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Entry of Epstein-Barr virus into lymphocytes and epithelial cells

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Entry of Epstein-Barr virus into lymphocytes and epithelial cells
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Miller, Nancimae
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xiii, 169 leaves : ill. ; 29 cm.

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Antibodies ( jstor )
B lymphocytes ( jstor )
Cell membranes ( jstor )
Cells ( jstor )
Epithelial cells ( jstor )
Epstein Barr virus infections ( jstor )
Fluorescence ( jstor )
Glycoproteins ( jstor )
Human herpesvirus 4 ( jstor )
Receptors ( jstor )
B-Lymphocytes -- virology ( mesh )
Cytopathogenic Effect, Viral ( mesh )
Department of Immunology and Medical Microbiology thesis Ph.D ( mesh )
Dissertations, Academic -- College of Medicine -- Department of Immunology and Medical Microbiology -- UF ( mesh )
Epithelium -- virology ( mesh )
Herpesvirus 4, Human -- pathogenicity ( mesh )
Membrane Fusion ( mesh )
Research ( mesh )
Viral Fusion Proteins ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph.D.)--University of Florida, 1991.
Bibliography:
Bibliography: leaves 150-168.
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Typescript.
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Vita.
Statement of Responsibility:
by Nancimae Miller.

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ENTRY OF EPSTEIN-BARR VIRUS INTO LYMPHOCYTES AND EPITHELIAL CELLS


By

NANCIMAE MILLER











A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

1991














DEDICATION





This dissertation is dedicated to my parents, John and Nancy Miller, who have

always encouraged all my endeavors and have provided me with love and support

throughout all that I have done.














ACKNOWLEDGEMENTS


I greatly appreciate the support and guidance from all those who helped me in

completing this work. My committee members, Drs. Sue Anne Moyer, William

Hauswirth, and John Dankert have been extremely encouraging and helpful. None of

this would have been possible without the excellent guidance from the chairperson of

my committee, Dr. Lindsey Hutt-Fletcher. She has been my teacher, my mentor, and

my friend. A special thanks goes to Dr. Alfred Esser for his input into the project and

the use of his spectrofluorometer. The time spent on this project has not been spent

alone, my thanks go to all the members of the laboratory past and present, especially

to Susan, Linda, Lisa, and Doug. Thanks are extended to my parents, my sister,

Michelle, and my brother, John, for always being there for me. Special thanks to

Dave for supporting me throughout the writing of this dissertation.















TABLE OF CONTENTS
page

DEDICATIO N ............................ ................... ii

ACKNOWLEDGEMENTS................. .... ............. iii

LIST OF ABBREVATIONS ...................................... vi

LIST OF FIGURES ........................................... vii

LIST OF TABLES ............................................ xi

ABSTRACT ................................................ xii

CHAPTERS

1 INTRODUCTION ......................................... 1

Discovery of Epstein-Barr Virus .............................. 1
Clinical Manifestations ..................................... 1
Description of EBV ........................................ 5
Entry of Enveloped Viruses into Animal Cells .................. .. 11
Measuring Fusion ......................................... 20
Purpose of This Work ...................................... 22

2 ESTABLISHMENT OF AN ASSAY TO MEASURE VIRUS FUSION .... 23

Introduction ............................................. 23
Materials and Methods ..................................... 24
Results ..................................... ............ 31
Discussion .............................................. 48

3 EFFECTS OF LYSOSOMOTROPIC AGENTS AND pH ON FUSION OF
VIRUS WITH LYMPHOCYTES .............................. 51

Introduction ............................................. 51
Materials and Methods ..................................... 52
Results ................................................ 56
Discussion .............................................. 83















4 MODIFICATION OF THE ENDOCYTIC PATHWAY TO DETERMINE THE
MECHANISM OF ACTION OF CHLOROQUINE ON VIRUS FUSION.. 86

Introduction ............................................. 86
Materials and Methods ..................................... 87
Results ................................................ 88
Discussion .............................................. 98

5 ISOLATION AND IDENTIFICATION OF EPITHELIAL CELLS EXPRESSING
A RECEPTOR FOR EPSTEIN-BARR VIRUS AND STUDIES OF VIRUS
ENTRY INTO THESE CELLS ............................... 101

Introduction ............................................. 101
Materials and Methods ..................................... 102
Results ................................................ 106
Discussion .............................................. 123

6 EFFECTS OF MONOCLONAL ANTIBODIES TO VIRUS MEMBRANE
PROTEINS ON BINDING AND ENTRY OF EPSTEIN-BARR VIRUS INTO
LYMPHOCYTES AND EPITHELIAL CELLS .................. ... 126

Introduction ............................................. 126
Materials and Methods ..................................... 127
Results ................................................ 129
Discussion ............................................. 144

7 SUMMARY AND CONCLUSIONS ............................ 147

Recapitulation ........................................... 147
Importance of Present Studies and Future Directions ............... 149

REFERENCES ...................................... ... 150

BIOGRAPHICAL SKETCH ..................................... 169














LIST OF ABBREVATIONS


AF 5-(N-octadecanoyl)aminofluorescein
AIDS acquired immunodeficiency syndrome
ASF asialofetuin
a.u. arbitrary units
ATP adenosine triphosphate
BL Burkitt's lymphoma
CMV cytomegalovirus
CR2 complement receptor 2
DMEM Dulbecco's modified eagle's medium
DNA deoxyribonucleic acid
EBNA Epstein-Barr virus nuclear antigen
EBV Epstein-Barr virus
FACS fluorescent activated cell sorter
FITC fluorescein isothiocyanate
HA hemagglutinin
HIV human immunodeficiency virus
HN hemagglutinin-neuramidinase
HSV herpes simplex virus
Ig immunoglobulin
I.M. infectious mononucleosis
LCL lymphoblastoid cell line
LSM lymphocyte separation medium
NaN3 sodium azide
NH4CI ammonium chloride
NPC nasopharyngeal carcinoma
OHL oral hairy leukoplakia
SCR short consensus repeat
SFV Semliki Forest virus
TPA 12-O-tetradecanoyl phorbol-13-acetate














LIST OF FIGURES


Figure page

2-1. Structural formula of octadecyl rhodamine B chloride (R. .......... 29

2-2. Excitation and emission spectra of R18-containing virions relieved of self-
quenching with Triton X-100 (infinite dilution). ....................... 32

2-3. Stability of self-quenching of R18-labeled virions. ................. 33

2-4. Relief of self-quenching of R1,-labeled virus bound to receptor positive
Raji cells and receptor negative Daudi cells. ........................ 39

2-5. Comparison of relief of self-quenching of R,,-labeled P3HR1-CI13 virus
bound to Raji cells, fixed Raji cells, or Molt 4 cells. ................... 40

2-6. Relief of self-quenching of R,,-labeled P3HR1-CI13 virus bound to tonsil
derived T-depleted leukocytes. ................................ 42

2-7. Comparison of relief of self-quenching of R8,-labeled P3HR1-CI13 virus
bound to tonsil derived B cells pre and post monocyte depletion by
adherance to plastic ........................................ 45

2-8. Relief of self-quenching of R,,-lab3led MCUV5 virus bound to fresh
T-depleted peripheral leukocytes. .............................. 46

2-9. Relief of self-quenching of R,,-labeled MCUV5 virus bount to BAT cells.. 47

3-1. Structural formula of 5-(N-octadecanoyl)aminofluorescein (AF). ........ 55

3-2. Relief of self-quenching of R,,-labeled MCUV5 virus bound to Raji cells
at pH 7.2 or pH 5.5. ....................................... 57

3-3. Relief of self-quenching of R18-labeled MCUV5 virus bound to BAT cells
at pH 7.2 or pH 5.5 ................. ....................... 58

3-4. Relief of self-quenching of R,,-labeled MCUV5 virus bound to T-depleted
leukocytes at pH 7.2 or pH 5.5. ................................. 59

3-5. Effect of preincubation of Raji cells with ammonium chloride or RPMI on
relief of self-quenching of R,,-labeled MCUV5 virus bound to cells........ 61









Figure DAe

3-6. Effect of preincubation of BAT cells with 20mM ammonium chloride or
RPMI on relief of self-quenching of R,,-labeled MCUV5 virus bound to cells.. 62

3-7. Effect of preincubation of T-depleted leukocytes with 20mM ammonium
chloride or RPMI on relief of self-quenching of R,1-labeled MCUV5 virus
bound to cells. ............................................ 63

