test setup was created with serial dilutions of the plasmid pCTSefs that were used as
templates for a conventional PCR with the total viral DNA primers. Additionally, plasmid DNA
of pPetAenv was used as positive control. As a result, a 316 bp amplicon could be detected
in all templates, except in the 1 pg dilution of pCTSefs (Figure 3.15).
81
Results
In order to find out if the plasmid detection was due to contamination of the plasmid or
recombination during bacterial cloning, a real-time PCR setup was designed. Therefore, a
reference primer-pair was designed that amplifies a part of the pCTSefs gag. A real-time
PCR with two simultaneous test-approaches was performed with a dilution series of pCTSefs
as templates; one approach was performed with the newly designed reference primers and
one approach was performed with the total viral DNA primers. The amplification was
detected by SYBR green. The result showed that both approaches can be compared,
because the real-time PCR reaction-efficiencies of both approaches were equal (Table 3.1).
The comparison showed lower CT-values obtained with the reference primers, demonstrated
by an 11 to 15 CT- difference between the total viral DNA and the reference assay (Table
3.1), indicating that plasmid molecules containing the gag target sequence were more
abundant than plasmids containing the reconstituted total viral DNA target sequence. This
suggests that indeed the plasmid preparation was contaminated a fraction of the plasmids
recombined during the bacterial culture. However, since the high CT-differences suggest that
only a small proportion of the plasmid preparation was contaminated. We decided that this
assay can be used for quantification in a time-course experiment.
Figure 3.15. Plasmid detection by the total viral DNA
assay: A dilution series of the plasmid pCTSefs was
used as template for a PCR with the total viral DNA
primer. A total viral DNA amplicon has a size of 316
bp and is indicated with an arrow. The bands show
Tda X^ra DSA unexpected amplification of pCTSefs as described in
316bp the text: lane 1: pCTSefs 100 ng; lane 2: pCTSefs 10
ng; lane 3: pCTSefs 1 ng; lane 4: pCTSefs 100 pg;
lane S: pCTSefs 10 pg; lane 6: pCTSefs 1 pg; lane 7:
negative control; lane 8: positive control - plasmid
pPetAenv;
Plasmid template CT
Total viral DNA assay
CT
Reference primer assay
Difference
Real-time PCR efficiency 0.7 0.75 ...
pCTSefs 1 ng 18.19 5.87 12.32
pCTSefs 100 pg 22.09 9.56 12.53
pCtSefs 10 pg 27.13 13.71 13.42
pCTSefs 1 pg 33.26 18.135 15.125
pCTSefs 100 fg 37.865 22.76 15.105
pCTSefs 10 fg 37.715 25.89 11.825
Table 3.1. Comparison of CTs between real-time assays with total viral DNA primers and reference primers
within the gagqene: A pCTSefs dilution series was used as template for two real time PCR assays: a real-time
82
Results
PCR assay for total viral DNA was performed concurrently with a real time PCR assay with primers that amplify a
part of the gag- region. The different CT values for the plasmid dilutions from the two assays are listed in the
table.
3.2.1.2. 2-L TR circle assay
The primers that are used for detection of 2-LTR circles should not be able to produce an
amplicon from the pCTSefs or pCT5efsD66V plasmid, because the unique U5-U3 site is not
present on the plasmid molecules. However, the primer could bind on the opposite 5' LTR
and 3' LTR and could theoretically amplify a larger amplicon. This should be prevented
because of the limited elongation time of the PCR.
Serial dilutions of the plasmid pCTSefs were used as templates for a conventional PCR with
the 2-LTR circle primers. No amplicon could be detected in any of the plasmids-templates,
but only in the positive control reaction containing a plasmid as template that harbours a US-
US junction (Figure 3.16).
2-LTR circle 83 bp
Figure 3.16. Absence of plasmid detection by tlie 2-
LTR circle assay: A dilution series of the plasmid
pCTSefs was used as template for a PCR with the 2-
LTR circle primer. A 2-LTR circle amplicon has a size
of 83 bp and is indicated with an arrow. As expected,
there is no band and no amplification of pCTSefs as
described in the text
lane 1: pCTSefs 100 ng; lane 2: pCTSefs 10 ng; lane
3: pCTSefs 1 ng; lane 4: pCTSefs 100 pg; lane S:
pCTSefs 10 pg; lane 6: pCTSefs 1 pg; lane 7: negative
control; lane 8: positive control - plasmid containing 2-
LTR junction;
3.2 1.3 Integrated DNA assay
The primers of the nested PCR approach can detect plasmids as well as all other viral DNA
forms as background amplification, as described earlier (Figure 3.17). Thus, the real-time
PCR assay is once done without Alu primers to detect all background amplification and to be
able to subtract it from the amplification accomplished by the real-time PCR assay with an
Alu primer. Thus, the amount of integrated DNA can be estimated.
83-
Results
Integrated DNA 160 bp
12 3 4 + Alu
Figure 3.17. Plasmid detection by the integrated DNA assay: A dilution series of the plasmid pCTSefs was used
as template for a nested-Alu PCR for integrated DNA. An integrated DNA amplicon has a size of 160 bp and is
indicated with an arrow. As expected, there is amplification of pCTSefs in both rounds of the nested PCR as
described in the text.
lane 1: pCTSefs 100 ng; lane 2: pCTSefs 10 ng; lane 3: pCTSefs 1 ng; lane 4: pCTSefs 100 pg; lane S: pCTSefs
10 pg; lane 6: pCTSefs 1 pg; lane 7: negative control; lane 8: positive control - HeLa^ integration standard:
lowest dilution
3.3. Viral vector production
The integration proficient FIV vector CTSefs and the integrase deficient FIV vector
CT5efsD66V were produced by transient co-transfection of the plasmids pCTSefs or
pCTSefsD66V and the plasmid pHCMV-G in 293T cells. The result showed that the RT
activity of the produced CTSefs vectors was lower than the RT activity of the produced
CTSefsD66V vectors. Thus, more CTSefsD66V virions were produced and thus the viral
supernatant contained a higher virion concentration (Figure 3.18).
Due to the higher PERT titer of CTSefsD66V, less volume of viral supernatant had to be used
for infection, in order to expose the cells to equal numbers of viral particles. In correlation to
the PERT titer, 1.68 times more viral supernatant of CTSefs was used to infect HeLa cells in
the time-course infection experiments with equal amounts of virus.
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Results
PERT titer
2,00E+11 1 1 one 111 3.
8,00E+10 1
FIV IN+ FIV IN-
|l PERT liter [pU/uH ,03267E+11 1,732E+11
Figure 3.18. PERT titer of the 2
different viral vectors produced by
transient transfection: PERT
measures the activity of the viral
enzyme reverse transcriptase (RT).
In general, enzyme activity is given
in units; here the PERT titer of the
two viral vectors is given in pico
units (pU) per |jl. The FIV IN- vector
CT5efsD66V shows a 1.68 higher
PERT titer than the FIV IN+ vector.
3.4. Time course infection study
Next to real-time PCR development, the aim of this study was to use the developed assays
to get information about abundance and stability of the different FIV DNA forms in a time
course infection study. Two time course infection studies were performed: one with the
integration proficient CTSefs and one with the integration deficient CT5efsD66V. The results
from the integration deficient virus were compared to the results of the integration proficient
virus in order to get information about the episomal DNA forms in both setups. Transduced
cells were harvested at time points 8h, 24h, 48h and 144h post infection. At each time point,
the DNA was extracted and analyzed with the developed real-time PCR assays. The cells
were transduced in duplicate and each real-time PCR assay was performed twice in order to
obtain statistically reliable results. Extracts were analyzed as described before (see 2.6.7).
Thus, each value that will be presented in this thesis is the average from four values from the
time course infection. All results were related to the cell number in order to compare the
results of the different assays. The cell number was determined with a human apo-B real-
time PCR. For that, the DNA extracts from the infection studies were analyzed with primers
and probe specific for the human single copy gene apoB. The HeLa integration standard
served as standard for this assay.
