Leukocytes Using an In Vitro Mode1 Aashish · 2020. 4. 7. · The Interaction Between Human Cytomegalovirus and Polymorphonuclear Leukocytes Using an In Vitro Mode1 of Study Aashish

The Interaction Between Human Cytomegalovhs and

Polymorphonuclear Leukocytes Using an In Vitro Mode1 of Study

Aashish Chakravertty

A thesis submitted in conformity with the requirernents

for the degree of Master of Science

Graduate Department of Laboratory Medicine and Pathobiology

University of Toronto

(9 Copyright by Aashish Chakravertty, 1999

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The Interaction Between Human Cytomegalovirus and Polymorphonuclear Leukocytes Using an In Vitro Mode1 of Study

Aashish Chakravertty Master of Science, 1999

Department of Laboratory Medicine and Pathobiology University of Toronto

ABSTRACT

Background: Human cytomegdovirus (HCMV) is a cornmon causative agent of

congenital viral infections in humans and an important pathogen in allograft recipients.

HCMV transmission has been associated with a number of body fluids, particularly

blood. The use of leukocyte filtered blood and blood products for transbion has

resulted in significantly reduced HCMV infection in allograft recipients. In patients with

active HCMV infection, detection of HCMV phosphoprotein pp65 inside

polymorphonuclear leukocytes (PMNL) is often used to follow the virus load. It is as yet

unresolved whether detection of pp6S and other viral antigens is a result of active HCMV

infection within PMNL, or detection is due to uptalce and accumulation of viral antigens

from infected cells through phagocytosis. The purpose of this study was to investigate

the interaction between HCMV and PMNL, usïng an in vitro mode1 of study.

Methods: H L 6 0 cells (human promyelocytic leukemia ce11 line) which are of rnyeloid

lineage, were stimulated with DMSO to obtain more mature cells. Unstimulatecl and

stimulated cens were inoculated with live and heat inactivated HCMV at various

multiplicities of infection (MOI). Cells were tested for the praence of HCMV

imrnediate-early (IE) antigen (Ag), pp65 Ag, and late Ag by immunofluorescence assays

(IFA).

They were M e r tested for HCMV DNA, mRNA, inféctiou viral progeny, and

examined for virai particles by electron microscopy (EM) and immunoelectron

microscopy (IEM).

Results: HCMV IE, pp65, and late Ag were detected in unstimulated and DMSO

stimulated HL60 cells inoculated with live or heat inactivated virus. Inoculated cultures

were positive for viral DNA. NASBA positive signals suggesting the presence of HCMV

late pp67 mRNA transcripts were detected withui inoculated cultures, as well as in the

original viral inoculurn. There was no evidence of infectious progeny virus within

inoculated cultures. Virus-like particles were detected by EM, but not to leve1s

characteristic of HCMV infection. Attempts at employing IEM for fÙrther identification

of viral particles was unsuccessful.

Conclusion: Our results provide some support for replication of HCMV in HL-60 cells

as demonstrate- by virai antigen and DNA detection, and support for the contrary (no

transmission of virus, EM work). Without M e r investigation, it is not possible to

definitively conclude the extent to which HCMV replicates in HL-60 cells.

ACKNOWLEDGMENTS

I would like to thank the following people in recognition of their contribution to this

thesis.

Firstly, my supervisor Dr. Tony Mamilli, who has provided me the opportunity to work

under his tutelage. Over the past few years, 1 have gained so much insight into the field

of virology and research, and would like to express my gratitude for the wealth of

h o wledge and guidance Dr. Maznilli has provided.

1 also wish to thank my cornmittee members, Drs. Joyce de Azavedo, Kelly MacDonald

and Martin Petric for their valuable advice and suggestions throughout my project.

Special thanks to George Moussa and Robert Chua for their laboratory expertise, Anna

Smargassio and the entire Department of Microbiology at Mount Sinai Hospital for ail

their assistance.

1 would like to close by thanking my parents Rita and Anin Chakravertty and my brother

Raja for their encouragement and support throughout my studies.

TABLE OF CONTENTS

LIST OF FIGURES ............................................................................................. *0008

LIST OF TABLES ............................................................................................... *.**9

........................................................................................ INTRODUCTION 10

............................................................................... PROJECT HISTORY ..IO

CYTOMEGALOVIRUS

............................................................................................ Characterization 1 4

................................................................................................. Epidemiology 14

............................................................................................. Clinical Disease 15

........................................................................................................ Latency 1 6

.............................................................................. HCMV Replicative Cycle 17

............................................................................... HCMV Gene Expression 18

...................................................................................... HCMV Inactivation -20

....................................................................... HCMV Permissive Ce11 Lines 22

TBE MYELOID LiNEAGE

.............................................................................................. Granulopoiesis -23

........................................................................................ Granule Production 25

....................................................................................... Neutrophil Function 27

Cytokines IL-8. IL.6. TNF-a and HCMV ..................................................... 30

.............................................................................. Culturing of Neutrophils 30

HL40 CELL LINE

............................................................. Establishment of HL-60 Ce11 Line 1

Cytologie Characteristics of HL-60 Cultured Cells ....................................... 31

.................................................................................. 1.4.3 General Differentiation 32

1.4.4 ModelofStudy .............................................................................................. 33

1 .4.5 Infectivity of HL-60 Cells .............................................................................. 35

2.0 OBJECTIVES m.m.......m..m...m........................m..mm...m.....m......m..m.....m..m....... ~ ~ o m o m m m o o ~ 6

MATERIALS AND METHODS

Propagation of MRC-5 Cells ......................................................................... 37

............................................................................. Propagation of HFF Cells -37

Propagation of HL-60 Cells ........................................................................... 37

.................................................. Induction of Differentiation of HL60 Cells 37

......................................................................................... Virus Propagation -38

........................................ Determination of Stock Viral Titre in TCIDSdml -38

...................................................... Inoculation of HL-60 Cells With HCMV 39

.............................................................................. HCMV Heat Inactivation -39

Detennination of Viral Titre in HCMV hoculated Cultures of .......................................... Unstimulated or DMSO Stimulateci HL-60 Cells 39

Immuno fluorescence Assa y (FA) For Detection of HCMV ........................................................... 1mmediate.Early. pp65. Late Antigen 40

HCMV Nucleic Acid Isolation and PCR ....................................................... 41

Determinhg the Lower Limit of Detection of PCR ...................................... 41

HCMV pp67 mRNA Detection ..................................................................... 42

........... Electron Microscopy (EM) Analysis of HCMV Inoculated Cultures 43

Emmunoelectron Microscopy (IEM) Analysis of HCMV Inocdated ......................................................................................................... Cultures -44

IL.6, IL.8. TNF-a Stimulation ...................................................................... 46

RESULTS

Effect of HCMV Inoculation on HL-60 Ce11 Cultures .................................. 47

Dose Dependent F A Response ..................................................................... 48

Variable lFA Success ..................................................................................... 48

pp67 mRNA Transcript Detection ................................................................. 48

hfectious Progeny Not Produced in IFA Positive Ce11 Cultures .................. 49

............................................................................ Electron Microscopy (EM) 54

............................................................. Immunoelectron Microscopy (IEM) .59

.................................... Effect o f IL-6, IL-8, TNF-a on HL-60 Ce11 Cultures 60

Figure t Proposeci differentiation sequence (restriction potentialities) of ............................. the pluipotential hematopoietic stem ce11 CFU-S 26

Figure 2 The effect of increasing MOI on percentage of cells IFA positive .......................................................................... for late HCMV Ag ..S 1

Figure 3 Success rate of HCMV late antigen detection within inoculated ............................................................................ HL-60 ce11 cultures 52

Figure 4 Electron micrograph of a HCMV inoculated HFF ceIl 4 days ................................................................................ ps t inoculation. -57

Figure 5 Electron micrograph of a HCMV inoculated HL-60 ce11 4 days .................................................................................. ps t inoculation 58

Figure 6 Immunoelecm>n micrograph dernonstrating non-specific binding ......................................... of conjugated gold particles to HFF cells. 62

Figure 7 Proposed mode1 of interaction between HCMV and a HL-60 ce11. ... 68

LIST OF TABLES

Table 1 Detection of HCMV antigens and DNA within inoculated HL-60 .................................................. ce11 cultwes 4 days post inoculation 50

Table 2 Sumrnary of IFA, PCR, and NASBA analysis using live or heat inactivated HCMV as inoculum with HL-60 ce11 cultures 4 days

........................................................ post inoculation. .................... ... 53

Table 3 Determination of viral titre in HCMV inoculated IFA positive HL-60 cells and DMSO stimulated HL-60 cells 4 days

................................................................................ post inoculation.. 55

Table 4 Defining features of HCMV infection by electron mimswpy.. ....... 56

Table 5 Summary of IEM atternpts using HFF cells uninoculated and Inoculated with HCMV, 4 days p s t inoculation ............................... 6 1

1 . Q ) I N T R O D U C T I O N

Background knowledge relating to the studies performed in this thesis is selectively

summarized in this introduction under the following headings :

Project History

Cytomegalovirus

The Myeloid Lineage

HL-60 Ce11 Line

1.1) HCMV PROJECT HISTORY

Leukocytes have been shown to be the main vehicle through which human

cytomegalovinis (HCMV) is transmitted during transfusion. The use of leukocyte

filtered blood and blood products for transfusion results in significantly reduced rate of

HCMV infection in the recipient (De Witte et al., 1990; Winston et al., 1980). In patients

with active HCMV infection, the presence of HCMV phosphoprotein 65 (pp65) inside

the nuclei of pol ymorphonuclear leukocytes (PMNL) (antigenemia assay) (The et al.,

1990; Veal et ai., 1996) is often used to assess the viral load (The et al., 1995).

Furthmore, quantitative detedon of pp65 antigen has been shown to be an effective

diagnostic approach for monitoring the progress of HCMV disease (Ranger-Rogez et al.,

1996). A number of studies have been wnducted on the detection of HCMV pp65 Ag in

PMNL fkom transplant patients and H N positive patients, with symptomatic HCMV

infection (Boeckh et al., 1992; Erice et al., 1992; Veal et al., 1996; Spaete et al., 1994;

van der Bij et al., l988a; Grefle et al., 1992; van der Bij et al., l988b; Zipeto et al., 1992;

Boland et al., 1992; Grefle et al., 1994). Since pp65 Ag was detected during active

HCMV infection within PMNL, it was hypothesized that HCMV wuld infect PMNL,

which would lead to active gene expression and production of viral pp65 Ag. Along with

detection of the early-late pp65 Ag, immediate-early (IE) Ag and HCMV late Ag have

been detected in PMNL from patients with active HCMV infection (Revello et al., 1992;

Gema et al., 1990; Sinzger et al., 1995). In addition, HCMV IE Ag has been detected in

fibroblast cells, inoculated with PMNL f5om viremic irnmunosuppressed patients (The et

al., 1990; Dankner et al, 1990). A more recent study has found pp65 Ag in PMNL upon

CO-culturing with HCMV infected fibroblasts or infect4 endothelial cells, and these

PMNL were shown to transmit infectious virus upon CO-culturing with fksh fibroblast

cells (Revello et al., 1998). Virus has also been isolated fiom PMNL preparations h m

infected patients (Gema et al., 1992).

Additional support for the concept of active HCMV infection of PMNL is

provideci by detection of viral DNA in the cells. HCMV DNA has been detected in

PMNL of post transplant recipients (VeaI et al., 1996; Dankner et al., 1990; von Laer et

al., 1995; Gerna et al., 1992; Saltzman et al., 1988; Zipeto et al., 1992; Boland et al.,

1992) as well as those h H N positive patients (Veal et aL, 1996; Gema et al., 1992;

Boivin et al., 1998; Zipeto et al., 1992; Gozlan et al., 1993). Detection of HCMV DNA

alone, does not correlate with active HCMV infection, since the presence of the virus in a

latent state is always a possibility. However, deteftion of viral DNA, dong with

detection of antigens or mRNA transcripts, provides stmng evidence for active Wal gene

expression. The best indicator for expression of the HCMV genome is the presence of

mRNA transcripts in PMNL. Gozlan and colleagues (1993) detected HCMV late mRNA

in PMNL fiom HIV-infected patients. In a subsequent study, IE mRNA and late mRNA

was detected in PMNL of rend transplant patients with active HCMV infection (von Laer

et al., 1995). Dankner and colleagues (1990) were able to detect HCMV IE, early and

late mRNA transcripts in PMNL of irnmunosuppressed patients, thus demonstrating gene

expression f?om al1 three phases of HCMV replication.

Along with detection of HCMV DNA, mRNA transcripts and antigens h m d l

phases of the replication cycle, intact virus particles have been obsewed in PMNL by

elecwn mimscopy (Martin et ai., 1984). Although the literature Qted thus far provides

strong support for active HCMV gene expression and viral replication within PMNL of

the susceptible host, other studies on the interaction of HCMV and PMNL, have fond

conflicting results.