3-8. Effect of preincubation of Raji cells with chloroquine or RPMI on relief of
self-quenching of R1,-labeled MCUV5 virus bound to cells.............. 64

3-9. Effect of preincubation of BAT cells with chloroquine or RPMI on relief of
self-quenching of R1,-labeled MCUV5 virus bound to cells.............. 65

3-10. Effect of preincubation of T-depleted leukocytes with chloroquine or RPMI
on relief of self-quenching of R8,-labeled MCUV5 virus bound to cells ...... 66

3-11. Effect of preincubation of Raji cells, BAT cells, and T-depleted leukocytes
with 5mM methylamine or RPMI on relief of self-quenching of R8,-labeled
MCUV5 virus bound to cells..................................... 67

3-12. Relative intensity of fluorescein isothiocyanate-dextran (FITC-dextran)
fluorescence as a function of pH.................................. 70

3-13. Excitation spectra at pH 7.4 of BAT cells containing FITC-dextran before
and after addition of monensin. ................................. 71

3-14. Excitation spectra at pH 7.4 and pH 7.0 of NH4CI treated BAT cells
containing FITC-dextran before and after addition of monensin ........... 72

3-15. Excitation spectra at pH 7.4 of chloroquine treated and untreated BAT
cells containing FITC-dextran before and after addition of monensin....... 74

3-16. Excitation spectra at pH 7.0 of methylamine treated and untreated BAT
cells containing FITC-dextran before and after addition of monensin....... 75

3-17. Fluorescence properties of virus labeled with AF at pH 6.0 to pH 7.4. .. 77

3-18. Relief of self-quenching of AF-labeled MCUV5 virus bound to Raji and
Molt 4 cells................................................. 79

3-19. Relief of self-quenching of AF-labeled MCUV5 virus bound to Raji cells
at pH 7.2 or pH 5.5........................................... 80

3-20. Relief of self-quenching of AF-labeled or R,1-labeled MCUV5 virus bound
to BAT cells at pH 7.2 ........................................ 81









Figure page

3-21. Effect of preincubation of BAT cells with ammonium chloride or RPMI
on relief of self-quenching of AF-labeled MCUV5 virus bound to BAT cells
at pH 7.2................. .......... ........................ 82

4-1. Effect of preincubation of Raji cells with sodium azide (NaN) or RPMI on
relief of self-quenching of R,,-labeled MCUV5 virus. ................... 89

4-2. Effect of preincubation of BAT cells with sodium azide (NaN) or RPMI on
relief of self-quenching of R18-labeled MCUV5 virus. ................... 90

4-3. Effect of preincubation of T-depleted leukocytes with sodium azide
(NaN), chlorpromazine, or RPMI on relief of self-quenching of R1,-labeled
MCUV5 virus................................................ 91

4-4. Effect of preincubation of BAT cells with chlorpromazine or RPMI on relief
of self-quenching of R,,-labeled MCUV5 virus. ....................... 93

4-5. Effect of preincubation of Raji cells with chlorpromazine or RPMI on relief
of self-quenching of R,,-labeled MCUV5 virus. ....................... 94

4-6. Effect of preincubation of Raji cells with leupeptin or RPMI on relief of
self-quenching of R1,-labeled MCUV5 virus. ......................... 95

4-7. Effect of preincubation of BAT cells with leupeptin or RPMI on relief of
self-quenching of R1,-labeled MCUV5 virus ................... ...... 96

4-8. Effect of preincubation of T-depleted leukocytes with leupeptin or RPMI
on relief of self-quenching of R8,-labeled MCUV5 virus .................. 97

5-1. Relief of self-quenching of R8,-labeled MCUV5 virus bound to parabasal
and basal epithelial cells ................... ................. 114

5-2. Fluorescence profile of HB5 antibody binding to basal epithelial cells. .... 116

5-3. Relief of self-quenching of R,,-labeled MCUV5 virus bound to unsorted
basal epithelial cells or basal epithelial cells from which HB5 (+) cells were
removed by cell sorting. ........................................ 117

5-4. Effect of preincubation of basal cells with chloroquine or RPMI on relief
of self-quenching of R,8-labeled MCUV5 virus bound to cells............. 119

5-5. Effect of preincubation of basal cells with methylamine, NH4CI, or RPMI
on relief of self-quenching of R1,-labeled MCUV5 virus bound to cells ...... 120

5-6. Effect of preincubation of basal cells with sodium azide or RPMI on relief
of self-quenching of R,8-labeled MCUV5 virus bound to cells............. 121









Figure pae

5-7. Effect of preincubation of basal cells with NH4CI or RPMI on relief of self-
quenching of AF-labeled MCUV5 virus bound to cells. ................. 122

6-1. Effect of preincubation with 100ug of monoclonal antibodies on relief of
self-quenching of R,,-labeled P3HR1-CI13 virus bound to Raji cells........ 133

6-2. Effect of preincubation of virus with monoclonal antibodies on relief of
self-quenching of R,,-labeled MCUV5 virus bound to BAT cells........... 136

6-3. Effect of preincubation with 100ug of normal mouse immunoglobulin or
antibody 72A1 on relief of self-quenching of R8,-labeled MCUV5 virus bound
to basal epithelial cells. ......................................... 141

6-4. Effect of preincubation with 100ug of normal mouse immunoglobulin or
antibody F-2-1 on relief of self-quenching of R,1-labeled MCUV5 virus bound
to basal epithelial cells. ........................................ 142

6-5. Effect of preincubation with 100ug of normal mouse immunoglobulin or
antibody E1D1 on relief of self-quenching of R,,-labeled MCUV5 virus bound
to basal epithelial cells. ...................................... .. 143









LIST OF TABLES


Table page

2-1. Effect of labeling with R,, on the ability of [3H] EBV to bind to receptor
positive and negative cells. ..................................... 35

2-2. Effect of monoclonal anti-EBV and anti-CR2 antibodies on the ability of
R18-labeled [3H] EBV to bind to receptor positive cells. ................. 36

2-3. Effect of labeling with R1, on the ability of MCUV5 virus to induce
immunoglobulin synthesis by fresh T-depleted human leukocytes ......... 38

2-4. Monocyte depletion of T-depleted human leukocytes by adherance
to plastic................................................... 44

3-1. Effect of labeling with AF on the ability of MCUV5 virus to induce
immunoglobulin synthesis by fresh T-depleted human leukocytes ......... 76

5-1. Cell counts and viability of cells recovered from infant foreskin epidermis. 107

5-2. Morphological distribution of epithelial cells in fractions from Percoll
gradient....................................... ............ 108

5-3. Reactivity of epithelial cells with the monoclonal anti-CR2 antibody HB5.. 110

5-4. Reactivity of epithelial cells with anti-CR2 antibodies. ............... 110

5-5. Microscopic analysis of virus binding and fusion with epithelial cells.... 112

6-1. Effect of antibodies F-2-1, E1D1, 72A1, and E8D2 on the ability of
MCUV5 virus to induce immunoglobulin synthesis by fresh T-depleted
hum an leukocytes............................................ 130

6-2. Effect of antibodies F-2-1, 72A1, and E8D2 on the ability of [3H] EBV to
bind to receptor positive cells. .................................. 132

6-3. Effect of antibody on the relief of self-quenching of R1,-labeled virus
added to T-depleted leukocytes. .................................. 135

6-4. Effect of antibody or unlabeled virus on binding of R,,-EBV .......... 137

6-5. Effect of soluble CR2 or 72A1 on binding of R,,-labeled EBV. ....... 139














Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy

ENTRY OF EPSTEIN-BARR VIRUS INTO LYMPHOCYTES AND EPITHELIAL CELLS



By

Nancimae Miller

May 1991

Chairman: Lindsey Hutt-Fletcher
Major Department: Immunology and Medical Microbiology

Epstein-Barr virus (EBV) is a human herpesvirus which causes infectious

mononucleosis and is associated with two cancers, Burkitt's lymphoma and

nasopharyngeal carcinoma. To understand the biologic activity of EBV, it is crucial to

understand how EBV infects cells, and what viral components are important to this

process. Epstein-Barr virus infects two cell types, B lymphocytes and epithelial cells.