The results of the total viral DNA assay showed a peak of total viral DNA accumulation at 24
hours post infection. After this timepoint, the abundance of viral DNA decreased until the end
of the experiment. This infection-kinetics could be seen with both viral vectors - the
integration proficient as well as the integration deficient. However, at all time points there was
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Results
significantly more total viral DNA in cells infected with the integration proficient CTSefs
(Figure 3.19).
a)
b)
1.20
1.00 -
to
0.80
0.60
40
0.20
0.00
total viral DNA
copies / cell mean std. dev. P*
8h pCTSefs 0.57 0.04
0.040 pCTSefs D66V 0.41 0.10
24h pCTSefs 0.92 0.26
0.027 pCTSefs D66V 0.58 0.11
48h pCTSefs 0.32 0.02
0.030 pCTSefs D66V 0.16 0.10
144h pCTSefs 0.20 0.01
0.000 pCTSefs D66V 0.04 0.01
NC HeLa — — — 2 sided T-Test
total viral DNA
FIVIN-i- -^- FIVIN-
8h 24h 48h 144h
time
Figure 3.19. Results of the total viral DNA assay: The DNA extracts of the time-course infection study were
analyzed twice and in duplicate per real-time PCR assay for total viral DNA. Results were normalized to cell
number, obtained by real-time PCR assay for the human ApoB gene. Statistics were calculated with a 2-sided
paired T-Test (p^0.05). Statistical significance with p^O.OS is indicated with stars, (a) Table showing means and
standard deviations (b) The copies / cell on the y-axis are plotted over the timeline of the infection-experiment on
the X-axis. The mean and the standard deviations of total viral DNA copy number / cell are indicated.
The 2-LTR circle kinetics were similar to the kinetics of the total viral DNA. The peak of 2-
LTR circle abundance was at 24 hours. Afterwards, the abundance of 2-LTR circles
86-
Results
decreased, similar to the total viral DNA. This infection kinetics of 2-LTR circles could be
seen in case of both viral vector types. In contrast to the results obtained from the total viral
DNA, there was no significant difference in abundance of 2-LTR circles between the two
vector types at any time-point (Figure 3.20 a/b).
The percentage of 2-LTR circles among the total viral DNA remained relatively constant in
case of the integration proficient vector: 24 hours to 144 hours post infection, about 14 % of
all viral DNA forms were 2-LTR circles. In sharp contrast to that, the infection with the
integration deficient vector led to a constant increase of 2-LTR percentage: 144 hours post
infection up to 70 % of all viral DNA forms were 2-LTR circles. Furthermore, at most time
points, except 24 hours after infection, the 2-LTR fraction was significantly higher in cells
infected with the integration deficient vector (Figure 3.20 a/c).
a) 2-LTR circles
copies / cell % of total viral DNA mean std. dev. P* mean std. dev. P*
8h pCTSefs 0.04 0.01
0.1 S3 6.38 2.06
0.004 pCTSefs D66V 0.05 0.02 12.54 2.68
24h pCTSefs 0.14 0.09
0.543 14.53 6.05
0.057 pCT5efs D66V 0.17 0.02 30.07 6.65
48h pCTSefs 0.04 0.02
0.193 13.19 5.19
0.017 pCTSefs D66V 0.08 0.04 50.34 11.25
144h pCTSefs 0.03 0.01
0.614 13.97 4.69
0.000 pCTSefs D66V 0.03 0.01 70.64 6.73
NC HeLa — ... ... .... ... ...
* 2 sided T-Test
b)
2 LTR circles
-^FIVIN+ FIV IN-
0.20 -
^1 1^
0.10 -
^^
<C\...^ 1
^^^^^ ^ ' 1-, --•
•--- •-. :^
^-^ 0.00 ' 1 \ 1
8h 24h 48h
time
144h
Figure 3.20. Results of the 2-LTR circle assay: (continued and explained on the next page)
87
Results
c)
2-LTR circle / total viral DNA [%]
|BFIVIN+HFIVIN-|
80 -
60
40
20
n -
-k
•k rh
8h 24h 48h 144h
time
Figure 3.20. Results of the 2-LTR circle assay (continued): The DNA extracts of the time-course infection study
were analyzed twice and in duplicate per real-time PCR assay for 2-LTR circles. Results were normalized to cell
number, obtained per real-time PCR assay for the human ApoB gene. Additionally, results were related to total
viral DNA obtained per real-time PCR as described before. Statistics were calculated with a 2-sided paired T-Test
(psO.05). Statistical significance with p^O.05 is indicated with stars, (a) Table showing means and standard
deviations (b) The copies / cell on the y-axis are plotted over the timeline of the infection-experiment on the x-axis.
The mean and the standard deviations of the 2 LTR-circle copy number/ cell are indicated, (c) The percentage of
2-LTR circles among total viral DNA is shown at every time point of the time-course infection study. The mean
and the standard deviations of the percentage of 2-LTR circles / total viral DNA are indicated.
The time course kinetics of integrated DMA showed hardly any DNA of both vector types was
integrated by 8 hours post infection. In CT5efsD66V infected cells virtually no integrated viral
DNA copies / cell could be detected at any of the following time points. In contrast to that,
CT5efs infected cells showed integration of viral DNA after 8 hours up to a maximum of
integration at 48 hours post infection, followed by a final decrease. At all time points, except
at 24 hours post infection, significantly more integrated virus could be found in cells infected
with the integration proficient virus (Figure 3.21 a/b).
After 8 hours post infection the proportion of integrated DNA in case of the integration
proficient vector increased until the entire viral DNA was integrated by 48 hours after
infection. In case of the integration deficient vector significantly less integrated DNA could be
detected at all time points (Figure 3.21 a/c).
-88-
a)
Results
integrated DNA copies / cell % of total viral DNA
mean std. dev. P* mean std. dev. P*
8h pCTSefs 0.02 0.02
0.047 4.S1 3.23
0.057 pCTSefs D66V 0.00 0.00 -0.77 0.68
24h pCTSefs 0.39 0.31
0.082 40.65 26.25
0.046 pCTSefs D66V 0.01 0.02 1.17 2.68
48h pCTSefs 0.49 0.2S
0.028 1S3.41 75.14
0.026 pCTSefs D66V 0.00 0.00 1.18 1.72
144h pCTSefs 0.16 0.09
0.034 82.04 45.30
0.027 pCTSefs D66V 0.00 0.00 10.49 10.49
NC HeLa — — — —- — —
b)
c)
8h
integrated DNA
-•—FIV IN-t- -*- FIV IN-
24h 48h
time
* 2 sided T-Test
•k
0.60
0.40 y ̂ ^ ^^ •
0.20 ^^ •k ^^
0.00 ?_ .-., A , f-..-..—^^^t, , A
144h
integrated DNA / total viral DNA [%]
240
220
200
180
160
140
120
100
80 -
60-
40
20
0
IFIVIN-1-BFIVIN-
8h 24h 48h
time
144h
Figure 3.21. Results from the integrated DNA assay: The DNA extracts of the time-course infection study were
89
Results
analyzed twice and in duplicate per real-time PCR assay for Integrated DNA: The first assay was performed with an
AluF setup and an -Alu setup. The second assay was perfonned with an AluR setup and a -Alu setup (see page 199-
124). Results were normalized to cell number, obtained per real-time PCR assay for the human ApoB gene.
Additionally, results were related to total viral DNA obtained per real-time PCR as described before. Statistics were
calculated with a 2-sided paired T-Test (pso.05). Statistical significance with p^O.05 is indicated with stars, (a) Table
showing means and standard deviations (b) The copies / cell on the y-axis are plotted over the timeline of the infection-
experiment on the X-axis. The mean and the standard deviations of the integrated DNA copy number / cell are
indicated, (c) The percentage of 2-LTR circles among total viral DNA is shown at every time point of the time-course
infection study. The mean and the standard deviations of the percentage of integrated DNA / total viral DNA are
Indicated.