In a study by Sinzger et al. (1995) only the 72K IE Ag but not early or late Ag

were detected in PMNL consistent with an abortive infection. Turtinen and colleagues

(1987) were able to detect HCMV DNA in the cytoplasm of PMNL but were unable to

detect any viral mRNA transcripts. In a study reported by Grefie et al. (1992) both IE Ag

and pp65 Ag were detected in PMNL, but only IE mRNA was detected in PMNL and

there was no detection of pp65 -A. By contrast, both IE and pp65 mRNA were

detected in late stage HCMV infected endothelid cells (Grefte et al., 1994). Since pp65

is an early-late HCMV protein, this finding is suggestive of restricted expression of the

viral genome and acquisition of pp6S by means other than de novo synthesis. As

mentioned previously, HCMV virions have been detected in PMNL by electron

microscopy (Martin et al., 1984; Turtinen et al., 1987). Specifically, detection has been

localized to phagosomes and there has been no detection of intranuclear virions or

nucleocapsids, suggesting HCMV was not actively repticaîing inside PMNL.

These studies have provided important insights in our attempt to trying to explain

the observed in vivo interaction between PMNL and HCMV. in this context, there are

findings which support the concept bat PMNL are conducive for complete HCMV

replication and gene expression (Revel10 et al., 1992; Gerna et al., 1990; Suizger et al.,

1 995; Gozlan et al., 1 993; von Laer et al., 1 995; Danhier et al., 1990). Other f idhgs

support the concept that PMNL are conducive for only resmcted viral gene expression

(Sinzger et al., 1995; Gerna et al., 1992; Grefte et al., 1994). Therefore, while the

interactions between HCMV and P M N L rernains undear, m e r investigations are

warrant ed .

1.2.1) CHARA CTERI24 TION

Cytomegalovinrs (CMV), formally designated as human herpesvirus 5 (HHV-5),

is a rnember of the betaherpesvinis group. This DNA virus has the largest gemme of the

herpesvinises (230kb-240kb). It is characterized by a relatively slow replication cycle

which lads to the production of large, often multinucleated cells (Mocarski, 1996).

Similar to other herpesvinises, primary infection is followed by a latent infection.

Ubiquitous in nature, CMV is highly species-specific. A wide range of animal species

including humans, other primates, domestic animals and rodents have an associated CMV

(Mocarski, 1996). Humans are the only known reservoir for human cytomegalovinis

(HCMV). Transmission of HCMV has been associated with close personal contact

(saliva, sexual contact), vertical transmission (in utero, breast milk), organ and tissue

transplant, including blood transfusion. HCMV has been isolated fiom a number of

body fluids including oropharyngeal secretions, urine, cervical and vaginal secretions,

semen, breast milk, tears, feces and blood (Britt and Alford, 1996).

1.2.2) EPIDEMIOLOGY

In North Arnerica, HCMV infects approximately 50% of the population, with

infection rates rising to 90% within some urban centres. HCMV is the most common

congenital viral infection in humans. Data fkom the United States indicates that 0.2%-

2.2% (-40 000) of infants bom each year are infected in ufero. In cases where HCMV

disease ensues, damage to perceptual organs accounts for the majority of morbidity

associated with intrauterine HCMV infection. Long-term neurologic sequeiae may occur

in up to 10- 15% of these infected infants, with hearing loss comprising the most cornmon

neurologic abnorrnality. Between 8% and 60% of al1 infants are infeçted during the first

6 months of life, as a result of HCMV transmission via breast milk (Bntt and Alford,

1996).

1.2.3) CUNICAL D I S E S E

HCMV infection is usually asymptomatic in the healthy host. Occasionally,

infection may present as a mononucleosis-like syndrome, similar to that resulting from

infection with Epstein-Barr Virus (EBV) @ritt and Alford, 1996). Infiequent

complications of HCMV mononucleosis include pneunonia, hepatitis, CNS involvement

(Guillan-Barré syndrome) as well as aseptic meningitis. HCMV disease, resulting fiom

primary infection or reactivation of latent infection, within the immunocompromised and

irnmunosuppressed host, is a serious concern. With severe disseminated disease, HCMV

involvement can be identified in virhially al1 organ systems. Vinuia, resulting fkom

urinary tract viral replication is a consistent feature of HCMV infkction in al1 age groups.

Viruria occurs in both noxmal or immunosuppressdi~~l~~luno~~rnpromised hosts and can

occur during primary HCMV infection, or reactivation of latent HCMV.

HCMV is an important pst-transplant pathogen in allograft recipients. Sources

of infecting virus include the transplanted organ, blood products used during the

procedure, and reactivation of the recipient's endogenous virus. The degree of

immunosuppression in the recipient correlates with an increased risk of HCMV infection.

HCMV infections have been described in solid organ allografts involving the

kidney, liver, heart and heart-lung. Between 60% and 100% of renai, cardiac and hepatic

allograft recipients develop HCMV infection (Bntt and Alford, 1996). A numba of

clinical syndromes can arise due to HCMV infection in the pst-transplant period.

Prolonged fevers, leukopenia, as well as life-threatening complications such as severe

infection of the GI tract, hepatitis and pneumonia have been documenteci. Within bone

marrow transplant @MT) patients, HCMV is a significant cause of death. Mortality rates

can approach 80-90%. Pneumonîa is the most commonly associateci syndrome resulting

in mortality when left untreated (Bntt and Alford, 1996).

HCMV is a major opportunistic infëction encountered by patients with AIDS. In

the era before highly active antiretroviral therapy (HAART), autopsy studies have shown

90% of patients with AIDS develop active HCMV infection and up to 40% may develop

sight andor life-threatening HCMV disease. HCMV has been proposed as a cofactor in

the pathogenesis of HIV infections (Barry et al., 1990). Clinical syndromes with HCMV

have included disease in essentially every organ; the most clinically significant involve

the eyes, Iung, CNS, and GI system.

1.2.4) LA TENCY

ï o date, much work has been conducted in trying to identie the site(s) of latency

of HCMV. Early studies suggested T-lymphocytes were the primary site of latency (Rice

et al., 1984). The wai genome has been detected in peripheral blood monocytes and

macrophages of healthy carriers, thus suggesting these cells rnay also be a primary site of

latency (Taylor-Wiedeman et ai., 1991, Fish et al., 1995). Animal models have been

used to try and characterize CMV latency. Using a mouse model, endothelid cells, lung

alveolar macrophages and bone marrow cells have been identified as harbouring murine

CMV DNA in latently infected animals (KofEon et al., 1998). A recent study has

identified HCMV latency-associated transcripts within a small percentage of myeloid-

type progenitor cells and dendritic cell progenitors in the healthy seropositive host @abn

et al., 1998). Thus, hematopietic cells may be an important reservoir for HCMV and of

significant importance in reactivation of latent virus.

1.2.5) H C W REPLICA TIVE CYCLE

There are a number of key steps involved in the HCMV replicative cycle, which

have been elucidated from studies of HCMV infection of permissive fibroblast cell lines.

This process is initiated by attachment of the virus to a host cell receptor followed by

penetration and uncoating of the viral nucleocapsid (White et al., 1994). In fibroblast

cells, viral envelope glycoprotein B (gB) and glycoprotein H (gH) have been shown to

interact with host proteins annexin 11, heparan sulfate proteoglycans, and a 92.5 kDa

receptor (Compton, 1995; Pietropaolo and Compton, 1997; Keay et al., 1989; Keay and

Baldwin 1982). Virus penetration proceeds by pH independent fusion of the viral

envelope and the host cell plasma membrane, thus releasing the nucleocapsid into the

cytoplasm (Compton, 1995). The HCMV genome has three temporally distributed

classes of genes. Once the HCMV DNA is released inside the cell, transcription of

HCMV immediate early genes (IE) within the nucleus is initiated by the host cell RNA

polymerase 11. The temporal distribution of HCMV gene expression is M e r discussed

in section 1.2.6. Transcription of late genes is dependent on viral DNA replication. In

fibroblast cells, HCMV DNA replication is observed 14-1 6 hours post inoculation @.i.)

and is mediated by a v d l y encodeci DNA polymerase (Mocarski, 1996). Transcription

and translation of late proteins provides the structural components necessary for virion

assembly. The HCMV genome is packaged into prefomed nucleocapsids in the nucleus

(Mocarski, 1 996).

Vinons budding fiom the nucleus probably acquire an envelope from either the

i ~ e r nuclear membrane and accumulate in the perinuclear space, then move into

cytoplasmic vesicles (Mocarscki, 1996). The mechanism by which infectious virions are

released upon assembly and maturation remains unclear. It is known that HCMV is a

relatively cell-associated virus and at very late stages post infection, roughly half of the

progeny virions may be found in the culture media, with the remainder being cell-

associated.

1.2.6) HCW GENE EXPRESSION

Specific genes of HCMV are expressed at different times throughout infection.

These genes fa11 into three major classes. The earliest genes to be expressed are the a or

imrnediate-early (IE) genes which are transcribed independent of both viral DNA

replication and translation of other viral gene products. Similar to al1 herpesvinises,

expression of f3 (early) genes, and y (late) genes requires synthesis of fünctional IE gene

products. The principle XE gene product is a 491 amino acid phosphoprotein of molecdar

weight 72 kd (pp72). pp72 regulatory activities include autostimulation of its own

synthesis as well as a tram-activator of early and late gene expression (Mocarski, 1996).

In fibroblast cells, pp72 has been detected as early as 20-30 minutes (pi.). It has been

shown to associate with cellular chromatin and to be synthesized throughout infection.

However, transcript levels are lowest at the onset of viral DNA replication.

Generally, f3 gene transcripts are expressed later and expression continues into

late stages of infection. This class of genes cm be divided into subclasses based on their

time of expression. pi genes have been detected as early as 4-8 hours p.i. while $2 genes

can be detected 8-24 hours pi. Phosphoprotein 65 @p65), e n d e d by a f3 gene is an

early-late protein produced in the replicative cycle of HCMV. It is located in the

tegument region between the capsid and envelope of HCMV virions (Mocarski, 1996). It

has 561 amino acids and has been shown to be highly immunogenic. Furthemore, it is

the most abundant tegument protein, accounting for 95% of the mass of this structure.

Within infected cells, pp65 serves as a major phosphate acceptor and it is the primary

target for phosphorylation in vitro by the viral protein kinases. in addition, pp65 has

been hypothesized to have an important role in vivo since it is the principle target of

cytotoxic T lymphocytes (Da1 et ai., 1996). pp65 carries two nuclear localization signais

and its accumulation within the nucleus suggests a role within the nuclei of infected cells

(Mocarski, 1996). In vitro studies using fibroblast cells have shown that pp65 is

nonessential for growth of HCMV (Schmolke et al., 1 995). However, studies involving

astrocytoma cells indicate that detection of extracellular progeny was alrnost completely

blocked by inhibiting pp65 expression @al et al., 1996). Thus, the intracellular h c t i o n

of pp6S remains unclear.

As with early genes, y genes c m also be divided into subclasses. y1 transcript~ can

first be detected 12-36 hours p.i. and y2 transcripts at 2448 hours p.i. The majority of y

genes encode structural proteins. At least 30 proteins have been detected in mature

HCMV vinons, distributed in the envelope, tegument and capsid. Phosphoprotein 67

(pp67) is an example of a viral structural protein produced late in infection. The role of

pp67 is unclear, however this 102 amui0 acid tegument protein may have some

involvement with protein kinase activity of the Mnis (Mocarski, 1996).

The virion envelope carries a minimum of 8 glycoproteins organized in three or 4

giycoprotein complexes while the tegument contains as many as 20 proteins (Mocarski,

1996). In HCMV the major capsid protein (MCP) is a 1,370 amino acid protein of

molecular mass 150 kd. While MCP shares substantial arnino acid sirnilarity with the

major capsid protein (VPS) of HSV- 1, the HCMV MCP exhibits low imrnunogenicity.

1.2.7) HCMV INACTIVA TION

The HCMV envelope is a lipid bilayer membrane composed of host lipids and

viral proteins. The envelope is derived fiom either the nuclear or cytoplasmic

membranes, including early endosomal membranes (Mocarski, 1996). Biological

membranes are not rigid; lipids and membrane proteins are constantly in lateral motion

(Stryer, 1988). Membrane fluidity is mediatecl by the fatty acid composition and

cholesterol content of the membrane. Lipid bilayer membranes c m exist in an ordered or

rigid state, as well as a disordered or fluid state. The transition between the two states is

determined by the transition temperature, which is dependent upon the length of the fatty

acyl chains, and their degree of unsaturation. Shorter fatty acyl chains md double bonds

interfere with a highly ordered packing of fatty acyl chains and thus the transition

temperature is lowered. Membrane fluidity is moderated by choloesterol. Cholestml

prevents the crystallization of fatty acyl chains by fitting between them, and sterically

inhibiting large motions of fatty acyl chains, remdering the membrane more rigid (Stryer,

1988).

Heat treatment has been used as a mode of inactivating a wide variety of viruses.