To examine the early events in virus infection, binding and fusion, we have adapted an

assay that measures membrane fusion. Virus membranes were labeled with

concentrations of octadecylrhodamine (R8) or 5-(N-octadecanoyl)aminofluorescein

(AF) at which fluorescence is self-quenched. The fluorescence of AF is also sensitive

to changes in pH. Fusion and mixing of virus and cell membranes was measured in

terms of relief of self-quenching and was monitored kinetically.

The assay was used to compare virus fusion with lymphoblastoid cell lines,

lymphocytes recently transformed with EBV, normal B lymphocytes and epithelial









cells. Entry of EBV into all cell types occurred independent of exposure to low pH.

However, virus fusion with normal and recently transformed lymphocytes occurred

from within endocytic vesicles, whereas fusion with lymphoblastoid and epithelial cells

occurred at the plasma membrane.

The contribution to fusion made by virus envelope proteins to fusion was

studied with monoclonal antibodies that neutralized virus infectivity. Antibody to

glycoprotein gp85 inhibited fusion with all cells except epithelial cells. Antibody to

glycoprotein gp350, responsible for virus attachment to CR2 on lymphocytes, only

partially inhibited virus binding to epithelial cells and the remaining bound virus did not

fuse. Soluble CR2 inhibited virus binding to lymphocytes but only partially inhibited

binding to epithelial cells.

These studies document clear differences between virus entry into lymphocytes

and epithelial cells and suggest that the virus proteins involved in fusion with the two

cell types may be distinct.














CHAPTER 1
INTRODUCTION


Discovery of Epstein-Barr Virus

Epstein-Barr virus was discovered by electron microscopy during investigations

undertaken with lymphoblastoid cells cultured from a biopsy of an African Burkitt's

lymphoma in the early 1960s (Epstein and Barr, 1964). The virus was identified

morphologically as a member of the herpesviridae, but extensive virologic

investigations proved it to be distinct from any previously known herpesvirus; it could

not be transmitted to host cells known to be susceptible to herpes-simplex virus

(HSV), cytomegalovirus (CMV), or varicella-zoster virus (VZV) and was given the name

Epstein-Barr virus (Epstein et al., 1965). The uniqueness of EBV was confirmed

serologically when antisera to known herpesviruses failed to react in

immunofluorescence tests with cells carrying the virus (Henle and Henle, 1966).

Seroepidemiologic studies established the worldwide distribution of the virus in normal

healthy people.



Clinical Manifestations

Burkitt's Lymphoma

In the 1950s Dr. Dennis Burkitt became interested in a children's tumor in

Africa that was not only the most common children's tumor in Africa but also more

common than all other children's tumors added together (Burkitt, 1987). Also at that










time, M. A. Epstein was in search of a human cancer caused by a virus and became

interested in the tumor that Dr. Burkitt described. EBV fulfilled many requirements

used to define an oncogenic virus. Virus was present in all tumor cells but not normal

tissue from the same patient, patients had extremely high antibody titers to EBV, EBV

could immortalize human B lymphocytes in vitro, and EBV was capable of inducing

tumors in subhuman primates. However, there could be no simple causal relationship

between EBV and BL because the virus was found to infect humans worldwide at a

frequency of 90-100% (Evans, 1984). The virus was considered likely to play the role

of a cofactor in development of African BL, but additional cellular changes were

assumed to occur to create malignant BL cells. The presence of phenotypic

differences between EBV-genome containing BL cells and EBV-immortalized

nonmalignant cells provided support for this theory. All cell lines derived from BL

contained chromosomal translocations (Klein, 1983). The characteristic translocation

found in BL is a translocation of chromosome 8 with chromosome 14, but can also

involve 2 or 22 (Miyoshi et al., 1981). The c-myc oncogene has been localized to

chromosome 8 in humans. The translocations involve the juxtaposition of c-myc with

the immunoglobulin heavy chain gene cluster on chromosome 14, the kappa light-

chain genes on chromosome 2 and the lambda light-chain genes on chromosome 22

(Lenoir, 1987). It is likely that the c-myc oncogene plays a role in the development of

BL, but that the translocation is not induced by EBV. Rather, the appearance of

malignant cell clones that have altered c-myc may be facilitated when EBV causes

unrestricted proliferation of B cells in cooperation with immunosuppression from

persistent malaria infections that are holoendemic in central Africa.










Infectious Mononucleosis

Epstein-Barr virus (EBV) persists in those individuals it infects and induces

permanent seroconversion. The virus is transmitted horizontally and primary infection

usually takes place in childhood without apparent disease (Henle and Henle, 1979). if

the primary infection is delayed until adolescence or young adulthood, which happens

at a higher incidence in developed countries, infection leads to infectious

mononucleosis (I.M.) in about 50% of cases (Niederman et al., 1970). The first

suggestion that EBV was the cause of I.M. came when a laboratory technician (in the

laboratory of Drs. W. and G. Henle) who had previously lacked EBV antibodies,

seroconverted in the course of I.M.. Her circulating lymphocytes failed to grow in vitro

prior to the illness, but gave rise to permanent cultures when collected during the

acute phase or during early convalescence (Henle et al., 1968). Following this

discovery, a prospective study was conducted at Yale where it was found that all pre-

I.M. sera collected lacked antibodies to EBV, while the corresponding acute and

convalescence phase sera contained EBV antibodies (Niederman et al., 1968).

The initial step in pathogenesis of any primary infection with EBV, whether

symptomatic or not, is entry of the virus into the oropharynx and subsequent

replication at that site. The clinical manifestations of EBV-induced I.M. are thought to

be caused by a rapid polyclonal T and B cell proliferation. Primary replication of virus

occurs in pharyngeal epithelium (Sixbey et al., 1983, 1984) from which circulating B

cells are infected and transformed allowing their rapid proliferation (Rickinson et al.,

1987; Svedmyr et al., 1984). The symptoms that the I.M. patient experiences are

thought to result from the conflict in the immune system as an aggressive T cell

response is mounted in order to keep the B cell proliferation in control. Since the










immune system is so important for keeping the abnormal cell proliferation in control,

lymphoproliferative disorders can occur in the immunocompromised patient during

primary infection or thereafter due to failure to control the persisting latent infection.

Particularly at risk are immunosuppressed organ transplant recipients and those with

acquired immunodefiency syndrome (AIDS) (Cleary et al., 1986; Fauci, 1988; Hanto et

al., 1985; Purtilo, 1985).

Nasooharvnaeal Carcinoma

Epstein-Barr virus has also been associated with another human cancer,

nasopharyngeal carcinoma (NPC) (Anderson-Anvret et al., 1979; de-The, 1982). The

association was initially based on the finding of high antibody titers to EBV in all NPC

patients examined (de-The and Zeng, 1987; Henle and Henle, 1976). The antibody

levels for viral capsid antigen were unusually high, only paralleled by BL sera, which

increased the likelihood of involvement of EBV with the carcinoma. In 1976, EBV DNA

was found in all the undifferentiated carcinomas of the nasopharynx studied (Henle

and Henle, 1976). Subsequently, EBV DNA has been consistently found in all

undifferentiated carcinomas of the nasopharynx and has also been detected in

differentiated forms of the carcinoma as well (de-The and Zeng, 1987; Raab-Traub et

al., 1987). Nasopharyngeal carcinoma occurs throughout the world, but occurs with a

much higher incidence in populations of southern Asia. The high frequency of NPC in

the Kwantung Providence of southern China (de-The and Zeng, 1987) suggests that

other factors, perhaps genetic or environmental, are acting with EBV in the

development of the cancer (Henderson et al., 1976; Klein et al., 1978). Despite

considerable effort to identify carcinogenic substances and cultural patterns which

might operate as cofactors, no firm identification of such a factor has yet been made.










Oral Hairy Leucoolakia

Epstein-Barr virus is also associated with oral hairy leucoplakia (OHL), a

proliferative lesion of the lateral tongue epithelium found in persons infected with HIV

(Greenspan et al., 1984). The presence of OHL indicates that patients are severely

immunocompromised and has proven to be a valuable prognosticator of the onset of

AIDS (Greenspan et al., 1987; Schiodt et al., 1987). Studies of OHL lesions reveal

EBV particles within the nucleus, cytoplasm and the intercellular spaces of epithelial

cells (Sciubba et al., 1989). In the basal layers, the BZLF1 gene is expressed, which

activates the switch from latency into replication. In the upper third of the epithelium,

structural proteins and viral envelope components are found ibidd). There is

temporary regression of the OHL lesions when the patients are treated with acyclovir,

but the lesions recur weeks or months after cessation of acyclovir therapy, indicating

that EBV plays an active role in development of the lesions (Resnick et al., 1988).