A summary of the kinetics of all viral DNA forms during the time course infection is shown in
Figure 3.22.
a)
1.40
1.20
_ 1.00
» " 0.80 M 4)
•Q. 0.60 O o
0.40
0.20
0.00
b)
FIV IN+
- integrated -*- total viral -*^ 2-LTR |
FIV IN-
8h 24h 48h
time
144h
-»- integrated •*- total viral -*- 2-LTR 1
0,60 V
8 \^^^^ \ 8 0"0 ä o u \ T
0.20-
0.00 - . —, t , c ̂ ====:=:=,
Figure 3.22. Abundance and
stability of the viral DNA forms
during time-course infection: A
summarizing figure of all results
obtained from the time-course
infection as described in Figures
16 - 18. The abundance of
different viral DNA forms in copies
per cell is plotted against the
timeline of the time-course
infection study. The kinetics of the
infection study is shown in one
figure for the integration proficient
vector CTSefs (a) and the
integration deficient vector
CT5efsD66V (b).
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Results
3.5. Southern blot
For evaluation of the results of the real-time PCR assays, a Southern blot setup with a DIG
labelled probe was developed to detect all different viral DNA forms: by digestion with
different restriction enzymes, the DNA extracts could be analyzed for total viral DNA in one
blot, and for all the other viral DNA forms in a second blot. Thus, only one probe was needed
and had to be designed. Probe 1 was 1094 bp long and was spanning over the 5'LTR and
parts of the gag. The probe was labelled and tested with the DIG High Prime DNA Labelling
and Detection Starter Kit II (Roche). The probe showed a good labelling efficiency compared
to the reference standard and thus was used for the Southern blot (Figure 3.23).
Figure 3.23. Labelling efficiency of probe 1:
Probe 1 was 1094 bp long and was labelled by
DIG High Prime DNA Labelling and Detection
Starter Kit II (Roche). The labelled probe was
serially diluted and was blotted in spots (upper
spot series) together with a reference DNA
standard dilution series provided with the DIG
High Prime DNA Labelling and Detection Starter Kit II (Roche) (lower spot series). 6 spots of probe 1 and 5 spots
of the reference DNA were visible. The intensities were comparable.
The concentrations of the reference DNA spots were, from left to right: spot 1 - 1 ng/pl, spot 2-10 pg/pl, spot 3 -
3 pg/pl, spot 4 - 1 pg/jjl, - spot 5: 0.3 pg/pl, spot 6-0.1 pg/pl, spot 7 - 0.03 pg/pl, spot 8 - 0.01 pg/pl, spot 9 -
negative control.
However, the Southern blot with probe 1 showed no bands of the DNA extracts (data not
shown). Normally, 1 pg of plasmid should be visible on a blot. However, in this case not even
the plasmid was detected. Thus, the sensitivity of the probe was tested by Southern blot with
probe 1 on a serial dilution of pCTSefs ranging from 1 pg to 100 ng. The result showed only a
visible band from the lowest dilutions (lOng and 100 ng) (Figure 3.24).
12 3 4 5 6 7 8 Figure 3.24. Sensitivity of probe 1: Southern blot of a
serial dilution of pCTSefs. The detection was
•-'^ ^ ^ performed with the DIG labelled probe 1 as described
in the text. Only the Ing band and the 100 pg band of
pCTSefs were detected, as well as the marker in lane
1 and 8.
Lane 1: 1 kb marker, lane 2: 1 pg pCTSefs, lane 3: 10
^ • pg pCTSefs, lane 4: 100 pg pCTSefs, lane S: 1 ng
•V - pCTSefs, lane 6: 10 ng pCTSefs, lane 7: 100 ng
^m ^ pCTSefs, lane 8: 1 kb mari<er
91
Results
As a consequence, another probe with a length of 619 bp was designed within the envgene.
The probe was again labelled and tested by the DIG High Prime DNA Labelling and
Detection Starter Kit II (Roche) and showed again a good labelling efficiency (Figure 3.25).
Unfortunately, the Southern blot, analyzing the samples, again showed no detection of
samples from the time course infection experiment (data not shown).
5 6 7 8 9 - j, Figure 3.25. Labelling efficiency of probe 2:
Probe 2 was 619 bp long and was labelled by
«H . DIG High Prime DNA Labelling and Detection
Starter Kit II (Roche). The labelled probe was
9 serially diluted and was spotted (upper spot
series) together with a reference DMA standard
dilution series provided with the DIG High Prime DNA Labelling and Detection Starter Kit II (Roche) (lower spot
series). 6 spots of probe 1 and 6 spots of the reference DMA were visible. The intensities were comparable.
The concentrations of the reference DNA spots were, from left to right: spot 1 - 1 ng/(jl, spot 2-10 pg/pl, spot 3 -
3 pg/pl, spot 4-1 pg/pl, - spot 5: 0.3 pg/pl, spot 6 - 0.1 pg/pl, spot 7 - 0.03 pg/pl, spot 8 - 0.01 pg/pl, spot 9 -
negative control.
Finally, probe 3 was produced by PCR with the PCR DIG Probe Synthesis Kit (Roche).
However, in the Southern blot again only the 100 pg plasmid dilution together with shady
marker bands were detected by the probe (Figure 3.26, arrow), while the digested samples
from the time course infection experiment showed no bands.
2 3456789 10
t
Figure 3.26. Southern blot with probe 3: Southern blot
of samples from the time course infections and of
pPetAenv dilutions with the DIG labelled probe 3 as
described in the text.
Lane 1: 1 kb marker, Iane2; 5pg IN+ 24h, lane 3: 5 pg
IN+ 24h, lane 4: -, lane 5: 2.5 pg IN+ 24h, lane 6: 2.5
pg IN+ 24h, lane 7: -, lane 8: pPetAenv 1 pg, lane 9:
pPetAenv 100 pg, lane 10: 1 kb marker
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Discussion
4. Discussion
4.1. Development of real-time PCR assays
The real-time PCR assay to detect the total viral DNA in FIV-infected cells that was
developed in this project amplifies a fragment that is common to all different viral DNA forms
and spans over a part of the LTR plus part upstream the gag. The principle behind this assay
was already described elsewhere (Zack et al., 1990; Butler et al., 2001; Pierson et al., 2002;
Brüssel and Sonigo, 2003). The standards for absolute quantification should also contain this
fragment. Therefore, a serial dilution of the viral FIV plasmid pPetAenv with known
concentrations was chosen as reliable standard. The developed assay showed a good
correlation of 0.9975 and was able to quantify templates over a linear range of 7 log decades
with an efficiency of 99.6 %. These values qualified the assay for quantification of total viral
DNA. Furthermore, the reliability of the assay was shown by its use in the time-course
infection study. Since the integration deficient vector cannot form the most stable viral DNA
form that is integrated DNA, there should be a difference in the amount of total viral DNA in
comparison to the integration proficient vector. This expectation was verified, since the assay
showed a significantly higher amount of the total viral DNA in the CTSefs-infected cells at all
time-points.
The real-time PCR assay to detect 2-LTR circles in FIV-infected cells that was developed in
this project amplifies a fragment that spans over the U5-U3 region of 2-LTR circles. The
principle of the real-time PCR assay for 2-LTR circles was already described elsewhere
(Sharkey et al., 2000; Butler et al., 2001; Pierson et al., 2002; Brüssel et al., 2003; Brüssel
and Sonigo, 2003). A serial dilution of a plasmid that contains a 2-LTR circle junction served
as reliable standard for absolute quantification. The developed assay showed a good
correlation of 0.9903 and was able to quantify over a linear range of 7 log decades with an
efficiency of 89.9 %. These values qualified for quantification of 2-LTR circles.