Hurnan imrnunodeficiency virus (HTV) infectivity has been reduced to minimal levels

(several log decrease in titre) when subjected to 56OC heat treatment for 20-30 minutes

(McDougal et al., 1985; Spire et al., 1985; Resnick et al., 1986). Similarly, Sindbis

virus, polio virus, pseudorabies and H N were inactivated when subjected to heaî (Estep

et al., 1988). Heat treatment has been used to inactivate HCMV viral stocks (Almeida-

Porada et ai., 1997; Valyi-Nagy et al., 1988). Heat treatment of 56°C for 30 minutes

results in membrane fluidization of H N (Aloia et al., 1988). increasing the membrane

fluidity above a critical minimum decreases HIV viability. in a study conducted using

vesicular stomatitis virus, treatment which removed cholesterol resulted in fluidization of

the viral membrane and reduced infectivity of the virus (Moore et al., 1 978). Sirnilarily,

butylated hydroxytoluene (BHT)' a fluidizing drug, inactivates lipid enveloped DNA and

RNA viruses in vitro (Aloia et al., 1988). BHT is an additive wideIy used in human and

animal foods to maintain freshness and avoid spoilage by delaying degradation of lipid

components of food (Kim et al., 1978). BHT has been previously shown to greatly

rduce numbers of infectious HSV Miions (Kun et al., 1978). BHT, used at

concentrations known to have no biologically adverse effects when used as an additive,

was able to inactivate HCMV. Thus, heat treatment, reducing the cholesterol content, or

the use of BHT results in membrane fluidization leading to a loss of integrity of the viral

envelope (and associated receptors and ligands), and the observed inactivation of virai

infectivity .

1.2.8) HCMV Pennksive Cell tines

Depending on its nature, the host ce11 may be eitber permissive or restrictive for HCMV

infection (Burd et al., 1996). In a restrictive or non-permissive ceii line, the viral

replication cycle may be blocked at any point nom attachment through to the final stage

of assembly and release (White and Fenner, 1994). HCMV exhibits a highly restricted

host range in ce11 culture with skin or lung fibroblasts being the preferred host ce11

(Mocarski, 1 996). Undi fferentiated as well as transformed ceil lines are generaily non-

permissive (Mocarski, 1 996). A number of reports have identified otha permissive ce11

lines for HCMV infection in vim, however, ce11 types other than fibroblasts do not

support viral replication to the same degree (Mocarski, 1996). Brain endothelid cells

have been shown to support complete viral gene expression and cytopathic effect (Poland

et al., 1990) whereas astroglial lines Vary in their ability to support HCMV replication.

For exarnple, the T98G ce11 line supported incomplete (immediate-early) gene

expression, HS-683 supported extensive virus replication with minimal viral CPE, while

A-172 did not support any detectable gene expression (Poland et al., 1990). Neuronal

ce11 lines (SK-N-MC) were found to be fully permissive to HCMV infection (Poland et

al., 1990), and this was probably related to the state of differentiation of the cell, and to

the transcriptional regdatory molecules present in the ce11 at the time of infection (Burd

et al., 1996). Infectious virus was detected in glioma and neuroblastoma cells inoculated

with HCMV in vitro (Ogura et al., 1 986). HCMV infection of astrocytoma cells has been

shown with expression of IE, early and late antigens as well as production of extracellular

infectious virus (Duclos et al, 1989). While HCMV antigens have been detected within

PMNL, it is unclear whether these cells support active HCMV replication.

13) THE MYELOID LINEAGE

Z. 3.1) GRANClzOPOIESIS

In the life of the neutrophil, it passes through three phases: bone m m w , blood

and tissue. Within the bone marrow, there is a mitotic cornpartment and a non-mitotic

storage compartment. Most of the neutrophils in the body reside in the bone m m w

(Newburger and Parmley, 1 99 1 ). Once neutrophils are released into the blood, they have

a half-life of 6-9 hours and irreversibly move fiom circulating to marginating pools

(Newburger and Pannley, 1991). These granulocytes irreversibly leave the blood by way

of diapedesis between endo thelial cells and penetration of the basement membrane.

Arnong the mechanisms responsible for neutrophil ce11 death is programmed ceIl death or

apoptosis (Newburger and Parmley, 199 1).

Granulopoiesis begins with the pluripotential stem ce11 CFU-S (an acronym for

colony foxming unit-spleen) (Keeling, 1987). Most of these cells are in the resting phase,

but of these, a small fiaction differentiate into committed stem cells. A proposed

differentiation sequence of the pluripotential stem ce11 is outlined in Figure 1.

Granulopoiesis follows successive differentiation fiom the pluripotential stem ce11 to

committed stem ce11 CFU-GM (colony fonning unit-granulocyte-macrophage), followed

by further differentiation to committed stem cell CFU-G (colony fonning unit-

granulocyte).

The myeloblast is the first recognizable ceIl of the granulocytic series (McDonald

et al, 1 98 8; Keeling, 1 987; Jandle, 1 987). Differentiation of the myeloblast results in the

successive development of the promyelocyte, myelocyte, metamyelocyte, band

neutrophil and finaliy the terminal1 y di fferentiated segrnented neutrophil. The myeloid

maturation sequence is characterized by progressive decrease in nuclear size, early

presence of nucleoli and subsequent disappearance, early appearance of azuophilic

granules, late appearance of specific granules, as well as nuclear indentation and

segmentation (Beck, 1985)-

Myeloblasts represent the earliest stage of myeloid development within the

mitotic cornpartment of the bone mamw (Newburger and Panniey, 1991). They can

give rise to neutrophils as well as eosinophils and basophils and hence lack morphologic

feahires that predict their differentiation into one of the granulocytic lines (Newburger

and Parmley, 199 1). Myeloblasts do not contain any granules and have a roundoval

nucleus occupying 415 of the total ce11 area (McDonald et al., 1988). Pmmyelocytes are

at the midstage of development within the mitotic cornpartment, and resemble

myeloblasts except for the presence of azurophilic granules in the cytoplasm (McDonald

et al., 1988). Myelocytes are the first cells containing secondary or specific cytoplasmic

granules which are either neutrophilic, eosinophilic or basop hilic in nature (McDonald et

al., 1988; Keeling, 1987; Beck, 1985). Myelocytes also represmt the 1 s t stage of

development in the mitotic cornpartment (Newburger and Parmley, 1991). The

myelocyte is smaller than the promyelocyte and nucleoli are no longer present (Beck,

1985). Metamyelocytes as well as band and segmented neutrophils represent nondividing

cells within the bone m m w (Newburger and P a d e y , 1991). Metamyelocytes are

characterized by a slightly indented nucleus and persistence of secondary granules

(McDonald et al., 1988; Keeling, 1987). Further nuclear diffaentiation is evident in the

band neutrophil by the presence of a "u-shaped" nucleus. The band neutrophil is the first

of the neutrophilic line that can be identified in normal circulating blood (Keeling, 1987).

Finally, the temiinally differentiated segrnented neutrophil is the end product of the

granulocytic series. It is distinguished fiom its predecessor by its multi-lobular nucleus

~ ~ ~ e ~ t e d by thh strands of chromatin (keiing, 1987).

1.3.2) GRANULE PRODUCTION

D i f f m t types of hurnan neutrophil granules are formed sequentially d u d g

maturation of neutrophils h m the promyelocyte stage, to the band ce11 stage, and thus

c m be used as markers of myeloid differentiation. Specifically, two types of proteins are

stored within these granules: intragranular proteins which are liberated fiom the ce11

during exocytosis of the granule, and granule membrane proteins which are incorporated

into the plasma membrane (Le Cabec et al., 1997).

In a classification system based upon the content of myeloperoxidase, there are

two types of granules that are distinguishible in human neutrophils: peroxidase-positive

granules and peroxidase-negative granules (Beck, 1985). The peroxidase-positive

granules, othenvise termed azurophilic or primary granules, contain lysosomal hydrolytic

enzymes, neutral proteases, myeloperoxidase, glycoaminoglycans, acid phosphatase,

cationic bactericidal proteins, and lysozyme (Beck, 1 985).

1 Diff eren tiation

CFU-E

CFU-M

Figure 1. Proposed differentiation sequence (restriction of potentialities) of the pluripotential hematopoietic stem cell CFU-S. Diffmtiation follows a specific sequence of successive restriction of potentialities with each of the unlabeled cells on the left retaining al1 potentialities except those lost above it. CFU, colony forming unit; M, macrophage; EO, eosinophil; GM, granulocyte-macrophage; G, granulocyte; MEG, megakaryoc yte; E, erythroid. (Modi fied fiom Nicola NA and Johnson GR: Blood l982;60(4): 101 9- 1029.)

They are subdivided into those which are defensin-rich or defain-poor (Le C a b et

al., 1997). There are two subsets of peroxidase-negative grandes, classifieci according to

their lactoferrin and gelatinase content: specific or secondary granules, and gelatinase or

tertiary granules (Le Cabec et al., 1997). Specific grandes contain lysozyme, l ac to feh

and vitamin Bit-binding protein while the gelatinase or teritiary granules are named as

such due to their gelatinase content (Le Cabec et al., 1997; Bak, 1985).

Synthesis of granular proteins is dependent upon the matunty of the myeloid d l .

Azurophilic granular proteins are synthesized at the promyelocyte stage, specific granular

proteins are synthesized at the myelocyte/metamyelocyte stage, and gelatinase is

synthesized mainly at the band ce11 stage (Le Cabec et al., 1997; Newburger and

Parrnley, 199 1 ).

1.3.3) NEUTROPHIL FUNCTION

Neutrophils are responsible for much of the body's defense against bacterial

invaders through the process of phagocytosis (Keeling, 1987; Beck, 1985). The essential

hc t ion of the neutrophil is to rapidly move to a site of rnicrobial invasion, engulf, and

kill the microorganism (Newburger and Pannley, 1991). Phagocytosis is a cornplex

series of events involving chemotaxis, rewgnition, engulhent, and killing of the

organism (Keeling, 1987). Chernotaxis of neutrophils is an inducible interaction arising

fkom immunologie as well as nonimmunologic pathways (Keeling, 1 987). Activated

complement wmponents C3a and CSa, generated as part of the cascade of the classical

complement pathway or the alternate complement pathway, are chernotactic for the

neutrophil (Keeling, 1987; Abbas et al., 199 1 b).

The kinin-generating system, as well as the clotting system dso provide appropriate

stimuli for neutrophil recniitment (Jandle, 1987). Neuîrophils respond to chernotactic

stimuli by following a direct path towards the stimulus with ameboid movement

(Keeling, 1987).

A critical step in the phagocytic process is recognition of the foreign particle.

Phagocytosis occurs more rapidly and efficiently when particles to be engulfed are

opsonized (Keeling, 1987). Neutrophils possess surface receptorj for c~mplement C3 as

well as receptors for the Fc fiagrnent of s e m antibodies (Gallagher et al., 1979). Fc

receptors appear to trigger ingestion while C3 receptors are more involved in binding of

the opsonized particle to the neutrophil (Beck, 1985). Once attachrnent occurs, the

neutrophil extends pseudopods which surround the foreign particle thereby engulfhg it in

a temporary storage vacuole or phagosome (Keeling, 1987; Beck, 1985). Killing of

ingested organisms is achieved by the fusion of cytoplasrnic granules with the

phagosorne, and subsequent discharge of granular contents into the phagosome, a process

known as degranulation (Newburger and Parmley, 199 1 ; Keeling, 1987; Beck, 1985).

A number of pathways exist for killing ingested organisms. There are oxygen-

independent mechanisms which are darnaging to various bacteria. Some include the

acidic pH of the phagosome, exposure to lysozyme which hydrolyzes the glycosidic

bonds of ce11 wall peptidoglycans, and secretion of bactericidal proteins which can alter

the membrane permeability of bacteria (Newburger and Parmley, 199 1 ; Beck, 1985).

Also, defensins, which are cysteine-nch cyclic polypeptides, are cytotoxic to

metabolically active bacteria, fun@ and enveloped Wuses (Newburger and Pannley,

199 1 ). By in large however, the killing of ingested organisms is primarily achieved by an

oxygen-dependent process (Newburger and Parmiey, 199 1; Keeling, 1987; Beck, 1985).

During ingestion, neutrophils actively metabolize oxygen to produce reactive

products that are toxic to ingested bacteria (Newburger and Pannley, l99 1 ; Beck, 1985).

Upon activation of neutrophils, by chernotactic stimuli or attachment of particles, there is

a dramatic increase in oxygen consumption referred to as the "respiratory burst"

(Keeling, 1987). Al1 of the oxygen taken up during the respiratory burst initially foxms

superoxide (O2>. 0; is the first reduction product of oxygen and is highly reactive

resulting in m e r reduction to hydrogen peroxide (H202) (Beck, 1985). While

superoxide inactivates Mnxses, damages plasma membranes and is capable of killing

cells, hydrogen peroxide is probably more important for bactericidal activity (Be&

1985). H202 is directly bactericidal and is also involved in bacterial killing via the

myeloperoxidase-halide ion system (Keeling, 1987). Reactions between hydrogen

peroxide, myeloperoxidase and chloride produces toxic hypochlorous acid as well as

toxic chloroamines (Newburger and Pannley, t 99 1 ; Keeling, 1987). Therefore, the

neutrophil is structurally and fùnctionally equipped for its important task of rnigrating to

infections, recognizing the infectious agent, and killing the invading organism. Defects

in any of these pathways result in altered defense against pathogens (Newburger and

Parmley, 199 1).