Description of EBV

Classification and Morpholoav

Epstein-Barr virus is classified as human herpesvirus 4 and as a member of the

gamma herpesvirus subfamily (Roizman, 1982). Morphologically, EBV is

indistinguishable from other members of the herpes family. The diameter of the

mature virus particle is about 150 to 180 nm. The virus envelope is acquired as the

virus buds through the nuclear membrane. The envelope consists of at least five

proteins that are encoded by the virus, four of which are glycosylated. The lipid

component of the envelope is derived from host cell membrane in which cellular

proteins have been replaced by those encoded by the virus (Spear, 1980). Within the










envelope is a nucleocapsid exhibiting isosahedral symmetry which contains 162

capsomeres arranged in hexagonal and pentameric array. An amorphous tegument

fills the cavity between the nucleocapsid and the envelope. Inside the nucleocapsid is

the core virus particle consisting of core proteins and a large double-stranded

deoxyribonucleic acid (DNA) genome of approximately 172,000 base pairs in length

(Kieff et al., 1982). The viral mRNAs are translated in the cytoplasm and many of the

translational products then return to the nucleus where the nucleocapsid is

assembled.

Tropism and Latency

An unique feature of the gamma herpesviruses is their limited host range. All

members of this group infect lymphoblastoid cells in vivo and in vitro. The only

human member of the group is EBV and was originally identified as having tropism for

human B lymphocytes. Other members of the group include Marek's disease virus of

chickens and Herpes ateles and Herpes saimiri virus of new world monkeys. These

viruses infect T cells (Fleckenstein and Desrosiers, 1982; Nonoyama, 1982). The host

range of EBV in vitro is restricted to B lymphocytes of humans and new world

primates. EBV establishes latency in these cells and immortalizes them. Latently

infected lymphocytes, but not those fully permissive for virus replication, have been

demonstrated in vivo. The infected cells retain the complete viral genome and

express a restricted set of viral genes necessary to maintain latency (Hayward and

Kieff, 1976; Pritchett et al., 1975).

Three types of latently infected cells have been extensively studied,

lymphoblastoid cell lines (LCLs), Burkitt-lymphoma cells (BL) and nasopharyngeal

cells (NPC). All three types express Epstein-Barr nuclear antigen 1 (EBNA-1), which is










one of the latently transcribed genes that is necessary to maintain the episomal form

of the EBV DNA (Fahraesus et al., 1988; Rowe et al., 1987). Additional latently

transcribed genes EBNA-2, EBNA-3-6 and latent membrane protein (LMP) are

expressed in LCLs, but are down-regulated in BL and NPC cells (Klein, 1989; Rowe et

al., 1987).

For a long time it was generally accepted that EBV infected only B cells in vivo.

Recently, a second target for EBV, the undifferentiated epithelial cell, has been

identified (Greenspan et al., 1985; Lemon et al., 1977; Sixbey et al., 1984, 1987; Wolf

et al., 1984). The epithelial cell is permissive for replication and is thought to be the

source of virus that is shed in the oropharynx. Cultures of human epithelial cells have

been transfected (Grogan et al., 1981) and directly infected in vitro (Sixbey, 1983), but

the only cell currently available for studying the virus replication cycle in vitro is the

lymphocyte. Lymphocytes latently infected with EBV provide an unique system for

studying the biochemistry of herpes virus latency.

The ability of EBV to infect B lymphocytes is initiated by attachment of virus to

the 145-kilodalton (kDa) cell membrane glycoprotein, CR2, which also binds the C3d

fragment of complement (Fingeroth et al., 1984; Nemerow et al., 1985b). Recently it

has been shown that epithelial cells also express a receptor for virus attachment but it

is lost during differentiation of the epithelium (Sixbey et al., 1984, 1987; Young et al.,

1986). The B cell CR2 receptor is also lost during differentiation to the plasma cell

(Tedder et al, 1984). Immunoprecipitation from the surface of epithelial cells with an

anti-CR2 antibody yielded a 200-kilodalton membrane protein (Young et al., 1989).

The CR2 receptor has been detected on three T-lymphoblastoid cell lines (Fingeroth et

al., 1988) and on a fraction of normal human peripheral blood T lymphocytes (Fischer










et al., 1991), these findings suggest that the tropism of EBV for B lymphocytes may

rely on factors other than receptor specificity.

Permissiveness of Virus Replication in Vitro

Cultures of EBV-infected lymphocytes vary in their permissiveness for viral

replication, most cultures being nonpermissive, but replication does occur in a small

fraction of cells in some cultures. The nonpermissiveness of EBV infection has made

it difficult to study virus replication and also limits the amounts of purified virus

available for studying the components of mature virus particles. Clones of infected

lymphocytes that are more permissive of virus replication have been selected (Miller

and Lpman, 1973) and have facilitated studies of the virus replication cycle and

biochemical analyses. Two isolates of virus have been extensively studied, B95-8 and

P3HR1. The B95-8 strain is produced by a cell line derived from a clone of marmoset

lymphocytes that were infected with virus obtained from a culture of lymphocytes from

a patient with infectious mononucleosis. The B95-8 viral DNA has been completely

sequenced (Baer et al., 1984) and has been the prototype used for gene mapping.

The P3HR1 cell line is a clone of the Jijoye Burkitt-tumor derived cell line (Hinuma et

al., 1967). The P3HR1 cells are more permissive than the parent clone for virus

replication and the virus produced by P3HR1 cells lacks the ability to growth-transform

noninfected B lymphocytes (Miller et al., 1974).

Genome Structure

The linear double stranded EBV genome contains nonrandom single stranded

breaks (Pritchett et al., 1975). When the genome is carried in the latent state it

circularizes via joining of the terminal repeated DNA sequences at either end of the

molecule (Dambaugh et al., 1980). The genome consists of five large regions of










unique DNA domains, U1-U5, which are separated by four regions of internal repeats,

IR1-IR4, and flanked on both ends with tandem repeats (Cameron et al., 1987;

Dambaugh and Kieff, 1982; Given et al., 1979). Latently infected cells usually contain

more than one copy of the complete EBV genome, which can be integrated, but is

most often found to exist as a covalently closed circular episome (Lindahl et al.,

1976). Episomes are replicated once per cell cycle by DNA polymerase early in S

phase (Adams, 1987; Hampar et al., 1974). Replication of the episomal DNA is

proposed to occur from a circular form in a manner similar to that of SV40 DNA

(Gussander and Adams, 1984).

At least two EBV types have been identified in human populations (Rowe et al.,

1989; Sculley et al., 1988). The two strains have significantly divergent EBNA 2

sequences. These were designated EBV type A and B, but are more appropriately

designated EBV-1 and EBV-2, so as to parallel the HSV-1 and HSV-2 nomenclature.

EBV-1 and EBV-2 are considerably more closely related to each other than are HSV-1

and HSV-2. Analysis of hosts shedding both EBV types in the oropharynx revealed

only type 1 in peripheral blood lymphocytes (Sixbey et al., 1989). Oral hairy

leucoplakia lesions consistently contain EBV DNA of the type 2 (Raab-Traub and

Sixbey, personal communication). The type 2 strain transforms B lymphocytes less

efficiently than the B95-8 type 1 strain and B cell transformants of the type 2 are more

difficult to maintain in culture (Rickinson et al., 1987).

Membrane Proteins

The membrane antigen complex was initially described by surface fluorescence

of EBV-producing cells using human immune sera. The complex was further resolved

by analysis of infected cell membranes into three major envelope glycoproteins of










300-350 kda, 200-220 kDa, and 85 kDa (Edson and Thorley-Lawson; Thorley-Lawson

and Edson, 1979). Three additional membrane associated proteins, p105, gp78/55,

and the product of the BDLF3 open reading frame, have also been studied. The p105

protein is not glycosylated and differs from the other membrane proteins in that its

synthesis is not influenced by the viral DNA inhibitor phosphonoacetic acid

(Balachandran et al., 1986). Glycoprotein gp78/55 is the product of the BILF2 open

reading frame (Mackett et al., 1990). Antibodies to a bacterially expressed BDLF3

protein reacted with virus and with the plasma membrane of virus infected cells.