The real-time PCR assay to detect integrated provirus in FIV-infected cells that was
developed in this project uses Alu-repeat sequences within the human genome. Alu sites are
approximately 300 bp long and are the most prominent repeated sequences in the human
genome (Stevens and Griffith, 1994). In order to use the Alu sites, which are dispersed in
primate genomes, FIV must be enabled to infect human cells. Thus, FIV vectors were VSV-G
pseudotyped and could therefore infect the human cell line HeLa. The possibility to detect
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Discussion
integrated viral DNA forms in such a PCR-based assay was already described earlier
(Benkirane et al., 1993; Courcoul et al., 1995; Sonza et al., 1996). The principle was adopted
for a real-time PCR assay by Butler et al (2001) and modified to a real-time nested PCR
assay by Brüssel and Sonigo (2003). In order to increase the sensitivity of the assay, it was
proposed to use two outward-facing Alu-primers, since Alu elements can be orientated
forward and reverse relative to the provirus (Sonza et al., 1996). In contrast to that, a test
setup in this project showed the lowest CT-values when only one Alu-primer was used. We
assumed that the decreased sensitivity with both Alu primers could be due to primer-dimer
formation or other intermolecular bindings that interfere with the PCR reaction. As a
consequence, only the reverse Alu-primer or the forward Alu-primer was used for the 1^' step
of the nested PCR together with the 1^' round forward primer. Furthermore, our observations
recommended the use of the reverse Alu-primer in context of constantly reliable results. A
serial dilution of infected HeLa DNA, diluted in uninfected HeLa DNA, was tested to be
appropriate as standard for absolute quantification of integrated DNA. This type of standard
was already suggested to be a good representative of a natural infection (Brüssel and
Sonigo, 2003).
The developed assay showed a good correlation of 0.9960 and was able to quantify over 3
logarithmic decades with an efficiency of 94.5 %. These values qualified the assay for
quantification of integrated DNA. The reliability of the assay was also shown by its use in the
time-course infection study, since significantly more integrated DNA was detected in cells
infected with integration proficient virus compared to integration deficient virus. The linear
range of a 3 log decade may be small, but quantification in this project was reliable, since the
sample concentrations from the time point infections were in this range. However, if sample
concentrations are not covered by the linear range of quantification, the developed assay
may not be appropriate. Thus, this assay should be refined in context of the used standard.
Another advancement may be considered reckoning the relatively high standard deviations
of the developed assay. This might be explained by the fact, that the results of this assay are
generated by subtraction of DNA amounts, obtained from two parallel real-time PCR assays
(+Alu/-Alu). Nevertheless, relatively high standard deviations impede absolute quantification
and explain results like 150 % integration. However, despite these higher standard
deviations, the results are coherent, show significance and thus affirm the accuracy of the
assay. Furthermore, in context of quantification, the assay is a marked advancement to
established methods like Southern blot or conventional PCR.
Quantification of 1-LTR circles by PCR-based assays leads to the problem that 1-LTR circles
have no unique sites for discrimination against other viral DNA forms. So far, the problem
was handled by amplifying the LTR region with primers that bind upstream of the LTR in the
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Discussion
gag reg\on and downstream of the LTR in the envregion. This concept was already used for
conventional PCR analysis (Farnet and Haseltine, 1991; Bukrinsky et al., 1992; Teo et al.,
1997; Frey et al., 2001; Wu and Marsh, 2003), was adapted for real-time PCR (Jacque and
Stevenson, 2006) and was also used for the development of the real-time PCR assay for FIV
in this project. However, with this setup no unique detection of 1-LTR circles is possible. Co-
amplified 2-LTR circles are discriminated from 1-LTR circles by amplicon-size, since they
contain 2 LTRs within the PCR product. We first developed a conventional PCR and could
observe the expected amplicon. Thus, 1-LTR circles in infected cells could be detected by
this assay. However, using different primer pairs and variations of the annealing temperature
could not prevent an unexpected false-positive amplification of the same size from the
plasmid controls. Sequencing analysis revealed that the primers correctly anneal to the
plasmid but then produce a somehow "impossible" plasmid-fragment spanning over one
LTR. Considering amplicon size (1510 bp amplicon assay) and PCR conditions, the 1-LTR
circle real-time PCR assay by Jaque and Stevenson (Jaque and Stevenson, 2006), perfectly
matches our developed assay. They did not mention any false positive amplification, but
reported the verification of amplicons by sequencing analysis. Additionally, it may be
considered that they used a Taqman probe for amplicon detection, which is more specific
than SYBR green in our assay (Jaque and Stevenson, 2006). However, this report along with
other publications (Farnet and Haseltine, 1991; Bukrinsky et al., 1992; Teo et al., 1997; Wu
and Marsh, 2003) displayed no control reaction, like a plasmid control in our assay.
Interestingly, Frey and co-workers validate our findings by observing a 1-LTR circle band
from plasmid p34TF10 by using a conventional PCR setup (Frey et al., 2001: Figure 2b).
However, they did not draw a conclusion out of this false positive amplification. Ultimately,
our observation is furthermore verified by Yoder and Fishel, who also found false positives
from cytoplasmatic PIC extract (Yoder and Fishel, 2006). They speculated that no specific 1-
LTR circle quantification is possible by PCR based-methods, since 1-LTR circle primers can
produce LTR containing linear amplicons from all different viral DNA forms. During a PCR
reaction, these linear amplicons can hybridize at the homologous LTR fragments and
subsequently act as primer for further amplification. Thus, all viral DNA forms can produce
amplicons indistinguishable from 1-LTR circle amplicons. They specify this, by observing
false positives from templates with 30 % to 66 % homology. In our experiments, the LTR
homology is 23.5 % in case of the 1510 amplicon setup and 77.3 % in case of the 459
amplicon setup. However, in context of the plasmid pCTSefs the homology is lower due to
the CMV-U5 hybrid. Finally, recombination of the plasmids during bacterial propagation might
also be the reason for the false positive amplicons in our 1-LTR circle assay. We already
speculate about plasmid recombination in context of the little plasmid detection during the
total viral DNA assay (as discussed below). This may be disproved, since our 1-LTR circle
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Discussion
assay also produced a 1-LTR circle amplicon from pCT25ein. Further tests with plasmids
from bacterial propagation in recombination deficient bacterial strains may be a tool for
further evaluation of this problem.
Concluding, it is clear that 1-LTR circles can be amplified by that assay, however, not
uniquely, since there is evidence that also other viral DNA forms are additionally detected.
That renders this assay unreliable for 1-LTR circle quantification.
4.2. Evaluation of real time PCR assays
4.2.1. Real time PCR specificity
Real-time PCR is a highly specific and sensitive method to quantify DNA. Therefore
specificity should be evaluated, since unwanted amplification easily influences results (Klein,
2002). The real-time PCR assays in this project were developed in order to gain the highest
specificity in context of the different viral DNA forms. Therefore, specific primers were
designed for different viral DNA forms as described before. Furthermore, specificity was
increased by using oligonucleotide probes instead of the intercalator SYBR green for
amplicon detection. Primers and probes were also constructed in such a manner as to
prevent plasmid-detection in case of plasmid carryover resulting from transient transfection.
Tests with conventional PCR confirmed this for the 2-LTR circle assay. In contrast to that, the
nested Alu-PCR-assay for integrated DNA can amplify plasmid DNA. However, this was
expected and accounted for, since this plasmid amplification is as well as linear amplification
from other templates is detected by using the -Alu setup and is then subtracted from values,
obtained with +Alu setup. A test run also indicated unexpected plasmid amplification in the
total viral DNA assay. No observations concerning this false-positive amplification are
published. Further real-time PCR tests with reference primers could reveal the problem.
Since the use of reference primers resulted in much lower CT-values, we assume that the
total viral DNA assay detects a small fraction of potentially recombined plasmids among the
plasmid preparation used for testing. It is possible that the target sequences of the total viral
DNA assay, normally not present on the plasmids, were reconstituted during bacterial
propagation. Additionally, a contamination during plasmid preparation cannot be excluded.