1.3.4) CYTOKlh?ES IL-8, IL-6, TNF- a AhD ACMV

Cytokine production in vivo has been associated with increased pathogenesis of

HCMV infection and disease (Humar et al., 1999). HCMV infection of fibroblasts and

endothelid cells have shown upregulation of interleulrin-8 (IL-8) levels (Gnindy et ai.,

1998; Craigen et al., 1997). increased IL-8 levels have resulted in recruitment of

neutrophils to sites of endotheliai infection as well as significantly enhanced

transendothelial migration (Gnrndy et al., 1 998; Craigen et ai., 1 997; Humar et al., 1999).

IL-8 induction has been described in vitro with a number of viruses such as hepatitis B,

influenza A, respiratory syncytial virus, measles virus, as well as herpes simplex virus

(Craigen et ai., 1997). HCMV Section has been shown to upregulate interleukin-6 (IL-

6) production. Primarily fhctioning as a B lymphocyte growth and diflerentiation factor

and an activator of T lymphocytes (Abbas et al., 199 1 a; Geist and Dai, 1 W6), IL-6 dong

with HCMV have been irnplicated in lung allograft rejections. Turnor necrosis factor

alpha (TNF-a) is a potent activator of neutrophils as part of the uiflamrnatory response

(Abbas et ai., 199 1 a). TNF-a induces the HCMV IE gene promoter and thus may be

involved in reactivation of latent HCMV infection (Humar et al., i999; Fietze et al.,

1 994; Stein et ai., 1993; Docke et al., 1994). Therefore, cytokine induction/production

appears to be closely associated to HCMV infection.

1.3.5) CUL TURING OF NEC/TROPHILS

Band neutrophils and segmented neutrophils can be detected in nomal circulating

blood (Keeling, 1987). Once neutrophils are released into the peripheral blood, they

have a half-life of 6-9 hours (Newburger and Parmiey, 1991), and have a defined

lifespan; 4-5 days in nomal tissue, shorter in sites of infection and inflammation

(Keeling, 1987). Since the replication cycle of HCMV in a known permissive fibroblast

ce11 line is slow, 48-72 hours (Mocarski, 1996), culNnng of neutrophils for in vitro

studies is problematic. Thus, identification and use of a myeloid ce11 line possessing

similar morphological and fimctional characteristics as neutrophils would be best suiteù

for conducting in vitro HCMV inoculation studies. The HL-60 ce11 line allows for the

development of such a modet.

1.4) KL-60 CELL LINE

1.4.1) ESTABLISHMENT OF HL-60 CELL LllVE

Peripheral blood leukocytes were obtained by leukophoresis fiom a 36-year-old

caucasian female diagnosed with acute promyelocytic leukemia (Gallagher et al., 1977).

Leukocytes were initially cultured in the presence of conditioned medium fiom human

ernbryonic lung cet1 cultures, but it was subsequently found that continued growth of

leukocytes did not require conditioned medium supplements (Collins et al., 1 977;

Gallagher et al., 1 979).

1.4.2) CYTOLOGIC CHARA CTERISTICS OF HL-60 CUL TURED CE=

HL-60 cells grow in single-ce11 suspension without a tendency to clump or adhere

to plastic or g las (Gallagher et al., 1979). These cells are generally round or ovoid with

a diameter varying fiom 9p to 25p; larger cells are generally binucleate. Wright-Giemsa

staining has shown that the predominant ce11 type within the HL-60 ce11 culture, is a

promyelocyte (Gallagher et al., 1979). This ce11 contains a large round nucleus with

distinct margins, fine chromatin and 2-4 nucleoli. The cytoplasm is deeply basophilie

containing multiple azurophilic granules. HL40 cells stain with myeloid lineage specific

stains such as myeloperoxidase, ASD chloroacetate esterase and Sudan black B however

they do not stain with alkaline phosphatase, a stain characteristically positive in normal

neutrophilic granulocytes (Gallagher et al., 1979). While the majority of cells in this

culture are promyelocytes, approximately 1 O- 12% are more mature myeloid cells,

predominantly myelocytes, with rare fully mature segmenteci neutrophils (Collins et al.,

1978). HL-60 cells express surface receptors for Fc hgment and complernent (C3)

which have been associated with di fferentiated granulocytes (Gallagher et al., 1 979).

1.4.3) GENERAL DIFFERENTC4 TION

HL-60 cells allow for the developrnent of a well-dehed in vitro experimental

system for examining aspects of the differentiation process (Yen and Albright, 1984).

Precursor cells and differentiated cells differ in two significant ways. Differentiated cells

are usually restricted to the Gil0 ceIl cycle phase while precursor cells are proliferating

and distributed throughout the ce11 cycle. in addition, differentiated cells are capable of

specialized metabolism, characteristic of the diffixentiated phenotype, whereas p r e c m r

cells are not (Yen and Albright, 1984). HL-60 cells exempli@ these characteristics

They are proliferatively active and have the capability to differentiate dong either the

monocytic or myeloid ce11 lineages, in response to different soluble factors (Yen and

Albright, 1984).

Agents such as 1,25-(OH)2 vitamin D3, sodium butyrate, and 12-0-

tetradecanolphorbol-13-acetate (TPA) induce terminal monocytic differentiation of HL-

60 cells (Yen and Albright, 1984; Yen et al., 1987; Fischkoff and Rossi, 1990). Butyric

acid stimulation of HL-60 cells has resulted in stable sublines consisting of neutrophils,

monocytedmacrophages, eosinophils, as well as a sublines containing a mixture of these

celi types (Collins et al., 1978; Fischkoff and Rossi, 1990). Retinoic acid, dimethyl

formamide and dimethyl sulfoxide (DMSO) induce myeloid differentiation in HL-60

cells (Yen et al., 1987; Skubitz and August, 1983). Addition of DMSO results in a

marked increase in the proportion of mature myeloid cells: myelocytes, metamyelocytes,

band neutrophils, and segmente- neutrophils (Collins et al., 1978). DMSO has been

shown to induce hct ional as well as morphological maturation in HL-60 cells as

demonstrated by an increase in the rate of superoxide production, degranulation,

ingestion of opsonized particles, and bacterial killing (Newburger et al., 1 979;

Newburger et al., 1984). The phagocytic and chemotactic functions of the HL-60 cells

were grealy enhanced by induction for differentiation with DMSO (Gallagher et al.,

1979; Collins et al., 1978). HL-60 cells exhibit phagocytic activity and responsiveness to

chemotactic stimulus cornmensurate with the proportion of mature cells in the culture

(Gallagher et al., 1979; Collins et al., 1978).

1.4.4) MODEL OF STUDY

HL-60 cells have been widely used as a mode1 of study for myeloid ce11 growîh

and differentiation (Skubitz and August, 1983; Collins et al., 1977). Their ability to

acquire characteristics of more mature cells of the myeloid fineage upon induction for

differentiation have made HL-60 cells an important ce11 line for investigation. Collins

and colleagues (1978) have show that mature HL40 cells are capable of phagocytosis.

Thus, this ce11 line may be used as a model to study neutrophil function. Studies trying to

determine lineage direction and cornmitment stages for differentiation in the myeloid

lineage have utilized HL-60 cells as an appropriate model of study (Fischkoff and Rossi,

1990). A study interestecl in understanding the involvement of adherence glycoproteins

on the surface of poIymorphonucleat leukocytes (Pm) with mobilization of PMNL to

sites of inflammation ernployed the HL-60 ce11 line as a model for their investigation

(Schrnalstieg et al., 1 986). HL-60 cells have also been used to study monocytes and their

interaction with endothelial cells involving HCMV infection at sites of vascular injury

(Guetta et al., 1997). This ce11 line has also been widely used for studying the

mechanism of apoptosis (Shimura et al., 1997). As well, HL-60 cells have been used to

study the rffects of ce11 aging in vitro (Levi et al., 1997). It has been shown that prion

protein (PrPC), involved in prion diseases in humam and animals, is present on ~ ~ 3 4 +

bone marrow stem cells (Dodelet and Cashman, 1998). Induction of HL-60 cells with

retinoic acid mimics the suppression of PrP in granulocyte difkentiation and thus the

HL-60 ce11 line provides a potential model to study PrP gene regulation and protein

function (Dodelet and Cashman, 1998). Potential for differentiation dong monocytic or

granulocytic pathways bas made HL-60 cells a model system for studying HIV-1

infection in early myeloid cells (Pise et al., 1992; Cannon et al., 1993).

1.4.5) INFECTIVITY OF HL60 C E U S

HL-60 cells have been shown to be permissive for infection by a number of

in fectious agents. Human granulocytic ehrlichiosis (HGE), s recently recognized disease

close1 y related to Ehrlichia equi and Ehrlichia phagocytophiïa and îransmiîîexi by ticks,

has been shown to replicate within HL-60 cells (Bedner et al., 1998). Infection r d t e d

in growth arrest of the cells and induction of apoptosis. Thus, HL-60 cells provide a

usefùl mode1 for invatigating the reproductive cycle and cellular changes induced by this

pathogen. Measles virus (MV) has been shown to infect bone marrow progenitor cells as

well as HL-60 cells. These ceIls were used to assess the effect of maturation on MV

infection (Helin et al., 1999). A number of studies have been conducted in which HL-60

cells were infected with human immunodeficiency virus type 1 (HIV-1). These studies

investigated the effect of HIV infection on di fferentiation of myeloid progenitor wlls and

whether chronically infected cells could differentiate upon stimulation, resulting in ce11

lineaga expressing productive HIV infection (Pise et ai., 1992; Cannon et aï., 1993;

Semmel et al., 1994). A study conducted by Jerome and colleagues (1998) has show

that herpes simplex virus 1 (HSV-1) infection of HL-60 cells inhibits DNA

fragmentation, a characteristic feature of apoptosis ofien studied in HL-60 -11s.

Therefore, this ce11 line has been shown to be permissive for infection with a mernber of

the herpesvirus family.

2 . 0 ) O B J E C T I V E S

The infection of a human promyelocytic leukemia ce11 line (HL-60) with HCMV was

investigated. Untreated and dimethyl sulfoxide (DMSO) shulated HL-60 cells were

infected with HCMV. The airn of this project was to study the extent to which HCMV

replicates in these cells, which are considered as a mode1 for virus Section of P m .

Specifically, this investigation included the following aspects.

The expression of viral antigens within HCMV inoculated HL-60 cells and DMSO

stimulated HL-60 cells was examined by irnmunofluorescense assay.

The presence of the viral genome and viral mRNA transcripts within HCMV

inoculated ce11 cultures was investigated using PCR and nucleic acid sequence-based

amplification (NASBA).

HCMV inoculated HL-60 cells and DMSO stirnulated HL-60 cells were screened for

the presence of viral particles by electron microscopy/immunoelectron microswpy.

The production of infectious virus in HCMV inoculated HL-60 and DMSO

stimulated HL-60 ce11 cultures was studied.

3 . O ) M A T E R I A L S A N D M E T H O D S

3.1) Propagation of MRC-5 Cells

Human ernbryonic lung fibroblast cells (MRC-5) (Biowhittaker) were grown to

confluency in tissue culture flasks using Eagles minimal essential medium (MEM)

(B iowhittaker) supplemented with 200 rnM L-glutamine, 1 m g h l gentamich, 250 pg/ml

fungizone, I O mg/ml vancomycin and 5% fm bovine serum (FBS). Media was replaced

every 5-7 days.

3.1.1) Propagation of WFF Ce&

Human foreskin fibroblast cells (HFF) (Biowhittaker) were purchased in tissue culture

tubes as confluent monolayers. For maintenance of ce11 cultures, Eagles minimal

essential medium (MEM) (Biowhittaker) supplemented with 200 mM L-glutamine, 1

mg/ml gentamicin, 250 p g / d fungizone, 10 m g h l vancomycin and 5% fetal bovine

serum (FBS) was use& Media was replaced every 5-7 days.

3.1.2) Propagation of HL-60 Cells

Human promyelocytic leukemia ce11 line HL-60 (ATCC #CCL-240) was cultured in

Iscove's modified Dulbecco medium supplernented with 200 mM L-glutamine, 1 m g / d

gentamicin, 250 &ml fungizone, 10 m g h l vancomycin and i 0% FBS. Cultures were

split every 2-3 days malntaining ce11 densities between 2x10' and 1 x106 cells/rnl.

3.1.3) Inducrioit tif Differentiation of HL-60 Celh

HL-60 cells were induced to differentiate into more mature cells of the myeloid lineage

by stimulation of 5x1 O' celldnil with 1.5% DMSO for 6 days (Collins et al., 1978).

Maturïty was assessed by microscopie determination of nuclav differentiation. Cytospin

slide preparations of 0.1 ml of ce11 suspension were made using a Shandon Cytospin 2

centrifuge (2ûûxg, 3 minutes). The preparations were subjected to Romanowsky staining

(Sakura RSG-6 1 stainer) and slides were observed by light rnicroscopy.