Additional membrane proteins are likely to exist since there are many unassigned

open reading frames which have characteristics of those encoding membrane

proteins.

Glycoproteins gp300-350 and gp200-220 are present in large amounts in the

virus envelope and have been extensively characterized. Glycoprotein gp350 and

gp220 are encoded by the same open reading frame from which an intron is

removed, without change in reading frame to produce gp220 (Beisel et al., 1985;

Hummel et al., 1984). Monoclonal antibodies that recognize gp350/220 are capable

of inhibiting virus binding (Nemerow et al., 1987). Binding of EBV to CR2 is mediated

by attachment of gp350 (Nemerow et al., 1987; Tanner et al., 1987) and possibly also

by attachment of gp220 (Wells et al., 1982). A common epitope in gp350 and gp220

has been identified as a primary region responsible for virus binding to B lymphocytes

by attachment to CR2 (Nemerow et al., 1989).

Glycoprotein gp85 is also present in the envelope but in less abundant

amounts than gp350/220. Glycoprotein gp85 has been recently mapped to the

BXLF2 open reading frame in two independent studies (Heineman et al., 1988; Oba








11

and Hutt-Fletcher, 1988). Although no function has been conclusively ascribed to this

molecule, antibodies to it can neutralize virus infectivity (Strnad et al., 1982), thus

implying that it may play a role in the initiation of cell infection. The function of

gp78/55 has not been determined; neither a monoclonal antibody nor a polyclonal

sera to the recombinant molecule neutralized the ability of virus to transform cells.

Preliminary studies with recombinant vaccinia virus expressing the gene

product from the BDLF3 open reading frame have immunoprecipitated a protein of

90kd using serum from a patient with chronic mononucleosis (L.C. Davenport and

L.M. Hutt-Fletcher, personal communication).



Entry of Enveloped Viruses into Animal Cells

The earliest events in the virus replication cycle are attachment, penetration,

and uncoating. The initial event, virus attachment to specific cell receptors, is a major

determinant of cellular tropism and pathogenesis of viruses. Virus membrane proteins

protruding from the virus envelope mediate virus attachment to host cells. These

membrane proteins have other functions in addition to cell recognition and

attachment, namely fusion, penetration, and possibly, direction of egress of the virus.

Enveloped viruses enter cells by fusing with cellular membranes (Lonberg-Holm and

Philipson, 1974; White et al,, 1983; White, 1990). Since fusion is an energetically

unfavorable process, viruses utilize specific proteins to fuse with host cells and

introduce their genetic material into the host cell (White, 1990). Two pathways of

entry are commonly utilized and viral fusion reactions fall into two classes, low pH-

dependent and pH-independent. Some viruses, such as Sendai (Scheid and

Choppin, 1976), deposit their nucleocapsids directly into the cytoplasm by fusing with










the plasma membrane at physiologic pH. The alternative route, adsorptive

endocytosis followed by vesicle membrane fusion, is utilized by Semliki Forest virus

(SFV) (Helenius et al., 1980a; Marsh and Helenius, 1980), influenza A (Matlin et al.,

1981; White et al., 1981; White et al., 1983), Sindbis (Boggs et al., 1989) and vesicular

stomatitis virus (VSV) (Matlin et al., 1982; White et al., 1983). In most cases studied,

fusion is induced by a specific viral membrane 'fusion protein'.

Adsorptive Endocvtosis

Adsorptive endocytosis, also known as 'receptor-mediated' endocytosis, is a

process by which macromolecules are taken into cells. This process is initiated by

binding of a ligand to a cell surface receptor followed by invagination of the

membrane forming a vesicle (Goldstein et al., 1979; Silverstein et al., 1977).

Specialized regions of the plasma membrane have been morphologically identified as

sites for adsorptive endocytosis of some viruses (Goldstein et al., 1979). These

regions, the coated pits, are thought to concentrate receptors and receptor-ligand

complexes at sites of internalization. The protein clathrin is a major component of the

coated pits (Pearse, 1975) and is thought to participate in the early stages of

endocytosis (Doxsey et al., 1987). The process of adsorptive endocytosis as a

mechanism for virus entry has been documented for several viruses (White et al.,

1981; White et al., 1983; White, 1990). Semliki Forest virus (SFV), a togavirus, has

been widely studied since its isolation in 1944 (Smithbum and Haddow, 1944). It is

one of the best characterized enveloped viruses due to its simple structure. The

nucleocapsid envelope is a host derived lipid bilayer in which virus encoded

glycoproteins are inserted. This virus gains entry into cells by accumulation in coated

pits that are endocytosed. The endosomes become acidified, providing conditions










that trigger fusion of the virus envelope with the vesicle membrane (Helenius et al.,

1980b; Kielian and Helenius, 1985; Marsh and Helenius, 1980; White and Helenius,

1980; White et al., 1980). The entry of vesicular stomatitis virus (VSV) has been

reported to resemble that of SFV (Clague et al., 1990; Matlin et al., 1982). Influenza

virus, an orthomyxovirus, is also taken into cells by adsorptive endocytosis followed

by fusion of the viral membrane with the endosomal membrane (Matlin et al., 1981;

White et al., 1981; Yoshimura and Ohnishi, 1984).

Fusion at the Plasma Membrane

Fusion directly at the plasma membrane is utilized by paramyxoviruses

(Choppin and Compans, 1975a). The best studied member of this group is Sendai

virus, whose glycoproteins have been extensively characterized. The entry of Sendai

involves initial attachment of virions to the cell surface and subsequent fusion between

the viral envelope and plasma membrane (Choppin and Scheid, 1980; White et al.,

1983). It is well established that binding is mediated by the HN protein and fusion is

initiated by the F protein, both of which are spike-like projections on the surface of the

virus (Choppin and Scheid, 1980). Fusion activity has been shown to be critically

temperature dependent, optimally occurring at 370C, while fusion is insignificant at

temperatures below 23C (Hoekstra et al., 1984).

The major entry mechanism for human immune deficiency virus (HIV), a T-

lymphotropic retrovirus, is reported to be fusion with the plasma membrane at the cell

surface (Maddon et al., 1988; McClure et al., 1988; Stein et al., 1987). Previous data

from Maddon et al. (1986), proposed that HIV entry into T lymphoblastoid cells

occurred after endocytosis because the virus receptor, CD4, was internalized. The

key findings of Stein et al. showed that the entry of HIV was not low-pH-dependent,










and although they found no evidence of an endocytic entry pathway, they did not rule

out the possibility that virus could enter by both pathways in a pH-independent

manner. Analysis of cells expressing a mutant form of CD4 that had impaired ability

to undergo endocytosis revealed that HIV infection did not require endocytosis of its

receptor, CD4 (Maddon et al., 1988).

For EBV, studies utilizing electron microscopy and immunoelectron microscopy

have reported direct fusion at the plasma membrane of EBV with the lymphoblastoid

cell line, Raji (Nemerow and Cooper, 1984a; Seigneurin et al., 1977). Virus

nucleocapsids were found in the cytoplasm directly beneath the cellular plasma

membrane, while virus was never found to be bound to the clathrin-coated areas of

the plasma membrane, nor observed in endocytic vesicles. The same studies using

normal B lymphocytes revealed transfer of membrane bound virus into vesicles.

These vesicles were distinct in size and appearance from clathrin-coated vesicles.

After 30 minutes at 370C very few virus particles remained in the vesicles.

Membrane Fusion Proteins

A virus envelope has a relatively simple protein composition that has three

main functions: facilitation of assembly and egress of virus particles, protection of the

genome during the extracellular transport of virus, and delivery of nucleocapsids into

host cells. The following viruses have proteins well characterized for ability to mediate

viral and cell fusion: Sendai, Semliki Forest, influenza, and vesicular stomatitis virus.

Fusion proteins identified to date are glycoproteins which span the bilayer and have

the bulk of their mass exposed externally. The transmembrane anchor region of the

glycoprotein is frequently composed of hydrophobic residues that favor alpha helix

formation.










The envelope of Sendai virus, a paramyxovirus, has two proteins. The

hemagglutinin-neuramidase (HN) protein is responsible for attachment of the virus to

cell surface sialic acid residues. The fusion (F) protein initiates fusion at the plasma

membrane allowing virus penetration, virus-induced cell fusion and hemolysis (Hsu et

al., 1981; Scheid and Choppin, 1974; Scheid and Choppin, 1976). The F protein

consists of two sulfhydryl-linked glycopeptides (F, and F) resulting from proteolytic

cleavage of an inactive precursor (F) by a host cell enzyme (Hsu et al., 1982).