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Discussion
4.2.2. Southern blot
The Southern blot method was already used for detection of the different viral DNA forms. In
this project, the Southern blot, as independent method, should have evaluated the results
from the time-course infection study as formerly described (Butler et al., 2001). The Southern
blot with DIG-labelled oligonucleotide probes should have detected all viral DNA forms.
Various probes in different lengths and with different binding sites were produced and
labelled with two different labelling methods. However, all performed Southern blots could
not detect any viral DNA form from the time-course samples. The reason for this is not totally
clear. The controls by ethidium bromide staining showed successful blotting. Additionally, the
UV-crosslinker was checked, without finding an error. The labelled probes were checked by
dot blots in comparison to standards and always showed usability. The dot blots additionally
showed that the correct concentration of probe was used. Moreover, the eligibility of the
probes was affirmed by a slight band of plasmid control in some blots. However, plasmids
were only detected in unusually high concentrations. Furthermore, also the marker was
infrequently detected. These findings could indicate that the probes were too insensitive or
were applied in wrong concentrations. Further tests with different probe concentrations would
be needed to evaluate this problem.
4.3. Viral vector production
The integration proficient FIV vector CTSefs (Poeschia et al., 1998) and the integration
deficient FIV vector CT5efsD66V (Saenz et al., 2004) were used for the time course infection
study in this project. The viral vectors were produced in 293T cells by transient co-
transfection and were pseudotyped with the vesicular stomatitis virus G (VSV-G) envelope
protein. The use of 293T cells to produce VSV-G pseudotyped lentiviral vectors to infect non-
dividing cells was already described and represents a commonly applied system (Naldini et
al-, 1996; Poeschia et al., 1998; Kafri et al., 1999).
The use of viral vectors in a basic research project could be seen controversial, since viral
vectors do not represent the total wild type virus. However, the used vectors were based on
the wild type 34TF10 Petaluma strain (Pedersen et al., 1987; Olmstedt et al., 1989; Talbott et
al., 1989) and matched with the wild type virus to a very high degree. The few changes that
differed from the wild type virus included a frameshift in the e/7/gene, a vesicular stomatitis
virus G (VSV-G) protein within the virus envelope and a 5'LTR-CMV promotor hybrid in the
proviral plasmid. These changes in fact beneficial for this study: The envelope frameshift
created replication deficient vectors. Thus, monitoring could be limited to only one single
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Discussion
replication cycle. Furthermore, the FIV vectors were VSV-G pseudotyped to enable the FIV
vectors to infect the human cell line HeLa. That was crucial for the ability to use the
integrated DNA assay, since Alu repetitive elements are unique in primate genomes. Another
difference to the wild type virus affected the proviral plasmids and not the resulting vectors.
The proviral plasmids contained a 5'LTR-CMV promoter hybrid that helped to distinguish
plasmid DNA from reverse transcribed DNA.
4.4. Time course infection study
The single cycle time course infection study was performed with the integration defective
CT5efsD66V and the integration proficient CTSefs, respectively. Using integration defective
viruses in comparison to integration competent viruses is a commonly used setup to study
properties of retroviral episomal DNA forms (Stevenson et al., 1990; Taddeo et al., 1994;
Ansari-Lari et al., 1995; Wiskerchen and Muesing, 1995; Pierson et al.; 2002, Saenz et al.,
2004; Steinrigl et al., 2007). Therefore, two types of integrase mutants can be differentiated:
Non-specific phenotypes or class II mutations contain truncations, various single amino acid
changes or deletions, whereas nonpleiotropic or class I mutations contain specific mutations
in the catalytic centre of the integrase. Consequently, class I mutants only disturb the DNA-
cleaving and -joining reaction, while other processes like Gag/Pol processing, particle
formation, virion morphogenesis, reverse transcription or PIC nuclear import are not affected
(Saenz et al., 2004). In this study, the already described and tested integrase class I mutant
CT5efsD66V was used (Saenz et al., 2004). Thus, all differences that appeared between the
two vector types could be uniquely ascribed to the lack of a functional integrase.
The vectors did not contain a transgene like egfp to obtain viral liters per FACS analysis.
Thus, the viral titer was determined by PERT assay, in order to transduce HeLa cells with
equal amounts of viral vector. The eligibility of PERT assay was already demonstrated
(Lovatt et al., 1999).
The developed real-time assays were used to analyze the DNA extracts from different time
points after infection including 8 hours, 24 hours, 48 hours and 144 hours post infection. The
results were normalized to the cell number, as described above.
The obtained results give new insights into the biology of the different viral DNA forms during
an FIV infection, since there exist only limited published data. Saenz and co-workers used
the same FIV vectors for in vitro infections, but mainly focused on 2-LTR stability in the
context of gene expression. Similar studies that monitored different lentiviral DNA forms in
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Discussion
vitro by real-time PCR assays were only performed for HIV-1 (Butler et al., 2001; Butler et al.,
2002; Pierson et al., 2002; Brüssel and Sonigo, 2003); only one also used an integration
deficient virus for comparison (Pierson et al., 2002).
The total viral DNA assay showed similar total viral DNA kinetics after infection with both viral
vector types: the total viral DNA peaked at 24 hours and decreased markedly until 144 hours
post infection. Published HIV-1 studies reported a total viral DNA peak already at 12 hours
post infection (Butler et al., 2001; Butler et al., 2002; Brüssel and Sonigo 2003). If our study
differs from these studies in context of the total viral DNA peak, can just be figured out by
analyzing the 12 hours post infection time point. Furthermore, the total viral DNA assay
showed that the integration deficient vector produces total viral DNA forms with similar
kinetics, but in significantly lower quantity, at all time points. This might indicate a lack in
stability of the cDNA forms in CT5efsD66 infected cells. The integrated DNA is suggested to
be the missing cDNA form in CT5efsD66V infections, since the only difference between the
two vector types is the IN-mutation in CT5efsD66V. The results of the integrated DNA assay
and the 2-LTR circle assay confirmed this suggestion. By comparison of the vector types
during the time course, the assays showed no significant difference in 2-LTR circle
abundance, but huge differences in provirus abundance.
A markedly decrease of total viral DNA after the peak could be seen in this study, but was
also described for HIV-1, in relation to proteasome-mediated degradation of unintegrated
linear DNA (Butler et al., 2002). Butler and co-workers showed that the proteasome acts after
reverse transcription and does not degrade already formed DNA circles or proviruses, but
destroys the substrate for these forms that is the linear unintegrated DNA. These
observations cannot be fully confirmed by this project, since an increase in integrated DNA
was observed. This would not be possible if the linear unintegrated DNA as substrate would
be eliminated. Furthermore, concurrent with the total viral DNA decrease, also the 2-LTR
circle amounts decrease in case of both viral vector types, thus seem to be degraded.
However, also the cell number rises during that time, so that 2-LTR circles may not degraded
but diluted out - as discussed below.
The integrated DNA assay confirmed that CT5efsD66V is integration deficient: as expected
hardly any integration of this vector could be observed during the time-course infection
experiment. This in turn also affirms the reliability and usability of this assay. Consequently,
integration proficient CTSefs produced significantly higher provirus amounts during the
experiment, with a peak at 48 hours post infection. These kinetics were also observed for
HIV-1 infections (Butler et al., 2001; Brüssel and Sonigo, 2003). The fact that the difference
at 24 hours post infection is not significant is probably caused by the high standard deviation.
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Discussion
Unfortunately, the Integrated DNA assay displays higher standard deviations compared to
the other assays at all time points measured as discussed above.
Indeed, small amounts of provirus were detected in cells infected with integration deficient
FIV. Already former reports observed replication and provirus formation by integration
deficient virus (Engelman et al., 1995; Wiskerchen and Muesing, 1995, Gaur and Leavitt,
1998). This is may be explained by a non integrase mediated process (Gaur and Leavitt,
1998).
The reason for the decrease of integrated DNA after the peak is not clear, since integrated
provirus cannot be diminished and should be propagated by cell division. However, a slight
decrease of integrated HIV-1 provirus was already published and was explained by dilution
due to slower cell division rate of infected cells or even cell death of infected cells (Butler et
al., 2001; Brüssel and Sonigo, 2003).