3.2) Virus Propagufi'on

HCMV Davis strain (ATCC #VR-807) was propagated in confluent monolayers of MRC-

5 cells. Maintenance media was decanted fiom the monolayer, viral inoculum was

added, followed by adsorption of the virus for 1 hour at 37OC. When infected ce11

cultures showed greater than 80% cytopathic effect (4+ CPE), HCMV infected MRC-5

cells were harvested, and cell-free supernatants were frozen at -70°C. Virus was titrated

on confluent monolayers of MRC-5 cells in a 96 well microtitre piate (6 welldten fold

dilution) and concentration expressed as TCIDso.

3.2.1) Determination of Stock Viral Titre in TCIDsdml

MRC-5 cells were grown to confluency in a 96 well microtitre plate. A h z e n aiiquot of

virus stock was thawed rapidly in a 37°C water bath and 100 pl of virus suspension was

added to 900 pl of MEM, resulting in a 1 : 10 dilution of virus inoculum. Serial ten-fold

dilutions were perfonned. For each dilution of virus, 100 pl of suspension was

inoculated into 1 well of MRC-5 cells (media fiom well was aspirated prior to

inoculation). This procedure was repeated 5 times for each dilution of virus thus

resulting in a total of 6 inoculations per dilution of virus. The plate was incubated (37OC

x 1 hour), virus suspension aspirated, and 200 pl of fiesh media was added. The plate

was observed for detection of CPE at successive 24 hour tirne points until no fiuther

increase in CPE was observed. Detexmination of TCIDso was performed by the Reed and

Muench method (1 938) for quantifjing viral titre. Stock virus titre ranged between

30 000 TCiD5~ml - 40 000 TCIDsdml. One infectious unit (IU) was dehed as 0.7

TCIDso (Poisson distribution).

3.3) Inocuiation of HL60 C e k WM W C W

Unstimulated HL-60 cells or DMSO stimulated HL-60 cetls were seeded in shell vials,

inoculated at multiplicity of infection (MOI) ranging from 0.1 to 5 infectious unitslcell

(IUIcell), and centrifùged (3500xg, 15 minutes) in a DAMON DPR-6000 centrifige.

CelIs were resuspended in tiesh media (Iscove's modifieci Dulbecco medium + 5% FBS)

and incubated for 4 days at 37°C in an atmosphere of 5% CO2.

3.3.1) H C W Heat Inactivation

An aliquot of viral supernatant from the original inoculum was heated to inactivate the

virus in a water bath (56°C x 30 minutes). MRC-5 shell viais were iiioculated with the

preparation pnor to and der heat inactivation to determine if infectious virus rernained

after heating.

3.4) Determination of Virai Titre in HCMYlnocufated Cultures ofClirstimitia&d or

DMSO Stimulated HL60 Ceils

Immediately afier inoculation and centrifugation with HCMV, 1 x 1 o4 cells were removed.

The rernaining cells were resuspended in fiesh media and incubated for 4 days at 37OC in

an atrnosphere of 5% CO2. The harvested cells were niptured by vortexing with glass

beads, and the suspension was titrated in MRCd cells to determine the initial viral load

present within the inoculated culture (as described in 3.2.1) . Following the incubation

period, cells were again harvested and virus was titrated to determine the fiaal viral load.

The lower limit of detection of the assay was 10 infkctious units.

3.5) Immuno/luorescence Aswy (TFA) For Deteciibn of R C W Immediritc-Eurb,

pp65, Late Antigen

Four days post inoculation (pi.), HL-60 cells were deposited on slides using a cytospin

(as described in 3.1.2). The preparations were initially fixed for 10 minutes in phosphate

buffered saline (PBS) containhg 5% formaldehyde, 2% sucrose, washed in PBS

containing 1% FBS for 5 minutes, and exposed to PBS containing 0.5% Nonidet, 10Y0

sucrose, 1% FBS for 5 minutes to pemieabilize them, and then washed with PBS

containing 1% FBS for 5 minutes. Monoclonal antibody directed against HCMV

immediate-early (IE) antigen (Bartels, Inc.), pp65 (Biotest Clonab), or late antigen

(Bartels, Inc.) was applied to the slide which was then incubated at 37OC for 30 minutes

in a humidified chamber. Slides were washed 3x in PBS, the secondary antibody (FITC

conjugate of goat-anti-hurnan immunoglobulin with Evans Blue) was applied and the

slides were incubated at 37°C for 30 minutes in a humidified chamber. Slides were

washed, mounted and examïned with a fluorescence microscope.

3.6) HCMV Nucieic Acid Isoluîion und PCR

Total nucleic acid was isolated fiom HL-60 cells and DMSO stimulateci HL-60 cells

inoculated with HCMV, 4 days pi. using the ~ u c l i ~ e n s ~ Isolation kit. Nucleic acid was

stored at -70°C until use. For amplification of a designated HCMV genome sequence, 2

primers were used (Pl: 5'-GGA TTC GCA TGG CAT TCA CGT ATG T-3', P2: 5'-

GAA TTC AGT GGA TAA CCT GCG GCG A-3') resulting in a 406-bp DNA amplicon

whose sequence corresponded to that of the unique short region of HCMV. As a positive

control for nucleic acid extraction, amplification of a 536 bp thgrnent of the human p-

globin gene was performed using primers HBG-l: 5'-GGT TGG CCA ATC TAC TCC

CAG G-3', HBG-2: 5'-GCT CAC TCA GTG TGG CAA AG-3'. DNA amplification was

conducted using the Perkin-Elmer GeneAmpQ3 PCR Reagent Ki t Total nucleic acid was

arnplified in a 100 pl reaction volume containhg 2.5 U of AmpliTaq@ DNA Polymerase,

200 p M each of the four deoxynucleoside triphosphates and 1.0 p M of each primer

buffered with 10X PCR buffer [50 mM KCI, 10 mM Tris-HC1 (pH 8.3), 1.5 mM MgC12,

and 0.01% (wt/voI) gelatin]. Each sample was amplified in 40 cycles of 94"C, 56OC.

72"C, with a final 5 minute extension at 72OC. Amplified products were visualized by

electrophoresis on 1.5% agarose gel and ethidium bromide staining.

3.6.1) Determining the Lower Lhif of Deteclion of PCR

A previousl y titrated virai preparation was used to determine the lower limit of sensitivity

of the PCR assay. Total viral nucleic acid was extracted ( ~ u c l i ~ e n s ~ Isolation kit) fkom

200~1 of virus infected culture supematant, which had a defined number of infectious

units, fiom which 30p1 was used as template for PCR. Amplification parameters are

those describeci in 3.6. The lower limit of detection of PCR was determineci to be

between 2 12-228 infectious units, as determined fiom three separate trials.

3.7) WCMVpp67 mRNA Deîecîion

Total nucleic acid was extracted h m HCMV inoculated HL-60 cells and DMSO

stimulated HL-60 cells as described in section 3.5. Nucleic acid sequence based

amplification (NASBA) and detection of HCMV pp67 mRNA transcripts was carried out

using reagents and protocols provided by ~ u c l i ~ e n s ~ ~ . The NASBA reaction mixture is

comprised of 3 enzymes (reverse transcriptase (RT), RNase H, and T7 RNA polymerase)

and two primers. Primer 1 (Pl) wntains a 3' temiinal sequence compiementary to a

sequence on pp67 mRNA (+), and a 5' temiinal sequence of a prornoter recognized by T7

RNA pol ymerase. Primer 2 (P2) contains a sequence complementary to the P 1 -primeci

DNA strand. NASBA proceeds by binding of Pl to pp67 mRNA (+) and extension by

RT and formation of a cDNA copy. The RNA strand of the RNA:cDNA hybrid is

degraded by RNase H, followed by annealing and elongation of P2. This results in

double stranded (ds) DNA containing a transcriptionally active promoter recognized by

T7 RNA polymerase. T7 RNA polymerase produces multiple copies of RNA banscripts

which are antisense to the original target RNA sequence @RNA (-). Each newly

synthesized antisense RNA can bind P2 and act as a ternplate for synthesis of a

complimentary cDNA strand. RNase H degradation of the RNA strand in the

RNA:cDNA hybrid, followed by Pl binding and elongation results in ds DNA with a T7

RNA pol ymerase promoter. Therefore, N ASB A al10 ws for exponential synthesis of

RNA products.

To briefly describe the protocol for pp67 mRNA amplification, 5 pl of nucleic

acid was incubated with 10 pl of primer solution (6S°C x 5 minutes) and allowed to cool

(41 OC x 5 minutes). 5 pl of enzyme solution was added to the mixture and incubated

(4 1 OC x 90 minutes). Amplification of mRNA transcripts was complete and detection

was conducted as follows. 10 pl of each amplification product was mixeci with 10p1 of

detection diluent. 5 pl of diluted amplfication product was mixed with 20p1 of system

control (SC) hybridization solution and 20 pl of wild type (WT) hybridization solution.

Hybridization mixtures were incubated (41°C x 30 minutes), 300 pl o f assay buffer was

added to each mixture and detection of mRNA transcripts was wnducted using the

~ u c l i ~ e n s ~ Reader which ernploys the electrochemiluminescense (ECL) principle.

NASBA was perfomed in triplicate for HL-60 cells and DMSO stirnulated HL-60 cells

inoculated with live or heat-inactivated HCMV. Uninoculated HL-60 cells and HFF cells

were used as negative controls while HFF cells inoculated with HCMV s m e d as a

positive control for pp67 mRNA production and detection.

3.8) Efectron Microscopy (EM) Anaiysik of HCMVInoculated Cultures

EM was performed on cells infected for 4 days with HCMV. HCMV inoculated HL-60

cells and DMSO stimulated HL-60 cells were fixed in a solution of 2% glutaraldehyde in

O. 1 M PO4 b u f k (pH 7.3) for 1 hour. Preparations were washed in buffer (O. 1 M Po4 / 5

minutes) and p s t - fixed in 1 % osmium tetroxide ( 1 hour). After washing in buffer (O. 1 M

PO4 / 5 minutes), samples were dehydrateci in a graded ethanol series followed by

propylene oxide, then ernbedded in Spurr epoxy resin. Ultrathin (100 nm) sections were

placed on copper grids and stained with m y I acebte (10 minutes) and lead citrate (10

minutes). Sections were then exarnined by electron rnicroscopy.

3.8.1) Immunoelecîron Microscorn (IEM) Anahsis of HCMVlnoculeted Cultvres

Infected ce11 cultures were prepared for IEM examination 4 days pi. with HCMV. Three

separate fixation techniques were utilized for IEM. 1. Standard EM Fixation as

described in Section 3.8. A modification of this procedure was dso wployed which

excluded a pst-fixation in osmium tetroxide. This cornpouad is a strong fixative and

may affect antigenicity of viral epitopes. 2. Immuno-Fixation: Inoculated cell cultures

were fixed in a solution of 1% glutaraldehyde, 4% paraformaidehyde in a 0.1 M sodium

cacodylate buffer (pH 7.3) for 1 hour. Samples were washed in buffer, dehydrated in a

graded ethanol series and embedded in K4M Lowicryl resin under UV light at -20°C.

Ultrathin sections were placed on fonnvar coated nickel grids. This procedure was also

modified by lowering the concentration of glutaraldehyde and fixing samples in O. 1%

glutaraldehyde, 4% paraformaldehyde in a 0.1 M sodium cacodylate buffer (pH 7.3) for 1

hou. 3. Ctyosubstitution: The technique employed was modified fiom Haller et al.

( 1992). Inoculated ce11 cultures were washed in 0.1M Po4 buffer (2 x 5 minutes) and

fixed in a solution of O. 1% glutaraldehyde, 4% paraformaldehyde in a O. 1M PO4 buffer

for 2-4 hours. Cells were washed in buffer (3 x 10 minutes) and infused with 2.3M

sucrose at 4OC oveniight. Samples were subject to plunge fieezing in liquid nitrogen,

dehydrated and stained with 0.5% uranyl acetate in 100% methanol at -80°C for 48-72

hours. Samples were flushed at -80°C with 100% methanol to rernove excess uranyl

acetate. Temperature was brought up to -20°C oveniight. Methanol was exchanged with

1 : 1 HM20 Lowicry1:rnethanol oveniight at -20°C. Pure HMSO Lowicryl was introduced

to the samples and stored at -20°C for 1-2 hours. A fresh change of Lowicryl was made

and stored at -20°C for an additional 1-2 hours. UV polymerization was conducted at

-20°C and samples were processed as ultrathin sections were placed on formvar coated

nickel grids.

Imrnunolabeling of grids was conducted utilizing two separate protoculs.

1. Grids were rinsed in a solution of 0.15% glycine, 0.5% BSA in PBS (2 x 10 minutes)

then washed in a solution of 0.5% BSAPBS (4 x IO minutes). Grids were labeled with

monoclonal antibodies to the IE antigen, pp65 antigen or monoclonal aotibody 284,

which recognizes the major capsid protein (MCP) of HCMV (1 hour). The following

dilutions of antibody were tested: 1 : 1, 1 : 10, 1 :20, 1 : 1 0 . Unbound primary antibody was

removed by washing in 0.5% BSA/PBS (4x10 minutes). Grids were labeled with a 1:20

dilution of rabbit anti-mouse IgG conjugated to Arnersham 10 nm gold particles, for 1

hour. Unbound gold conjugate was removed by washing in 0.5% BSAIPBS (10

minutes), PBS (4 x 10 minutes) and in distilled water (4 x 10 minutes). Samples were

then stained with uranyl acetate (1 0 minutes) and Iead citrate (5 minutes). Monoclonal

antibody 28-4 was kindly provided by W.Britt, Birmingham, Alabama.