Viruses produced by cells that lack a suitable protease for F protein activation are

noninfectious (Hsu et al, 1982). F2 corresponds to the N-terminus of F,, and the

protein is anchored in the bilayer through F,. The N-terminus of F,, resulting after

cleavage of F, has been found to be unusually hydrophobic (Gething et al., 1978) and

it was suggested that the hydrophobic terminal peptide might play a role in fusion.

Support for this role has been provided by experiments with synthetic peptides

corresponding to the hydrophobic amino-terminus of F, showing that such molecules

inhibit virus fusion (Richardson et al., 1980). The amino acid sequence in this region

is highly conserved among paramyxoviruses (Scheid et al., 1978).

Orthomyxoviruses also have two types of spike glycoproteins which have

neuraminidase, hemagglutination, and fusion activities. One of the glycoproteins is a

neuraminidase (NA) and the other, the hemagglutinin (HA), has the capability to bind

to cell surface sialic acid residues and to catalyze fusion (Choppin and Compans,

1975b; White et al., 1982). Unlike paramyxoviruses, orthomyxoviruses are

endocytosed and fuse with the endocytic vesicle. The HA consists of two disulphide

linked glycopeptide chains, HA, and HA2, resulting from proteolytic cleavage of a

precursor glycoprotein HA,. The cleavage is irrelevant to adsorption, but is a










prerequisite for infectivity (Lazarowitz and Choppin, 1975; White et al., 1983). The

cleavage generates a new N-terminus on HA2 which is hydrophobic and highly

conserved in different influenza strains and has partial homology with the N-terminus

of F,. Synthetic peptides analogous to the N-terminus sequence of HA2 inhibit

infectivity by influenza viruses (Gething et al., 1986; Richardson et al., 1980). The HA

molecule in its neutral form is a trimer and the hydrophobic fusion peptide in each

monomer is unexposed until the low pH of the endocytic vesicle causes partial

dissociation of the HA trimer, thus exposing the fusion peptide which can insert into

the target bilayer (Doms et al., 1985; Schlegel et al., 1982) and initiate endosomal

membrane fusion. Collective research findings suggest that the pH induced

conformation does not involve any changes in secondary structure and that the stem

region of the spike remains trimeric. However, elements of the spike change their

relative positions with the globular heads dissociating from one another by bending

about a hinge region. This movement of the three proteins composing the spike is

thought to release the terminal fusion peptide from the molecular interior (Doms et al.,

1990; Doms and Helenius, 1988; Harter et al., 1989; Ruigrok et al., 1988; Stegmann et

al., 1987, 1989; Wharton, 1987; Wharton et al., 1988; White et al., 1983; White and

Wilson, 1987; Wiley and Skehel, 1987). The HA is the only membrane fusion protein

for which a crystal structure is known (White, 1990).

The envelope spike of Semliki Forest virus (SFV), a togavirus, consists of a

complex of three glycopeptides, El, E2, and E3. El and E2 are transmembrane

glycoproteins; E3 is noncovalently associated with E2 and is external to the bilayer.

This virus does not fuse with the plasma membrane at physiologic pH (Helenius et al.,

1980a). Virions are endocytosed and a fall in pH within the endocytic vesicle activates










membrane fusion (Marsh et al., 1983a). Lysosomotropic agents, which elevate

endosomal pH, inhibit SFV penetration (Helenius et al., 1982). Semliki Forest virus

can fuse directly with the plasma membrane in vitro at low pH (White et al., 1980).

The SFV spike glycoproteins have been shown to be fusogenic in the absence of

other virus components (Marsh et al., 1983b). As far as the role of the glycopeptides

are concerned, it has been suggested that the peptide El may be directly involved in

the fusion activity (Kielian and Helenius, 1985). Both SFV and Sindbis, another

togavirus, have El proteins containing a hydrophobic peptide segment located close

to the N-terminus, and this segment has an external position in the virus membrane

(Garoff et al., 1980; White et al., 1983). Since El and E2 occur as a complex, E2 may

also participate in the fusion reaction. The role of E3 is not clear, it is a small

peripheral glycopeptide and there is no homologue in Sindbis virus (Welch and

Sefton, 1979).

Vesicular stomatitis virus (VSV), a rhabdovirus, has only one type of envelope

glycoprotein, designated the G-protein. The G-protein has a hydrophobic region near

the C-terminus forming the intramembranous domain. A small hydrophilic sequence

at the C-terminus is in contact with the cytoplasm. The larger N-terminal domain,

containing the oligosaccharide chains, is exposed to the exterior of the cell (Rose et

al., 1980; Rose and Gallione, 1981). The G-protein has been cloned and sequenced

(Rose and Gallione, 1981). Eukaryotic cells expressing the cloned G-protein gene

fuse, at low but not at neutral pH, indicating that this protein is both necessary and

sufficient for fusion activity (Reidel et al., 1984). In addition, at low pH, the G-protein

spikes reversibly aggregate at the ends of virus particles (Brown et al., 1988); this

observation may be potentially relevant to determining the mechanism of fusion for










this virus. The fusion activity has been shown to occur at the plasma membrane if

cells with VSV attached to their surfaces are placed in a low pH medium (Blumenthal

et al., 1987; Matlin et al., 1982).

Herpesviruses are considerably more complex. The best studied, herpes

simplex virus (HSV), has an envelope that contains at least nine glycoproteins, five of

them have been characterized and sequenced (Bzik et al., 1984; Frink et al., 1983;

Gompels and Minson, 1986; McGeoch et al., 1985; Pellet et al., 1985; Watson et al.,

1982). Studies indicate that the receptor molecules recognized in one of the initial

binding events are heparan sulfate proteoglycans (WuDunn and Spear, 1989).

Recently, it was determined that glycoprotein gC is principally responsible for virus

adsorption to cells (Herold et al., 1991). Glycoprotein gC bound heparin and virions

devoid of gC exhibited significant impairment in adsorption and penetration. Three of

the glycoproteins, namely gB, gD, and gH, induce antibodies capable of neutralizing

HSV infectivity in the absence of complement and have been implicated in virus

penetration (Fuller and Spear, 1987; Gompels and Minson, 1986, Sarmiento et al.,

1979). Evidence implicating gB in penetration comes from studies of temperature

sensitive HSV-1 mutants that fail to process precursor gB molecules to mature forms

at nonpermissive temperature. The virions produced are noninfectious but can bind

to cells and the block to their infectivity can be overcome by treating virus-cell

complexes with the membrane fusing agent polyethylene glycol (Little et al., 1981;

Sarmiento et al., 1979). Neutralizing anti-gD monoclonal antibodies have been shown

to block HSV infection by preventing virus-cell fusion at the plasma membrane (Fuller

and Spear, 1987) and antibodies to this glycoprotein also block HSV-induced cell-cell

fusion, a process which may be analogous to the virus-cell fusion required for entry










(Noble et al., 1983). Virus lacking gB (Cai et al., 1988) or gD (Johnson and Ligas,

1988) attaches but does not penetrate. The glycoprotein gH is present in the viral

envelope at concentrations at least 10-fold lower that gD (Richman et al., 1986).

Despite this fact, antibodies against gH have neutralizing activity comparable to that of

antibodies against gD (Minson et al., 1986). A monoclonal antibody to gH has also

been shown to exhibit anti-fusion activity ibidd). Thus three glycoproteins, gB, gD, and

gH, are likely either to induce or influence the fusion process which occurs in a pH-

independent manner at the surface of the cell. There is no evidence to suggest that

they act as a single functional heteropolymer. Homodimers of gB extracted from

virions or infected cells are not associated with other glycoproteins (Claesson-Welsh

and Spear, 1986), and gB and gD have been shown to form morphologically distinct

structures in the virion envelope (Stannard et al., 1987).

Entry of Epstein-Barr Virus

Infection of B lymphocytes and epithelial cells with EBV is initiated by

attachment of virus to a 145-kilodalton cell membrane glycoprotein, CR2, which also

serves as the receptor for the C3d fragment of the complement cascade (Cooper et

al., 1990; Fingeroth et al., 1984; Nemerow et al., 1985b; Sixbey et al., 1987).

Expression of the CR2 molecule on both cell types is linked to cell differentiation.