Relating the integrated DNA from the integration proficient vector to the total viral DNA
reveals that provirus accumulates over time until 100 % of total viral DNA is integrated 48
hours after infection. One could be sceptical about the accuracy of the assay, because the
assay showed 150 % integration, followed by a drop to 80 % at the end of the time course. A
statistical analysis revealed that there is no significant difference between the amounts of
integrated provirus against total viral DNA after 48 and 114 hours (data not shown; paired 2-
sided T-Test p > 0.05). This supports the suggestion that most of total viral DNA is integrated
at these time points. However, the 2-LTR circle amounts show a stable 2-LTR circle fraction
of 5% among the total viral DNA at these time points.
The 2-LTR circle assay showed that 2-LTR circles peak 24 hours after infection and
decrease until 144 hours in case of both viral vectors. The same kinetics were already
obtained for HIV-1 (Butler et al., 2001; Butler et al., 2002). The decrease of 2-LTR circles
after the peak at 24 hours, seen in our study as well as in HIV-1 studies, is still under
discussion. It could be due to cell division; in this case, 2-LTR circles are diluted out,
because they do not contain any replicative element (Butler et al., 2001; Butler et al., 2002;
Pierson et al., 2002). Others argue that 2-LTR circles are labile and instable DNA forms
(Sharkey et al., 2000; Sharkey et al., 2005). A long-time in vitro study showed stable 2-LTR
circles and therefore even non-specific integration of 2-LTR circles was suggested (Brüssel
and Sonigo, 2003). The reason for the 2-LTR circle decrease in this study cannot be
revealed with certainty: it could be due to instability, or due to dilution since the cell number
was steadily rising from 24 hours post infection onwards (data not shown). Further
investigations, for example by using cell cycle arresting agents like aphidicolin, could give
more concrete information about 2-LTR stability.
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Discussion
The 2-LTR circle assay showed no difference between the integration deficient and the
integration proficient vectors, neither in l<inetics, nor in abundance. This is in sharp contrast
to published results for HIV-1 that demonstrated that integrase mutated viruses produced
higher amounts of 2-LTR circles (Hong et al., 1991; Engelman et al., 1995; Wisl<erchen and
Muesing, 1995, Hazuda et al., 2000). For FIV, the results of Saenz and co-worker support
our findings: Indeed, they found significantly higher 2-LTR circle abundance, but only in a
non-dividing cell type and not in dividing cells like in this study. However, examining the 2-
LTR percentage showed that up to 70 % of all viral DNA forms are 2-LTR circles in case of
the integration deficient virus, while the integration proficient virus only showed a 2-LTR
fraction of 5 %.
There is published data that in HIV-1 1-LTR circles are the most abundant circular episomal
DNA form (Pauza et al., 1990; Farnet and Haseltine; 1991; Butler et al., 2001). Due to the
fact that the 1-LTR circles could not be quantified in this study, conclusions about this ratio
can just be drawn by observation of the 2-LTR circle fraction. The result is controversial: 144
hours after infection with the integration defective vector, 70 % of all viral DNA forms were 2-
LTR circles, so that a higher 1-LTR fraction seems impossible. However, under wild type
conditions with the integration proficient viral vector, a larger 1-LTR circle percentage might
be possible, since at 144 hours post infection only 5 % of all viral DNA forms are 2-LTR
circles. Anyway, it also has to be considered that the published ratios were obtained for HIV-
1 and by Southern blot, thus a comparison to these studies may not be appropriate.
4.5. Conclusion
We could develop a set of real-time PCR assays that can be used to study abundance and
stability of most of the viral DNA forms that occur in FIV infected cells: total viral DNA,
integrated DNA and 2-LTR circles. Although comparable assay sets were already described
and designed for HIV-1, little is published for FIV yet. In contrast to published reports, we
were not able to develop a real-time PCR assay that uniquely detects and quantifies 1-LTR
circles. However, this observation also supports an already existing assumption that 1-LTR
circles cannot be specifically detected by PCR based methods in context of the existing
setup. Therefore, the evaluation of the existing 1-LTR circle assay or the search of new
strategies or other methods are suggested.
We could test the developed real-time PCR assays in vitro in time course infection studies in
the context of an integration deficient and an integration proficient FIV vector. One the one
hand, the results showed the usability of the real-time PCR assay with regards to
101 -
Discussion
consequential and reliable results. On the other hand, the results could already give insights
into the biology of the different viral DNA forms of FIV. In case of the integration proficient
virus, most of the cDNA was shown to be integrated 48 hours after infection, while the
highest abundance of 2-LTR circles could already be observed 24 hours after infection. A
stable 2-LTR fraction of 5 % was observed during the complete time course. In contrast to
previous findings, integration deficiency did not generate higher amounts of episomal 2-LTR
circles. However, since the highly stable proviral cDNA form cannot be produced in cells with
integration deficient virus, significantly less total viral DNA could be observed, resulting in a
2-LTR circle fraction that accumulates up to 70 %.
Concluding, the real-time PCR assays show high correlation, efficiency and specificity to
obtain reliable results in terms of absolute quantification. Therefore, they can help to get
insight into the biology of FIV DNA forms and ultimately also allow conclusions about the
biology of HIV-1 and other lentiviruses.
4.6. Outlook
The real-time PCR assays for total viral DNA, integrated DNA and 2-LTR circles were
developed and tested for usability. The integrated DNA assay indeed showed high
correlation and efficiency, but could still be improved in terms of increasing the linear range
of quantification. The attempt to design an assay to quantify 1-LTR circles should be
continued by further investigation and improvement of the existing setup, since there is
evidence that the developed setup is not suitable in terms of specificity. The most important
next step would be the development of a successful Southern blot assay, as independent
method, to verify the results of the real-time PCR assays. A next step to increase the
statistical evidence of the obtained results would be the repetition of the time course
infection. Furthermore, variations in the time-course infection setup could give more or new
information in terms of abundance and stability of the different viral DNA forms; for example,
additional time-points within the time course, or an extension of the time course would be
possible. Furthermore, arresting the cell cycle by aphidicolin or similarly acting chemicals
could be used to evaluate the stability of 2-LTR circles in non-dividing cells.
- 102-
Appendix
5. Appendix
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Appendix
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Appendix
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116
Appendix
5.2. Publications
This work was presented on a poster on the 9th International Feline Retrovirus Research
Symposium 2008 in Vienna, Austria:
Establishment and Validation of Real-Time PCR Assays for the Quantification of Different Forms of Feline Immunodeficiency Virus DNA
Hof er M.. Klein D., Steinrigl A.
Institute of Virology, Department of Pathobiology, University of Veterinary /Medicine Vienna, Vienna, Austria
Feline immunodeficiency virus (FIV) belongs to the genus of lentiviruses and is associated with an AIDS-like disease in cats. An essential step in lentiviral replication is the creation of double-stranded viral DNA from the RNA genome by reverse transcription and the subsequent integration of the viral DNA into the host genome. However, linear viral DNA molecules can also become circularized by reactions that prevent integration. 1-LTR circles form by homologous recombination between the two LTRs that flank the linear genome, while 2-LTR circles form by nonhomologous end joining of the linear viral DNA. Thus, at least 4 distinct forms of retroviral DNA exist simultaneously in infected cells: linear unintegrated DNA, linear integrated DNA, 1-LTR circles and 2-LTR circles. It was found that 2- LTR circles are quite stable and decreased only in correlation with cell-death or cell division. Furthermore, transgene expression from integrase deficient lentiviral vectors was shown to be equivalent to wild type lentiviral vectors, indicating that unintegrated lentiviral DNA per se is not inaccessible with regard to transcription and protein expression. Thus, investigation of abundance and function of the different viral DNA forms can give new insights into retroviral replication. The aim of this study was to develop and evaluate reliable real-time PCR assays for the different viral DNA forms occurring in FIV infected cells. As a result, 2-LTR circles, total viral DNA and integrated proviral DNA could be successfully quantified in time-course infection experiments in the context of either proficient or impaired viral integration. However, we were not able to specifically quantify 1-LTR circles by PCR based methods. It was recently speculated that the reason for that could be the hybridization of the linear amplicons at homologous LTR regions.