2. Grids were incubated for 10 minutes in 2 5 ~ 1 of a 10% solution of heat-inactivated

goat serurn in 0.1% BSA/Tris. Grids were labelled with monoclonal antibodies as

described above. Unbound primary antibody was removed by tramferring the grid

through a series of 5 droplets of phosphate buffer for 5 minutes. Grids were then labelled

with a 1 : 10 dilution of rabbit anti-mouse IgG conjugated to Biocell 20 nm gold particles,

for 1 hour. Unbound gold conjugate was removed by subsequent washes in distilled

water (5 x 2 minutes). After immunolabelling, samples were stained with uranyl acetate

(10 minutes) and lead citrate (5 minutes). Sections were then examined by electron

microscopy.

3.9) IL-6, IL-8, TNFu Sk'mulaa'on

Cytokines are produced during the effector phase of natural and specific

immunity. They are produced by a number of different ce11 types and rapidly secreted, in

order to help mediate the immune response. Cytokines IL-6, IL-8 and TNF- a a p p r to

be closely related to active HCMV infection in the host. Thus, the effect of these

cytokines on HCMV inoculation of HL-60 cells and DMSO stimulated HL-60 cells was

investigated.

HL-60 cell cultures and DMSO stimulated HL60 ce11 cultures were inoculated

with HCMV as previously described (3.3). The effect of cytokine stimulation on the

percentage of IFA positive (late antigen) cells, was determined by resuspending HCMV

inoculated cells in media containing various concentrations of cytokines IL-6, IL-8, and

TNF-a. Cytokine containing media was prepared and tested in triplicate over the

following ranges for IL-6, IL-8, and TNF-a: (IL-6: 0,O. 1,0.5, 1,5, 10 ng/ml), (IL-8: 0,

1 0, 20, 50, 75, 1 00 ng/ml) (TNF-a: 0, 0.05, 0.1, 1, 5, 1 0, 20 ng/ml). Following use of

only individual cytokines, media was prepared using a combination of the three cytokines

(IL-6: 10 ng/ml, IG8: 100 nghl , TNF- a 20 ng/ml) and (n-6: 5 ng/ml, IL-8: 50

n g h l , T N F a : 10 ng/ml), and tested over six trials. HL-60 and DMSO stimulated ce11

cultures inoculated with HCMV, resuspended in media without cytokines, served as

controls. HCMV inoculated ce11 cultures were assayed four days pi. by IFA for presence

of late viral antigen, as previously described (3.5).

4.1) Effect of HCWlnocu[an'on on 112-60 Cell Culiures

HL-60 ce11 cultures and DMSO stimulated HL-60 ce11 cultures were inoculated

with HCMV at a multiplicity of infection (MOI) of 1 infectious unit/cell. Using an

indirect immunofluorescence assay (TFA), HCMV inoculated cultures were positive for

viral antigens fkom al1 three phases of HCMV replication 4 days pi. (Table 1). Similar

levels of antigen were detected in unstimulated and DMSO stimulated HLdO cells

suggesting that differentiation may not influence HCMV antigen detection in this ce11

line.

HCMV antigens were detected by IFA in HL-60 and DMSO stimulated HL-60

ce11 cultures inoculated with heat inactivated HCMV. There was no significant

difference in the quantity of cells positive for HCMV Ag when using live or heat

inactivated virus inoculum. No viral antigens were detected in control MRC-5 cells

inoculated with heat inactivated HCMV, 4 days pi.

PCR was conducted to determine whether the viral genome was present within

inoculated cultures. A 406bp ffagment fiom the unique short (Us) region of HCMV was

amplified from HL-60 and DMSO stimulated HL-60 ce11 cultures, inoculated with live

and heat inactivated HCMV, 4 days pi . (Table 1). Fibroblast cells inoculated with live

or 56OC heat inactivated HCMV were PCR positive for viral DNA 4 days pi.

4.1.1) Dose Dependent IFA Response

Experiments were conducted to determine if the presence of virai antigen in HL-

60 ce11 cultures was directly related to the inoculating dose of HCMV. Unstimulated and

DMSO stimulated HL-60 ce11 cultures were inoculated at an MOI of O. 1, 0.3, 0.5, 1 and

5. Four days p.i., cells were tested by IFA for detection of late HCMV Ag, to determine

the percentage of IFA positive cells. As the infectious dose increased, the percentage of

LFA positive cells also increased for al1 inoculated cultures tested (Fig.2). HCMV

antigens were detected in cultures inoculated at MOI'S of 0.5 or greater- There was a

significant increase in percentage of IFA positive cells when the inoculating dose was

increased fiom an MOI of 1 to an MOI of 5.

4.1.2) Variable IFA Success

While HCMV antigen detection appeared to depend on the size of the inoculum,

detection of viral antigen by IFA was not feasible on every occasion in contrast to

infected MRC-5 cells (Fig.3). As the MOI was increased, the percentage of IFA positive

cells also increased in al1 inoculated cultures, with the highest yields achieved at an

MOI= 1.

4.2) pp6 7 mRNA Transcript Detedon

To m e r characterize the interaction between HCMV and inoculated cultures of

HL-60 cells, pp67 mRNA transcript detection was conducted ushg nucleic acid sequeme

based analysis OJASBA). Results are outlined in Table 2. Positive signals suggesting

detection of pp67 mRNA transcripts were found in both IFA positive and IFA negative

HL60 cells and DMSO stimulated HL-60 ce11 cultures, using live or heat inactivated

HCMV. Viral mRNA transfnpts were also detected in the original viral iwculum. Viral

transcripts were not detected in uninoculated HL-60 cells and DMSO stimulated HL40

cells (data not shown).

4.3) Infctious Progeny Not Produced in IFA Positive Cell Culncns

Upon detection of Wal antigens and gmetic material in HCMV i n d a t e d HL60 ce11

cultures, it was necessary to detemine whether infectious viral progeny was being

produced in these cells. For this determination, virus nom the infected cultures was

titrated. Inoculated HL-60 cells and DMSO stimulated HL-60 ceils were hawested after

centrifugation and again after a 4 day incubation period. In al1 trials, no virus could be

detected at the limit of detection of the assay afier 4 days (Table 3). No virus could be

detected in HL-60 cells and DMSO stimulated HL-60 cells inoculated with heat

inactivated HCMV.

Y0 Cell IFA (+)

O. 1 0.3 0.5 1 5 MOI

Fig.2. The effect of increasing MOI on percentage of cells F A positive for late HCMV Ag. For each determination, between 1 x 1 o3 and 1 xlo4 cells were counted and 0-4 cells were IFA positive. Data points represent the mean of six independent trials f SD at each infectious dose. MRC-5 cells inoculated at the above MOI result in -100% IFA positivity.

O. 1 0 .3 0.5 1 MOI

Fig.3. Success rate of HCMV late antigen detection within inoculated HL-60 ce11 cultures. HL-60 cells and DMSO stimulated HL-60 cells inoculated with HCMV (live or heat inactivated) were assayed for late viral antigen, 4 days p i . Inoculations were conducted over the complete range of infectious doses, for each trial. Bars represent successfbl antigen detection expressed as a percentage, based upon six independent trials. As the infectious dose was increased, the percentage of trials positive for late HCMV antigen detection by IFA also increased to a maximum at a MOI=l.

4.4) Electron Microscopy

in order to explore the possibility that viral replicative processes may have k e n

initiated but at some point were prematurely inhibited resulting in production and

assembl y of noninfectious virions, electron microscopy (EM) was çonducted to

determine if HCMV nucleocapsids could be detected inside infected HL-60 ce11 cultures

positive for HCMV antigens by iFA. The HCMV permissive human foreskin fibroblast

(HFF) cells were used as a control for HCMV replication. EM examination of HCMV

inoculated cultures showed typical characteristics of HCMV infected cells (viral

nucleocapsids in the nucleus, budding fiom the nuclear membrane, multivesicular bodies

containing viral nucleocapsids) as had been shown in HCMV infected fibroblasts and

endothelid cells (Pande et al., 1 990; Depto and Stenberg, 1 989; Sing and Ruscetti, 1 990).

HCMV virus particles were detected in inoculated HFF cells (Fig.4A-C) and particles

consistent with viral morphology within inoculated HL-60 cells (FigSA-B). These

particles were approximately 120nm in HFF cells and 150nm in HL-60 cells. No HCMV

nucleocapsids were detected in uninoculated cultures. Observations of EM analysis are

summarized in Table 4.

Table 3. Determination of viral titre in HCMV inoculated IFA positive HL-60 cells and DMSO stimulated HL-60 cells, 4 days post inoculation.

HL-60 + HCMV DMSO Stimulated HL-60 + HCMV

Initial' Titre Final' Titre Initial Titre Final Titre

121 - 1 04 -

208 - 1 04 -

178 - 222 ..

103 - 78 -

Ti tre infectious unitslml h i a l = virus titre akr HCMV adsorption (-) Below 10 IU inal al = virus titre after 4 days post inoculation

Fig.4. Electron micrograph of a HCMV inoculated HFF ce11 4 days post inoculation. (A) Presence of virus particles in the nucleus as well as a degenerated nuclear membrane. (B) Vinons budding fiom the nuclear membrane. (C) Presence of a virus particle inside a cytoplasmic multivesicular body. Arrows indicate virus particles. Q = nucleus, Q= cytoplasm.

Fig.5. Electron micrograph of a HCMV inoculated HL-60 ce11 4 days post inoculation. (A) Cytoplasmic detection of a virus particle. (B) Presence of a virus particle inside a cytoplasrnic multivesicular body. Arrows indicate virus-like particles.

4.4.1) Immunoefeciron Mlcroscopy (IEW

In order to c o n w whether particles detected within inoculated HL-60 cells were

viral nucleocapsids, imrnunoelectron rnicroscopy (IEM) was attempted. Specificaliy,

three separate sample preparation techniques, as well as modifications within the

procedwe, were attempted (Section 3.8.1 ). immunocytochemical staining was also

perfomed using two different techniques. IEM analysis was perfomed initially upon

HFF celis, those inocuiated with HCMV, as well as uninoculated, to serve as a positive

and negative control respectiveiy. The findings are sumrnarized in Table 5. Prior to

processing for IEM analysis, HFF ce11 cultures were subject to IFA analysis to test the

sensitivity of monoclonal antibodies to be used for IEM. Positive control samples stained

with each of the antibodies and negative control sarnples did not stain (data not shown).

Al1 of the fixation protocols including modifications have shown detection of virus

particles within positive controls, and a lack of detection in negative control samples

(Table 5). None of the techniques were able to yield a positive result with

imrnunostaining for detection of virus particles. Figure 6 depicts an immunoelectron

micrograph of uninoculated and HCMV inoculated HFF cells processed under the

conditions highlighed in Table 5. Figure 6A shows the presence of gold particles within

the nucleus and cytoplasm of an uninoculated HFF cell. Virions were not detected in this

cell. Figure 6B shows detection of both virus particles and gold particles within the

nucleus of a HCMV infected HFF c d , however no gold staining of virions was observed.

Similar observations were found with each of the techniques and antibodies tested.

Since, it was not possible to obtain adequate controls for immunolabelling, HCMV

inoculated cultures of HL-60 cells were not tested.

4.5) Eneeî of IL-6, LL-8, TNF- a riRL60 Cell Cultures

Cytokines IL-6, IL-8 and TNF- a pztr to be closely related to active HCMV

infection in the host. These cytokines were utilized in HCMV inoculations of HL40 and

DMSO stimulated HL-60 ce11 cultures, to observe if there were any stimulatory effects

resulting in increased numbers of [FA positive cells. Individual cytokines were tested

over a range of their observeci biological effects (IL-6: O. lng/ml - 10 ng/ml, IL-8: 10

ng/ml- 1 00 @ml, RilF-cr : 0.05 ngM - 20 ng/ml) (Chernicon International inc.). There

was no significant increase in percentage of IFA positive cells using concentrations of the

cytokines within their specified range (+/O cytokines: 0-4 positive cells / 10 00). HCMV

inoculations of HL-60 cells and DMSO stimulated HL-60 cells were conducted, and cells

were incubated in the presence of the three cytokines (IL-6: 1 O ng/rnl, IL-8: 100 ng/ml ,

T N F a : 20 ng/rnl) or (IL-6: 5 ng/ml, IL-8: 50 nglml , TNF- a : îû nghl). There was

no increase in the percentage of IFA positive cells for late HCMV Ag with either

combination of cytokines, in HL-60 cells and DMSO stimulated H L 6 0 cells, when

compared to cytokine negative controls (0-4 positve cells / 10 000).

Table 5. Summary of IEM attempts using HFF cells uninoculated and inoculated with HCMV, 4 days post inoculation

Fixation Standard Immuno Fixation Cryosubstitution Protocol EM Fixation

Virion detection pp65 Pl

PZ IE Pl

PZ Late Pl

P2 MCP Pl

P2 OSO.,: osm

(+) (-) Control Control

lm tetroxide glut : glutaldeh yde

(+) (4 Control Control

(+) Control: HCL

(+) Control

1% glut.