CR2 expression on human B lymphocytes is lost at the plasma cell stage of

differentiation (Tedder et al., 1984). Immunofluorescent studies have demonstrated

expression of CR2 on epithelia in a differentiation-linked manner as it is on B

lymphocytes (Sixbey et al., 1987; Young et al., 1986, 1989). Binding of EBV to CR2 is

mediated by attachment of at least one virus membrane glycoprotein, gp350










(Nemerow et al., 1987; Tanner et al., 1987), and possibly also by attachment of gp220

(Wells et al., 1982).

Penetration of virus has been studied in normal B cells and lymphoblastoid cell

lines. Virus fuses with the membrane of the lymphoblastoid cell line Raji at the cell

surface and CR2 is not internalized (Nemerow and Cooper, 1984a; Tedder et al.,

1986). In normal B cells, both receptor and virus are endocytosed into thin-walled

nonclathrin coated vesicles before fusion occurs ibidd).

The virus envelope protein mediating the fusion event has not been

conclusively identified. The EBV envelope glycoprotein, gp85, which has been

recently mapped to the BXLF2 open reading frame of EBV DNA does, however have,

characteristics of a fusion protein (Oba and Hutt-Fletcher, 1988; Heineman et al.,

1988). Computer assisted analysis of the sequence indicates that it is overall a

hydrophobic molecule with a potential N-terminal signal sequence and a C-terminal

anchor sequence. The sequence also includes a stretch of 16 extremely apolar amino

acids that could be a fusion sequence (Oba and Hutt-Fletcher, 1988). The gp85

glycoprotein has homology with the herpes simplex virus glycoprotein gH, and the

varicella-zoster virus gplll, which are involved in cell to cell fusion.



Measuring Fusion

The common procedures used to examine fusion of biological membranes,

such as microscopic or cytochemical techniques, are frequently difficult to quantitate

and have low sensitivity; extensive fusion activity may be required before it can be

detected. The use of radioisotopes to measure fusion does not permit continuous

monitoring of the fusion process and it is necessary to separate fused and nonfused










membranes in order to quantitate fusion events. Electron spin labels have been used

extensively with virus systems (Maeda et al., 1975, 1981, Lyles and Landesberger,

1979) but the extent of fusion is difficult to quantitate and continuous monitoring of the

fusion event is technically challenging. Assays utilizing fluorescent probes are much

faster and easier to perform than assays using electron spin probes and easily permit

continuous monitoring of the fusion events. The assay presented and utilized

throughout this work relies upon the relief of fluorescence self-quenching of the

fluorophore octadecyl rhodamine B chloride.

Quenching of fluorescence intensity can occur by a variety of mechanisms.

These include collisional processes with specific quenching molecules, excitation

transfer to nonfluorescent species, and complex formation or aggregation that forms

nonfluorescent species, also known as concentration quenching. Quenching of

fluorescence by added substances or by impurities can occur by a collisional process.

Molecular oxygen is one of the most widely encountered quenchers. This is because

02 is a triplet species in its ground electronic state and is able to transfer unpaired

electrons to the fluorescent species which is in the singlet state. The fluorescence

quenching of octadecyl rhodamine B chloride (R,) is due to complex formation and is

dependent upon the concentration of the fluorophore in the lipid-containing

membrane. The self-quenching is concentration dependent because of the formation

of excimers (excited dimers) when interactions of the excited-state species occurs.

Most excited fluorophores emit fluorescence from a singlet state. The formation of

dimers results in quenching since the doublet species is not fluorescent (Tinoco et al.,

1985). The efficiency of self-quenching is directly proportional to the ratio of R,1 to

total lipid. When the fluorophore is incorporated into a lipid bilayer at concentration










up to 9 mol% with respect to total lipid, the efficiency of the self-quenching is

proportional to its surface density (Hoekstra et al., 1984). When fusion of a labeled

membrane with a nonlabeled membrane occurs, there is a decrease in the surface

density of the fluorophore and this results in a proportional relief of the self-quenching.



Purpose of this Work

The overall objective of this dissertation is to understand how Epstein-Barr virus

enters its two target cells, the B lymphocyte and the epithelial cell. A greater

understanding of the conditions for successful virus penetration into both epithelial

cells and lymphocytes, as well as the viral components necessary to mediate these

events, will help to understand the unique tropism of EBV for B lymphocytes and

epithelial cells. This work presents experiments undertaken to develop an assay for

measuring fusion of EBV with cell membranes and application of the assay to follow

fusion with lymphoblastoid cells, B lymphocytes, and epithelial cells.














CHAPTER 2
ESTABLISHMENT OF AN ASSAY TO MEASURE VIRUS FUSION


Introduction

Membrane fusion is an effective process for delivering membrane-bound

contents from one cellular compartment to another. Viruses take advantage of this

important process and utilize membrane fusion for entry into cells. The mechanism of

fusion has become one of the most intriguing questions in cell biology, and viruses

provide a natural experimental system for studying the fusion process. Fusion of two

lipid bilayers is an energetically unfavorable process, and the fact that viruses, which

have relatively simple membranes, use this process for entry into cells makes them a

very interesting model for studying membrane fusion.

Studies of membrane fusion have, in the past, been largely morphological and

descriptive due to lack of techniques for measuring and analyzing the fusion process

in isolation. Many assays used involved radioisotopes (Haywood and Boyer, 1982;

White et al., 1983) and electron spin probes (Maeda et al., 1975, 1981; Lyles and

Landesberger, 1979) or involved use of indirect techniques such as hemolysis and

infectivity (White et al., 1983). Of all these techniques only electron spin resonance

permits the continuous monitoring of the fusion process, which is desirable in a fusion

assay. More recently, the assay described in this work has been widely adopted for

measurement of membrane fusion in isolation from other events in the virus life cycle

that precede or follow it. This method not only allows for continuous monitoring of








24

fusion between membranes but also provides an opportunity to analyze the kinetics of

fusion between membranes, which can be useful for comparing the kinetics of virus

fusion with different cell types.



Materials and Methods



Lymphoblastoid Cell Lines

Cell lines were grown at 37C and diluted at least biweekly in RPMI 1640

(Sigma Chemical Co., St. Louis, Missouri) supplemented with heat-inactivated fetal

calf serum (5-10%, depending on cell type), 100 IU of penicillin and 100ug of

streptomycin per milliliter. The cell lines used include four human EBV genome-

positive B lymphoblastoid cell lines, Raji (Pulvertaft, 1964), Daudi (Klein et al., 1968),

P3HR1-CI13 and P3HR1-C15 (Heston et al., 1982). Raji is a latently infected virus

nonproducing cell line expressing CR2. Daudi is a genome positive nonproducing cell

line that currently, in our laboratory, does not express CR2. P3HR1-CI13 is a

superinducible virus producing cell line and P3HR1-CI5 is a genome positive cell line

derived from the same parent line as P3HR1-CI13, but currently in our laboratory does

not produce virus. Also used were MCUV5, an EBV producing marmoset cell line and

Molt 4 (Minowda et al., 1972), an EBV genome negative human T cell line that

expresses CR2, but cannot internalize virus (Menezes et al., 1977).

Virus Production and Radiolabelina

A small percentage of the P3HR-CI13 cells spontaneously produce low levels

of virus, but this amount can be increased after induction with 30ng of 12-0-

tetradecanoyl phorbol-13-acetate (TPA) per milliliter (Sigma). The virus obtained from










the MCUV5 cell line will transform fresh human B cells and induce them to secrete

immunoglobulin (Gerber and Lucas, 1972), whereas the P3HR1-CI13 virus is a non-

transforming lytic strain of virus. Virus was obtained from producer cells by harvesting

the virus from culture supernatant 7 days after induction with TPA. Cell culture

supernatant was cleared of cells by centrifugation at 4,000 x g for 10 minutes.

Bacitracin (Sigma) was added to the clarified supernatant (100 ug/ml) to reduce virus

aggregation, and the virus was pelleted by centrifugation at 20,000 x g for 90 minutes.

The virus pellets were resuspended in 1/250 original volume of medium containing

100ug per ml bacitracin, reclarified of cell debris by centrifugation three to four times

at 400 x g, and filtered through a .45um-pore filter (Acrodisc, Gelman Sciences, Inc.,

Ann Arbor, Michigan).