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Appendix
Establishment and Validation of Real-Time PCR Assays for the Quantification of Different Fornns of Feline Innmunodeficiency Virus DNA
Matthias Hofer \ Dieter Klein ^^, Adotf Steinrigl'
1) Instrtute of Viroiogy. Department of Pathobioiogy, University of Veterinary Medicine, A-1210 Vienna. Austria
2) Vetomics Core FaciKty for Research, University of Veterinary Medicine, A-1210 Vienna, Austria
Introduction
tuboquanT inW)rMKin ot ma vni DNA mlo Vw no« g«noms H
n^M pravani rria^alior 1 -LTR cOa* lorm tiy hcmologiui 'BC
ma by ravarta ktnacnpaon and ihs
Jao bacoma arculanlaa by nadioni
lo LTRi Dial tlanK Itia linaar gsnyna wMa 2-LTR
ncl totmt ol ralrovrai DNA aotl •mültanaoutly in
orcla» Sinca apnoina lanlivrral DhIA km« iwfa
>t Iha «ftarant viral DNA lortni could
Figure 4 a TotMvirMDNAaiaay
Fidelity of real-time PCR assays
cinaea kiirn by nonnomologDui end (Oining 01 ma tnaai vial DNA Thut al M
inladaO c4>t linaw unrugraMd DHA tmav mavMM DNA 1-LTR orcM a
rgportaä lo ahrw a canain MaMity ard anabW gana anpraaaon invadigaton of
give naw intigMt inio WnBvir« raphcation WtMa nvMtgaaor o> ijrtarani virN DMA torma by raal-tma PCfl la aalaOShaa 1« HIV-1. httla
mlotmation ii pubtiiTiM tor RV
rna am ot ir» alujy nia* k> davalop and avituala raliabta raal-Vna PCR •ai^i tor dllarani vitaJ DNA torma occurring in RV inlaCWd call*
loU vir« DNA. lin*M (ilagraMd DNA. 1-LTR orcM »id 2-LTR circtea Smilai ait^i tiava otraaOy baan Oaacnbad kx HIV-1 mdwaraadvlad
tor f IV Thia abundvic« and nabilily <A m» V(al DNA tormi «rars dawrmiriad n time-coina intaction «Kparimanla in the conlaxl 01 »t\m
pioliciam or MipMad vral inMgralon Tint wai laolitalad by ttie uae ol Itie inte^ation protioarit FIV-vactor CT5Ms anO Iha inMgraiion datoant
F(V-v«ctorCT6at»D66Vtlial contains «namino acid »ut>ttilu»on in the viral sniyme iriB^ase
Viral vectors and re^-time PCR setups
Figure 1
Viral vectors were produced in 2637 caMt tiy tranaiant
tranalaction pl^rrud pHCMV-G {Burnt ai al. Proc r4atl Acad Sci
1993) was co-transtecteO wifi the vir« FIV plavniOi pCTMs
(Poeschia el al f^al Med 1998) ana pCT5e'sD66V (Sunz al
al . J Virol 2004). raapectivaly pCTE^ts na> a CMV-LTR hybrid
and containt an envelope Vrameshitl (ets) pCTSettOefiV is
idenncal witti the enceplior a( a D-V miflalion w\ codor No 66
(D66Vj and theralore is mlegratian OatioerTt All plasmids ware
kindy provided by Dr Ere PoBSCMa
rd diutont ot luiIaMa plaamidi (a and b) or mtecled DNA {c) The loganinmic copy
nkimDar of the tvnplale input it ploned against fie CT values on tm y-au* Tlie mean and the slarvMud dovialion of the duplicates are ndcaied
Th« corraltfion coatticient |r') ot the «andvd curvs the slope ot the ilandara cune and in« real-tiTTie PCR eltioency |E) are litlad in Iha insed
boiaa AH aaaayt sTiowed tugti aenaitivily and gaoa correlalion |a. b and c) Thw IDUI viral DNA ast^ {a) and the 2-LTR circle aasay |b) car
quantify over 7 loganthirc decades The intogratad DNA aaaay (c) shows a lower ranoe o< quarvAcalKin ovar 3 loganlhrnc dacadaa
Application of real-time PCR assays in a time course infecöon experiment
Figure 5a Toia viral DNA
Figure 2a Figure 2c
Figure 5b
Figure 2b
Haia celia were intectad wilti equal «nounit ot CTSelt and
CT5elsD66V. raspactivaly Cats were haivealad at attarsnl tma
pants post inlaclion Sn pi. 24n p.i. «Sn p i and 144h pi DNA
aMracts ware analyzed hvice and in duplicate tiy the developed real-
tme PCR assays Reautts wgra normatiBd to cell number, obtained
par real-tme PCP assay lor hirnan ApoB garw The mean and the
standen} deviations of the mulls are indicated StalisticE were
calculated vnth a 2-sided paired T-Tast |(c^OS) Statistical
significance IE indcated with stars The DNA lorm copes/ cell on the
y-aiis are pbtted over 1t>e timeline ol ttie intectiori-eiiperiment or the
i-a»s AddticnaMy. the percentage ot viral DNA lorms among total
viral DNA is shewn at every tme pont of the time-course inteclcn
2-LTfi crcJes / total vir* DNA (%]
•n^kwMnaid
Rgura2
(a) Ttw total viral DNA assay is shown «nth pnmors (t and r) and probe |p) on a viral DNA IraGTient containing ttie 51TR and part of the gag The
assay delects all viral DNA torms and thus amplities the long Mrminal repeat {LTR) reckon, tound in al viral DNA torms Residual plasmid from
Iranaiart transtection cannot be deMctsd due lo Itie Cti^V-U5 riybnd on the viral plaxnid (b) The 2-LTR circM assay a shown with primers (t arid
r) and piate (p) on a 2-LTR orcle fragment with both LTRs The assay Oatecls 2-LTR cirdas via ttie unique US-U3 |uncticn (c) TT« integrated
DNA assay is a real-time nastad PCR assay Both rounds c^ ttie assay are shown on a tra^enl containing a part ot the provirus integrated into
t>ost DNA containing an Ati repetitve site Ttie assay uses repetitive Alu-sitss. dspeised within the liuman genome, lo delect integrated provinis
tn ttie '• round, the lonniard primer (lambda-M). tagged wnti an lambda phage heel sequence and the reverse Alu prmei (Alu-r) amplily a
tra^enl spanning tram the integtated provinjs to the tial adiacent Alu-sita In order to exclude unspecific amplification the 2" round specifically
ampliftas a proviral DNAha^enlby ii«ng alambda-ftael specific tonvanjpnma (12) nom »the rawerae pnmet(r2) and the probe (p2)
Figure 5c
Figure 3a
No unique 1-LTR circle detection by real-time PCR
Figure 3d
Figure 3b
Figure 3c
I laiafciii i-afc» I
(a) A revarta pnmar f) <h the gag and a tcniiwd prmv <f) m ma •ni'
regnn are uaed lor t-LTR ciicte dataction. smca 1-LTR aiclee have no
unique sH lor real-time PCR assay developmant In pnnocM these
pnmars amplity boffi circular episomal DNA lorms. bul t-LTR otclas can
be distinguished »cm 2-LTR orclM by amplicon sis (b) A PCP with the
1-LTR circle prmerswas partormadwith dflererl amsalirig temperatures
DNA aidracts of mtecled cells show correcl amplifcaton of an 1510 bp
fragment Plasmid controls shew an uriexpected false positive
•mplitication fragment of ttie same siz« (1 infection CTSafs IN« Bh pi 2
inlaction CTSe's IN< 144h pi 3 negative control * [iaimid pCTSals 1
ng. £ plumid pCTSats 1 pg) (c) A SYBR-^asn ran time PCR with the t-
LTR arOe prwners was pertormad A i 10 dikitcn senes of pCTSefs |1pg
- too ng) sanred as lemctfate Agan ampldtcaaon from the plasmids was
obaarvad The mMlng cume ana^sis it shown The change m
fluorescence with tamperalure on the y-ans is plotled agnnsl Vie
tvnperatura on the >-axis The melling curves do not show single peaks
associated with single meflmg temperatures ot ampHxns This inctcMes