(-) Control: uninocu

0.1% glut.

1 1

V inoculated HFF cell Pl : labelling protocol 1 lated HFF cell P2: labelling protoc012

Fig.6. Imrnunoelectron micrograph demonstrating non-speci fic binding of conjugated gold particles to HFF cells. (A) Uninoculated HFF cell. Presence of gold particles in both the nuclear and cytoplasmic regions, with no detection of virus particles within the cell. (B) HCMV inoculated HFF cell. Detection of numerous virus particles, few gold particles and non-specific association of the two within the nucleus. Arrows indicate gold particles. a = nucleus, Q = cytoplasm.

5.O)DI S C U S S I O N

HCMV IE, pp65 and late antigen were detected in HL-60 cells and DMSO

stirnulated HL-60 cells inoculated with live and heat inactivated virus, 4 days p.i. Under

similar conditions, cultures of the known HCMV susceptible fibroblast ce11 line (MRC-

5) were 100% infected with live virus and uninfectexi with heat inactivated inoculum,

while in the HL-60 ce11 line, viral antigens were detected in very few cells using both live

and heat inactivated HCMV. To M e r investigate these findings, experiments were

conducted with the MRC-5 fibroblast ce11 line and HCMV (data not shown). In

fibroblasts inoculated with HCMV, there was detection of IE, pp65, and late Ag 1 hour

post inoculation (pi.). Considering the infectious cycle of HCMV in fibroblast cells,

virus penetration has been shown to occur 5 minutes p.i. and IE protein has been detected

as early as 20 minutes p.i. (Mocarski, 1996). The switch fiom early gene transcription to

late gene transcription has been shown to occur approximately 24-36 hours pi.

Therefore, detection of IE, pp65, and late viral antigens in HCMV inoculated fibroblasts

1 hour p.i. are probably due to antigens acquired fiom the initial virus inoculum, and not

fiom de novo synthesis. Similar findings have been reportai by 0th- investigators

(Revello et al., 1992; Grefte et ai., 1992; Revello et al., 1998). Antigens were also

detected at 24,48, 72, and 96 hours p.i. as gene hanscription and translation were taking

place within infected fibroblast cells.

Investigating antigen detection in MRC -5 cells using 56OC heat inactivated virus

inoculum, it was observed that at 1 hour p.i., al1 3 viral antigens were detected. However,

observation at 24, 48, 72, and 96 hours p.i., viral antigens could not be detected. Thus,

viral antigens present as part of the inoculum can be initially detected in fibroblasts upon

56°C heat treatment of the inoculurn. The disparity between why Wal antigeas can be

detected 1 hour p.i. using heat inactivated inoculum but not at later times may be

explained as follows. Heat treatment results in inactivation of the virus (see section

1.1.5) rendering the virus incapable of gene transcription and translation. The initial

detection of viral antigens (1 hour p.i .) is due to uptake, and localizatioa/conceatration of

specific antigens. Over tirne, the initial antigens present may disperse, become extmded

fiom the nucleus, or degraded by enzymes present within the c d , and become

undetectable. Since transcription and translation of the viral genome are not occurring,

new viral antigens are not being produced. T'us, after initial viral antigen detection at 1

hour p.i, Wal antigens are not detected in fibroblasts inoculated with heat inactivated

virus inoculum.

halogous flndings have been described with HCMV antigen detection in

polymorphonuclear leukocytes (antigenernia assay) (The et al., 1990). Delayed

processing of blood samples has shown a significant decrease in the number of HCMV

antigen positive cells detected (Gema et al., 1 992). Quantitative antigenemia has been

shown to drop between 40% to 55% when samples have been stored for 24 hours; with a

more significant decrease in samples stored at room temperature, than at 4OC (Bush and

Sluchak-Carlsen, 1998; Boeckh et al., 1994; Landry et al., 1995; Schafer et al., 1997).

While the process/mechanism(s) involved in accounting for the di fferential antigen

counts in polymorphonucIear ledcocytes is unclear, these studies suggest that HCMV

antigens may not be indefinitely stable within these cells. Thus, sirnilar processes may be

invo lved for explaining initial deteaion of HCMV antigens, followed b y their subsequent

disappearance, in fibroblast cells using heat inactivated virus inoculum.

In the case of HL-60 cells and DMSO stimulated HL-60 cells inf'ted with

HCMV, the maximum number of cells positive for viral antigens was found to occur 4

days p.i. using live or heat inactivated virus inoculum. No Wal antigen was detected

when cells were examined at 1,24, 48 or 72 hours p.i. No increase in the number of cells

containing viral antigen could be seen by incubation beyond four days. A distinct

difference was evident when comparing fibroblast cells with HL-60 cells and their

interaction with heat inactivated HCMV. HL-60 cells and DMSO stimulated HL-60 cells

were HCMV Ag positive while fibroblast cells were negative for HCMV Ag by F A 4

days pi . (Table 1). Apart fkom the fact that these are two different ce11 lines challenged

with HCMV, the observed antigen detection in HL-60 cells may be accounted for by the

possibility that the interaction between HL-60 cells and HCMV may be a longer process

than that described for fibroblast cells (Mocarski, 1996). The route of virus entry into

HL-60 cells is unclear. Receptor binding as well as phagocytosis of virus particles

remain possible routes of entry. Considering phagocytosis of virus particles, perhaps

only a small fraction of HL-60 cells are in a state in which they can actively phagocytose

these particles. Accordingly, it takes time for these few cells to acquire enough viral

antigen fiom the inoculum to produce a signal detected by FA. Specuiative and not

previously reported in the literature is the idea that the binding/phagocytic interaction

between HL-60 cells and HCMV involves very few virions at a time. Thus, the entire

process of virus uptake may require 4 days before any appreciable detection of viral

antigens can take place by IFA. Furthemore, upon virus entry, perhaps virions remah

stable within the cytoplasrn for a period of tirne. Secondary signalling systems may not

have been triggered and enzymes involved in degrading foreign particles may not have

been activated (Keeling, 1987; Newburger and Pannley, 1991; Beck, 1985). Such

signailing systems when finally triggered result in breakdown of the virus particle and

subsequent nuclear locaiization of viral antigens. These ideas are illustrated in Figure 7.

Observing HL60 cells and DMSO stimulated HL-60 cells, virai antigen detection

(IFA) in HCMV inoculated cultures did not consistently yield positive results, as was

observed with inoculated fibroblast cells (Fig.3). Since the route of virus entry is unclear,

explanation of this observation can be attempted by considering both reccptor-ligand

binding and phagocytosis, as a means of virus entry.

Consider k t a binding interaction between HCMV and HL40 cells. Perhaps

there are very few putative host cell receptors for HCMV ligands to bind on the surface

of the cell. Furthemore, these receptors may be distributed widely apart. This is of

concern if productive binding and attachment of the virion requires binding to multiple

receptor(s) thus involvhg a cascade of interactions between viral and cellular

components (Compton, 1995; White and Femer, 1994).

In accordance with the describeci postulates the following analysis of Fig.3 can be made.

There is no detection of viral antigen when inoculating HL-60 cells at an MOI of 0.1-0.3.

Since the initial interaction between the virus and host ce11 is one of chance, bringing the

virion into close vicinity with the host c d , at this low MOI, opportunity for interaction is

very limited. ïncreasing the infeftious dose to an MOI of 0.5, the probability of

interaction between virions and receptors increases, allowing for attachment and

penetration of the virus (Fig. 7A) followed by Ag detection by IFA. Our results indicate

that HCMV inoculation at an MOI. of 1 allows for optimal binding and subsequent

detection of Wal antigens, with the most wnsistency. Increasing the infectious dose and

inoculating at an MOI of 5, there is a distinct drop in fiequency of trials successfùi for Ag

detection. It is unclear why this correlation exists.

Similady, if a phagocytic interaction was involved, what may be o c c u r ~ g is that

inoculating at an MOI of 0.1-0.3, the viral load is t w low and appreciable phagocytosis,

degradation and antigen detection does not occw. Increasing the infectious dose to an

MOI of 0.5 and 1 increases the chances of this interaction occuning by virtue of a greater

number of virions in the surroundings. By increasing the infectious dose to an MOI of 5,

there is an unexplainable observai drop in frequency of trials successful for Ag detection.

An important finding fkom this set of experiments was at the best of thes,

HCMV Ag detection only appproached -80% with a range of percent positive trials fiom

-20% - -80% for al1 HL-60 ce11 cultures. Fibroblasts are a known HCMV permissive

ce11 line in vitro (Compton, 1 995; Mocarski, 1 996). Inoculation with an infectious dose of

0.1 MOI was sufficient to observe Ag detection in each trial (100% success, Fig.3).

Observing less than 100% Ag detection success with HL-60 ce11 cultures is not entirely

unexpected as HCMV has a highly restricted host range in ce11 culture (Momki , 1996).

What is worthy of investigation is that employing the same technique(s) for inoculating

cells, viral antigen detection which occurred in one trial, did not necessarily occur the

ma-

l 2 3 4

Fig.7. Proposed mode1 of interaction between HCMV and a HL-60 ceIl. (A) Receptor binding interaction between HCMV and a HL-60 d l . (B) Phagocyîic interaction between HCMV and a mature HL-60 cell. 1. HCMV virion with intact envelope binds to a receptor on HL40 d l / DMSO stimulated HL60 ceIl (A), HCMV virion is phagocytosed by a mature HL-60 ceIl (B). 2. Vims is internalized and rernains stable wiWwithout envelope. 3. Additional virions attach (A), phagocytosed (B) and penetrate into the cell. 4. Signalling pathways are activated, virions are degraded releasing viral antigens which localize in the nucleus and are detected by IFA 4 days post inoculation.

following time the experïment was conducteci. These factors as welI as other techniques

which could be used will be addresseci towards the end of this section.

Detection of HCMV DNA within PMNL has been described using DNA

hybridization techniques (Veal et al., 1996; Dankner et al., 1 990) and PCR. (Gerna et al.,

1992; Boivin et al., 1998; Zipeto et al., 1992). in this study, PCR was used to detect viral

DNA in HL-60 ce11 cultures and fibroblast cells 4 days pi., using live virus inoculurn as

well as heat inactivated virus inoculum. Heat inactivation of HCMV (section 1.2.6)

results in membrane fluidization (Aloia et al., 1988). Viral ligands implicated in early

events of HCMV infection such as binding and penetration are associated with the

envelope of the virion (Compton, 1995). Thus, heat inactivation results in loss of

integrity of the viral envelope and correspondhg ligands for host ce11 receptors.

Detection of the viral genome in fibroblast cells and HL-60 ce11 cultures inoculated with

heat inactivated virus inoculum, cannot be explained by the conventional mode1 of virus

attachent and penetration (Compton, 1995). It was necessary to determine other

possible routes of entry of HCMV.

Binding of beta-2 microglobulin (P2m) to the tegument of HCMV (Stannard,

1989) could provide an altemate route of entry into permissive cells for unenveloped

HCMV nucleocapsids. P2m has been identifie. as the light chah (12 000 MW) of class 1

HLA (Cresswell et al., 1974). Class 1 HLA is a dimeric molecule composed of a

pol ymorp hic, trammembrane a-chah (44 000 MW, heavy chain) noncovaientl y linked

with a B-chah (12 000 MW, light chain) anchored outside the plasma membrane

(Cresswell et al., 1974; Hyafil and Strominger, 1979). Heat inactivation of HCMV at

56°C resuits in fluidization of the lipid membrane envelope (Aloia et al., 1988) exposing

the nucleocapsid and associated tegument (see section 1.2.6). Synthesis of &m by cells

of the body (Gnindy et al., L 98%) as well as exogenous addition of &m in fetal bovine

s e m (Grundy et al., 1987a) allows for binding of B2m to the tegument of HCMV

vinons (Stannard, 1989). Binding of Ptm-coated nucleocapsids to class I HLA molecules

on the surface of host cells (Grundy et al., 1987b) followed by displacement of the fb

chain of HLA and association with P2m nom the f32m-coated nucleocapsid (Hyafil and

Strominger, 1979) could provide an alternate route for attachent and entry for

unenveloped HCMV parricles. While it is unclear what interaction is occurring between

HCMV and HL-60 ce11 cultures, attachent of heat treated virus rnay occur in this

manner.

Detection of viral DNA in HL-60 cells and DMSO stimulated HL-60 cells has

significant implications. Promyelocytes are normally found only in the bone marrow and

our results raise the possibility that in vivo, HCMV may penetrate into these neutrophil

precursor cells. Other reports have shown HCMV DNA detection in bone marrow cells

including C ~ 3 4 ' hematopoietic progenitor cells (Maciejewski et al., 1992; Meyer-Konig

et al., 1997). The replication cycle of CMV is slow, 48-72 hours to yield detectable levels

of viral progeny (Mocarski, 1996). Neutrophils have a defined lifespan, 4-5 days in

normal tissue, shorter in sites of infection and inflammation (Keeling, 1987). Thus, virus

may penetrate into promyelocytes and other immature cells in the bone marrow and

following fûrther maturation and release into circulation, mature HCMV DNA harbonng

cells may possibly transmit the gemme to other susceptible cells (Gmdy et al., 1998) or

upon proper stimulation, active transcription may ensue within the mature PMNL. Thus,

promyelocytes could be an important site of HCMV latency.