detection ot (MIerenI tamplalaa and creation of various amplicons (d)
Saquancmg of plasmid pCT&efs with both t-LTR pnmars |l and r)
toMowM by an alisrvnant of ttie resulting ampticans ll-ampMxin and r-
ampkcon) againsl the map ol pCTSels (onty a IraTnenf is shown) pnmar*
Integrated DNAf tot« viral ONA t%]
F191« E (cohtmuad)
|a) Ttie results ot ttie total viral DNA assay sfioviM a petfi of total vital DNA accumulalon at 2* hous post intec
alxjndance of viral DMA decreesod until the and of Itie atpemsnl At all Ime points there was si^rticanlly more to
infected with the inte^alion profCMnt CTSsfs (b) The 2-LTR cirds assay showed a peali of 2-LTR circles at 24 hours »
abundance ol 2-LTR circles decreased, srniila' to the total vral DNA The 2-LTR assay showed no difference between the inte^aon
delioent and ttie inte^ation profiaant vectors nd in lunelics nor in abundance Hcwner. ewmining ttie 2-LTR parcantage showed thai up
to 70\ ot all vral DNA forms are 2-LTR cirdas in case ol the integration deticnnt virus, while the mteg-ation prolioent vinjs only snowad a 2-
LTR traction ot 5% (c) Ttie rniejated CNA assay showed ttiat hartfy any DNA of both vector types was integrated by B hours p 1 In oontein
of It« ntegralion dedoeni vector virtually no integrated DNA could be delected at any ol ttie lollowing Dme points Consequently mte^ation
protiCMnt CTSsIt prodces sqmtcartly high« prcmrut amount dinng the bme-courss with a peaK at «8 hours p 1 Relatng the irM^ated
DNA trom the inlev-aton protoant «actor lo the total vntf DNA reveals that pn:ivrus accumulates over bma unbl ovar 100% ol total viral DNA
IS inlesialad by 43 hours Mar inlackon Ttw ntagralad DNA assay rttfUwfaa hi^wr standard deviations compared to the other assays, which
decreases the accuracy of »baolute quantilication
We could develop a selol real->ma r>CR assays that can be usad 10 study abuiKlanca and staMily ol most ol the viral DNA lorms tiat occur m
f IV inlactad calls told wM DNA nlegralad DNA aid 2-LTR ciicles Howwer we were not aCle lo develop a real-time PCR assay that
uniquely detects and quanWies t-LTR crUes We could last ttia daweloped real-tme PCR assays n vtro in tme course inlaclion ttucMs m
the conlexl of iniegraton dehcivif and inteTitKn ptoliciait FIV vacKirs Ttie results presented here show ttie usability of the raal-tma PCR
assays regaidngconaequantial and'«UtMrasults. inadOaon. mate raauKs provKle msi^ns into the Ciolo(y ot ttie dftarant FIV DNA torms
118
Appendix
5.3. Acknowledgements
Zu allererst möchte ich mich ganz herzlich bei meinem Betreuer Prof. Dieter Klein bedanken,
dass er es mir ermöglicht hat diese Masterarbeit zu schreiben. Weiters möchte ich Prof.
Klein für seine Anregungen und seine große Unterstützung danken, als auch für seinen
persönlichen Einsatz bezüglich meiner Zukunft in der Wissenschaft. Nicht zuletzt hat er auch
großen Anteil daran, dass es mir ursprünglich ermöglicht wurde dieses Masterstudium zu
beginnen.
Ich möchte mich auch besonders bei Dr. Adolf Steinrigl bedanken, da er mich wunderbar
durch diese Arbeit geleitet und begleitet hat. Er war mit seinem Wissen stets für mich da und
stand immer mit Rat und Tat zur Seite.
Ich bedanke mich bei Reinhard ErtI, der mit seinem Können vor allem bei der praktischen
Arbeit immer eine große Hilfe war. Den weitern Mitgliedern des „Zimmer 17" möchte ich für
die gute Arbeitsatmosphäre und für alle vergnüglichen Kaffeepausen danken. Natürlich
danke ich auch allen anderen Mitgliedern des Instituts für Virologie, die mich alle so
freundlich aufgenommen haben!
Ich möchte diese Masterarbeit meinen Eltern Mag. Manfred und Eva Hofer widmen, denn
ohne sie würde es diese Arbeit nicht geben. Ich danke Ihnen ganz besonders herzlich für ihr
immerwährendes Vertrauen und ihre unendliche Unterstützung, ohne die meine Ausbildung
und letztendlich diese Arbeit unmöglich gewesen wären!
Ich möchte meiner Schwester Julia Hofer für die vielen Gespräche über das
„wissenschaftliche Leben" und ihre wertvolle Unterstützung danken! Mein Großvater hat das
Ende meines Studiums leider nicht mehr miterlebt. Ihm gebührt jedoch auch Dank für die
großzügige Unterstützung meiner Ausbildung.
Zu guter letzt möchte ich meinen Freunden und Studienfreunden danken, die das
(Studenten-) Leben erst so richtig erträglich gemacht haben und daher auch einen Anteil an
dieser Masterarbeit haben!
119
Appendix
5.4. Curriculum vitae
Name Matthias Hofer Bakk.techn
Date of Birth \6'^ of September 1981 in Linz, Austria
Nationality Austrian
Address A-5302 Henndorf, Moosstraße 36 A
E-Mail [email protected]
Telephone H-43-650-3032123
Education:
From To Type of Education
1988/89 - 1991/92 Primary school in Henndorf am Wallersee (Province of Salzburg, Austria)
1992/93 - 99/2000 Gymnasium in Salzburg (Province of Salzburg, Austria)
2000-09 - 2001-09 2 semesters of study-programme "Business Informatics", Johannes-Keppler-University, Linz, Austria
2002-03 - 2006-09 Bakk.techn.; Bachelor study-programme of "Food Science and Biotechnology", University of Natural Resources and Applied Life Sciences Vienna, Austria
2006-01 - 2006-06 Stay abroad with "Erasmus-Programme" on Linköping University, Linköping, Sweden
since 2006-09 Master study programme " Biomedicine and Biotechnology", University of Veterinary Medicine Vienna, Austria
Scientific Experience Record :
From To Type of Experience
2004-07 2004-09 Internship, Institute of Microbiology- Division Microbial Ecology, University Innsbruck, Austria
2006-06 2006-09 Bachelor thesis "Influenza Vaccine- Methods for Manufacture" Institute for Applied Microbiology, University of Natural Resources and Applied Life Sciences Vienna, Austria
2007-02 2007-02
2007-07 2007-09
Internships, Research Institute of Virology and Biomedicine, University of Veterinary Medicine Vienna, Austria
2008-01 & 2008-04 Tutor of students in university laboratory courses "Molecular Methods for Quantification" and "Virology lab course". University of Veterinary Medicine Vienna, Austria
2008-02 2008-09 Master thesis "Establishment and evaluation of real-time PCR assays for quantification of different DNA-forms of feline immunodeficiency virus". Institute of Virology, University of Veterinary Medicine Vienna, Austria
2008-08 Poster Presentation: Hofer M., Klein D., Steinrigl A. (2008), Establishment and Validation of Real-Time PCR Assays for the Quantification of Different Forms of Feline Immunodeficiency Virus DNA, 9"' International Feline Retrovirus Research Symposium 2008 in Vienna (Austria), 24.08 - 27.08.2008
120-