Upon detection of viral antigens and viral DNA withui inoçulated HL-60 cell

cultures, search for viral mRNA transcripts was conducted using NASBA. Studies have

shown detection of late viral mRNA transcripts within PMNL using hybndization

techniques (Gozlan et al., 1993) and RT-PCR (von Laer et al., 1995; Dankner et al.,

1990). Recent studies have shown that detection of HCMV mRNA using NASBA is

more sensitive than RT-PCR (Blok et al., 1998) and thus qualitative NASBA was

perfoxmed on HCMV inoculated ceIl cultures. Our results indicate that a positive signal

was obtained using NASBA suggesting that mRNA was present within inoculated HL60

cell cultures as well as in the inoculum (uninoculated ce11 cultures were NASBA

negative). Since the NASBA assay for CMV pp67 mRNA detection is a qualitative

assay, it cannot be concluded whether detection of pp67 transcripts within inoculated ce11

cultures is a result of transcription within inoculated cells. Of significant importance is

that mRNA transcripts were detected in the inoculum, prior to and fier heat inactivation

(56°C/30minutes), while the instability of mRNA has been well documented. Total

nucleic acid was extracted (Section 3.6) and used for pp67 mRNA detection. It is

unlikely that mRNA would be present in the inoculum due to tirne and temperature

effects. The stability of HCMV DNA is unknown. NASBA has been show to amplie

both DNA and RNA sequences, with accumulation of both products occuning during the

first 45 minutes. AAer 90 minutes, the arnount of RNA product exceeds DNA product

20-fold (Sooknanan et al., 1995). Therefore, what may account for the positive signal

detected in tested samples is in fact single-stranded viral DNA which has entered the

cyclic amplification process. To firrther elaborate, viral DNA may dissociate during the

amplification process (6S°C), allowing for binding of Primer 1 to single stranded DNA.

Alternatively, pieces of fiagmented viral genome may be found within the stock virus

inoculum, origïnating h m ruphued, infeaed fibroblast cells. Binding of Primer 1 and

extension by reverse transcriptase would provide a template for binding Primer 2. As

discussed in Section 3.7, synthesis and subsequent detection of pp67 transcripts would

result. While measures were taken to ensure that contamination did not occur,

nonetheless contamination of reactions with only a few molecules of nucleic acids can

lead to false positive results. Thus, positive NASBA signals may have resulted h m

carry-over or contamination during nucleic acid isolation and amplification procedures.

Studies evaluated NASBA pp67 mRNA detection according to specificity and

predictive value for onset of HCMV infection, by cornparison to other assays such as

antigenernia and RT-PCR. However, these studies were conducted with whole blood

sarnples and NASBA has not been tested on ce11 culture. Thus the NASBA procedure

may be an inappropnate diagnostic tool for investigating whether active transcription was

occurring within inoculated HL-60 ce11 cultures. Confirmation of r e d t s employing RT-

PCR or the use of radio-labeled nucleotides for detection of progeny mRNA transcripts is

necessary in determinhg the extent to which HCMV replicates in HL-60 cells.

Studies have been conducted whereby IFA positive PMNL have been shown to

transmit infectious vinis (Grundy et al., 1998; Craigen et al., 1997). In this study, we

have shown that IFA positive HL-60 cells and DMSO stimulated HL-60 cells did not

produce v ins progeny that could be transmitted to fibroblast cells. This firrther

substantiates the hypothesis that active gene transcription and viral replication does not

occur within HL-60 cells.

To determine if HCMV virions could be detected in HL-60 cells, samples positive

for late HCMV Ag were processed for EM analysis and showed detection of particles of

approximate size and similar morphology to HCMV virions, within the cytoplasm

(FigSA), and within a multivesicular body (FigSB) of HL-60 cells inoculated with

HCMV. A dense concentration of nucleocapsids and virions within the nucleus was

observed in HFF cells inoculated with HCMV (Fig.4A, 4B). This finding is consistent

with Severi et al. (1988) who showed that starting h m 3-4 àays pi., there were a

multitude of nucleocapsids, both containhg an electron-dense core and coreless, in

HCMV infected fibrobhsts. Nuclmcapsid maturation has been shown to occur within

the nucleus where the HCMV genome is packaged into preformed nucleocapsids

(Mocarski, 1996). Thus, accumulation of nucleocapsids in the nucleus of infected cells is

characteris tic of HCMV infection and virion morphogenesis.

In cornparison to HFF cells, there appears to be no detection of intranuclear

nucleocapsids in HL-60 cells inoculated with HCMV. This finding suggests that HCMV

replicative processes may not be occurring in HL-60 cells, and thus further corroborates

the hypothesis that HL40 cells are not conducive for Wal replication. EM detection of

HCMV virions in PMNL has been previously described (Martin et al., 1984). Martin and

colleagues (1984) have shown that detection of HCMV virions has been iocalized to

phagosomes. There was no detection of intranuclear virions or nucleocapsids, suggesting

HCMV was not actively replicating inside PMNL.

In order to c o n m detection of virus particles, immunoelectron microscopy

(IEM) was utlilized. IEM was initially tested on the HCMV permissive HFF ceIl line.

HFF cells infected with HCMV were assayed by IFA and dernonstrateci typical nuclear

staining using antibodies directed against E, pp65, MCP antigens. Utilizing a number of

fixation and immunolabelling combinations we were unsuccessNl in obtainuig positive

immunolabelling of infected HFF cells. Our results do provide a foundation for

development of a fixation procedure for IEM detection of HCMV in culture.

Immunoelectron microscopy has been used for diagnosing herpes virus infections

(Folkers et a[., 199 1 ; Vreeswijk et al., 1988; Fokers et al., 1992). In these studies, virus

has been isolateci h m a lesion and characterized by binding of the antibody-gold

conjugate to core and envelope antigens. There was no need to utilize fixation

procedures which may denature protein molecules (Hayat M.A, 1970). Although

atternpts were made to minimize the toxicity (remove 0s04) and concentration of

fixatives (0.1 % glutaraldehyde), antigenicity could not be maintained. Cryosubstitution

has been shown to preserve pst-embedding antigenicity (Haller et al., 1992) which

appears to be problernatic with HCMV antigens. Thus, cryosubstitution was performed

and antigenecity to vira1 epitopes was not retained.

Cytokine production in vivo has been associated with increased pathogenesis of

HCMV infection and disease (Humar et al., 1999). Attempting to stimulate HCMV

inoculated HL-60 cells and DMSO stimulated HL-60 cells with various wmbinations of

cytokines IL-6, IL-8 and TNF-a, our studies have indicated that there is no affect on the

percentage of HCMV Ag positive cells. Simulation of the in vivo condition is not

entirely possible to replicate under in vitro studies, however, testing of inoculation

conditions utilizing cytokine concentrations fonsistently reported in clinically significant

HCMV infections, allows for some insight into interactions which may be occurring.

According to the published literature, this is the first study to our knowledge

investigating the interaction between the human promyelocytic ce11 line HL60 and

HCMV. Our results suggest that HCMV does enter HL-60 cells, while the exact physical

association remains unclear. Viral antigens have been detected fiorn al1 three phases of

replication, however antigen positive ce11 cultures were not capable of transmitting

infectious virus. Viral DNA was detected within inoculated ce11 cultures but

confirmation for active gene expression codd not be made, since the Wal mRNA

detection assay (NASBA) did not provide conclusive results. Finally, EM analysis has

shown that vinidvirus-like particles were not detected at levels which would characterize

HCMV infection. Therefore, there is some support for replication of HCMV in HL-60

ce1Is provided by viral antigen and DNA detection, and support for the mntrary (no

transmission of virus, EM work). It is not possible to provide a definitive conclusion

conceming the extent to which HCMV replicates in HL-60 cells without fùrther

investigation.

Our results indicate that there appears to be some interaction between HCMV and

HL-60 cells in vitro. For further experimentation, it is necessary to obtain a greater level

of consistency using this mode1 for investigation. Thus, fbture studies should focus

initially on detmining factors to enhance antigen detection within HL-60 cells and to

obtain consistent results. Factors accounting for discrepant obsmations can be broken

d o m into three broad categones; ce11 culturing, virus harvesting, and technique. HL-60

cells are a continuously proliferathg human myeloid ce11 line predominantly consisting

of promyelocytes (Gallagher et al., 1 979) with approximately 1 0- 12% spontaneously

differentiating to more mature cells of the myeloid lineage (Collins et al., 1978).

Culturing HL-60 cells involves splitting and passaging in culture media to maintain ce11

densities between 2x 1 0' and 1 x 1 o6 cells/ml (Section 3.1.1). Therefore, a few factors

arise which may result in slight differences during HCMV inoculations. Some of these

include, the passage numbedage of cells which cm affect ce11 viability, membrane

composition, susceptibility to stress, as well as receptor interaction. A study conducteci by

Shimura and colleagues ( 1997) has shown that passaging of HL-60 cells predisposes the

ce11 population to apoptosis at room temperature. Rutter and colleagues (1W6) employed

pro ton magnetic resonance spectroscropy to show that earl y passage MRC-5 cells

compared with late passage MRC-5 cells demonstrate an increase in cholesterol and lipid

content. Yuan et al. (1 996) has shown that late passage MRC-5 cells are more susceptible

to oxidative stress than early passage cells. Thiele and colleauges (1987) first

demonstrated the influence of culture age on the susceptibility of MRC-5 cells to HCMV

infection. They found that monolayers beyond 3-1 1 days old were less susceptible to

HCMV, possibly due to older monolayers losing receptors for HCMV. Such findings

were confirmed by Fedorko et al. (1989).

The length of time cells had been in culture before medium renewal may affect

membrane properties and thus interaction with HCMV. A study by Levi et ai. (1997) has

shown that in HL-60 cells, there is decreased membrane lipid fluidity due to increase in

cholesterol concentration, upon incubation in culture medium. Another factor to consider

may be that, at the time of harvesting cells for inoculation, ce11 cultures which have

proIiferated to the upper limit of suggested cell density may behave differently than cells

which are cultureci at roughly 1 log lower ce11 density.

Virus culturing rnay also affect individual innoculation trials. HCMV was

propagated in confluent monolayers of MRCJ cells, and harvested when infected ce11

cultures showed greater than 80% CPE (4+), which was evident 4-6 days p.i (Section

3.2). Thus, virus hawested, titrated, and fiozen at different times rnay be a factor.

Finally, slight differences in technique or experimental conditions rnay affect

independent inoculation trials. For example, ce11 pelleting, resuspension, and transfer

during the inoculation procedure can provide stress upon the cells. Thus, cells in a given

tnal rnay respond differently to such situations, possibly amibutecl to their age and

cultunng specificities, as described earlier. Another factor accounting for differences

between independent trials is visualization by immunofluorescent microscopy. Since the

number of cells showing fluorescence is quite low, it is possible that an aliquot observed

rnay not contain any positive cells, or while scanning, positive cells rnay be missed.

Once this mode1 is working consistently, an important direction would be to

elucidate the route of virus entry. Is uptake of vinislvinis constituents occurring by way

of phagocytosis or is there a specific binding interaction between the virion and a host

ce11 receptor. Addressing the issue of a phagocytic phenornenon, HL-60 cells exhibit

phagocytic and chemotactic fiinctions cornmensurate with the percentage of mature cells

in the culture (Gallagher et al., 1979; Collins et al., 1978), however our studies have

indicated that there is no significant difference in IFA positivity between immature and

mature HL-60 cells. This suggests that phagocytosis rnay not be involved in at least the

early stages of myeloid ce11 differentiation. Considering viral ligand-host receptor

binding between HL-60 cells and HCMV, a number of viral envelope proteins which rnay

act as putative receptors have been studied in depth using known susceptible fibroblast

ce11 lines. Glycoprotein B (gB) is the major envelope protein and has been the focus of

most attention as a viral receptor (Mocarski, 1996). gB binds to heparin sulfate

proteoglycans (HSPG) (Compton, 1995; Compton et al., 1993) as well as annexin II

(Pietropaolo and Compton, 1997; Adlish et al., 1990) and is involved in virus

penetration, cell-to-ce11 spread, fusion of infected cells however is not involved in

attachent of the virus (Compton, 1995; Boyle and Compton, 1998; N a v m et al.,

1993). Similarily, studies involving glycoprotein H (gH) have shown that this envelope

protein binds to a 92.5 kDa receptor on fibroblast cells (Compton, 1995; Mocarski, 1996;

Keay et al., 1989; Keay and Baldwin, 1992) and it too is primarily involved in virus

penetration, and fusion of infected cells with no apparent involvement with virus

attachent (Compton, 1995; Fuller et al., 1989; Keay and Baldwin, 199 1; Keay and

Baldwin, 1995). Thus, it is still unclear which Wal receptor(s) are involved in

attachent of HCMV to fibroblast cells. Defining putative receptors involved in

attachent to HL60 cells may provide insight into virus attachent occuring in vivo.

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