P3HR1-CI13 virus was intrinsically labeled with (3H) thymidine (3HTdR;

Amersham Corp., Arlington Heights, Illinois) by feeding cells with medium containing

100uM hypoxanthine and 0.4uM aminopterin (Sigma), inducing them with TPA when

they reached confluency (day 0) in the presence of 2 uCi of 3HTdR (specific activity 5

Ci/mmol) per ml, adding an additional 2uCi of 3HTdR (specific activity 52 Ci/mmol)

on day 3, and harvesting the virus on day 7 in the same manner as described above.

All virus stocks were stored at -700C.

Monoclonal Antibodies

Monoclonal antibodies were purified from hybridoma culture supernatants by

chromatography on protein A-Sepharose (Genzyme, Boston, Massachusetts). The

antibody 72A1 (Hoffman et al., 1980) is an IgG1 antibody that recognizes the viral

glycoprotein gp350/220. Two monoclonal antibodies that react with CR2 were used,










OKB7 (Rao et al., 1985), which blocks virus binding, and HB5, which does not block

the virus binding site of CR2 (Nemerow et al., 1985a).

Virus Binding Assay

The ability of intrinsically radiolabeled virus to bind specifically to CR2 was

determined by use of receptor positive and negative cells that had been briefly fixed

with ice-cold 0.1% paraformaldehyde. Virus was incubated with 2 x 106 fixed cells for

60 minutes at 4C, cells were washed five times to remove unbound virus and the

acid-precipitable radioactivity that remained attached to cells was counted. The ability

of antibody to interfere with virus binding was determined by preincubation of virus

and antibody for 1 hour at room temperature.

Isolation of B-cell Enriched Leukocvtes

Heparinized human peripheral blood was separated by flotation on

Lymphocyte Separation Medium (LSM; Utton Bionetics, Charleston, South Carolina).

T cells were depleted from the leukocyte fraction by a double cycle of rosetting with 2-

aminoethyl isothiouronium bromide-treated sheep erythrocytes (Pellegrino et al., 1975)

and centrifugation over 60% isotonic Percoll (Pharmacia Fine Chemicals, Piscataway,

New Jersey). The nonrosetting cells remain at the RPMI-Percoll interface and are

collected and washed free from remaining Percoll.

Human tonsil tissue was teased apart with forceps and rinsed with RPMI to

collect single cells. Cells were washed and resuspended in RPMI and separated on

LSM. T cells were depleted by rosetting as stated previously.

The T-depleted leukocytes were also depleted of monocytes in some

experiments by one of two methods. Monocytes were depleted by adherence to

plastic petri dishes in 10% RPMI for 1 hour at 370C and the nonadherent cells were








27

collected. Alternatively, cells were incubated with iron filings in a 15 ml polypropylene

tube at 370C on a rotator for 1 hour and the iron containing cells were removed with a

magnet. The remaining cells were layered on lymphocyte separation medium for

additional removal of iron containing cells. The extent of monocyte depletion was

determined by cell counts and nonspecific esterase staining of cells prior to and after

depletion.

Nonspecific Esterase Stain

Nonspecific esterase is contained in the granules of monocytes and was

stained with a solution of Sorensen's buffer, hexazotised pararosaniline and alpha-

naphthyl butyrate (Li et al., 1973). Sorensen's buffer consists of 0.2M Na2HPO, and

0.2M NaH2PO, at pH 6.3. Hexazotised pararosaniline was prepared by mixing equal

volumes of pararosaniline HCL and 4% sodium nitrite. One gram of alpha-naphthyl

butyrate was dissolved in 50ml of dimethyl formamide and stored at -200C, protected

from the light. To prepare the primary stain, 0.25ml of hexazotised pararosaniline and

3.0ml of alpha-napthyl butyrate were added to 44.5ml of Sorensen's buffer and the

solution was filtered through a Whatman #1 filter and 5 X 106 fixed cells were stained

for 30 to 45 minutes at 370C. The slides were rinsed with deionized water and

counterstained in methyl green for 15 seconds. The slides were rinsed again in

deionized water and air dried. Monocytes were identified by the brown coloration of

their cytoplasm.

Initiation of an Immortalized B Cell Line

T-depleted leukocytes were isolated from peripheral blood as previously

described. The cells were plated in a 24-well tissue culture plate at a concentration of

2 X 106 per milliliter and 100ul of virus were added to each well using a twofold










dilution series starting at 1:10. Clonal outgrowths were selected and propagated in

RPMI 1640 with 10% fetal calf serum. A cell line initiated in this manner, designated

BAT, was utilized for comparing virus fusion with immortalized B cells, freshly isolated

human B cells and lymphoblastoid cell lines derived from tumor tissues that had been

in culture for many years.

Virus Titration and Neutralization

Infectivity of EBV was measured in terms of its ability to induce human

peripheral B lymphocytes to secrete immunoglobulin in culture (Kircher et al., 1979).

T-depleted leukocytes were incubated with or without virus at 370C in 96-well round-

bottomed tissue culture plates at concentrations of 10s cells per well in 100ul of RPMI

1640 supplemented with 10% heat-inactivated fetal calf serum, 100IU of penicillin per

milliliter, and 100ug of streptomycin per milliliter. After 6 days in culture, 100ul of

medium were added to each well. On day 12, the culture supernatants were collected

and the immunoglobulin concentrations were measured. The ability of antibody to

neutralize infectivity was determined by preincubating virus for 1 hour at room

temperature with an equal volume of normal rabbit antibody at 100ug per ml, or with

mixtures of rabbit antibody and test antibody adjusted so that the total amount of

immunoglobulin remained constant at 100ug per ml. All antibodies were heated for

30 minutes at 560C to inactivate complement prior to incubation with virus.

Incorporation of Octadecvl Rhodamine B Chloride (R,,) into Virus Membranes

Octadecyl rhodamine B chloride (R,,,, is a fluorescent amphiphile that can be

readily inserted into biological membranes (Figure 2-1). A stock solution of 13

nmoles/ul of R, (Molecular Probes, Inc., Junction City, Oregon) was prepared in

chloroform/methanol (1:1) and stored at -200C. The probe was incorporated into virus





















(C2 5


COO(CH2) -CH3
'17


Figure 2-1. Structural formula of octadecyl rhodamine B chloride (R1,).








30

membranes by modification of the method of Hoekstra et al. (1984). Three microliters

of stock probe were dried under nitrogen and solubilized in 39ul absolute ethanol and

15ul of this solution, containing 15nmole R,,, were added to 250ul of concentrated

virus under vortexing. For mock-labeled virus the same volume of absolute ethanol

was added to the virus as used in the labeling procedure. Probe and virus were

incubated at room temperature in the dark for 1 hour. Virus and nonincorporated R,,

were separated by chromatography on Sephadex G-75 (Sigma Chemical Co., St.

Louis, Missouri). Labeled virus was aliquoted and stored at -70C.

Fluorescence Deauenching Assay

An Aminco-Bowman spectrofluorometer (SLM Amino Bowman Instrument Co.,

Urbana, Illinois), equipped with a chart recorder was used for continuous monitoring

of fluorescence. The cuvette chamber was equipped with a magnetic stirrer and held

in a temperature controlled circulating water bath. For fluorescence measurements,

the instrument was calibrated such that any residual fluorescence of membranes at

time zero was set at the zero level. At the end of the assay, Triton X-100 (Sigma)

(1.0% v/v, final concentration) was added to allow measurement of the maximum

obtainable fluorescence for the virus bound upon infinite dilution of the fluorophore.

R,,-labeled virus (volume not exceeding lOOul) were added to pellets of 2 x 106

cells and incubated for 1 hour at 40C on ice and in the dark. Cells were washed four

times with ice cold Dulbecco's saline and transferred to the microcuvette of the

spectrofluorometer. The principle of the assay relies upon the self-quenching

properties of R,1 when inserted into the virus membrane. When the two fusing

membranes come into molecular contact their lipid components must mix and this

mixing dilutes the R,8 allowing relief of the self-quenching. The relief of self-quenching










of the R,8 was continuously monitored at excitation wavelength of 560nm and

emission wavelength of 585nm and documented with a chart recorder.

Immunoolobulin Assay

Immunoglobulin in culture supernatants was measured by a double sandwich

micro-enzyme-linked immunosorbent assay (Voller et al., 1976) using rabbit anti-

human immunoglobulin as the immobilized antigen. Antibody in the culture

supernatants was allowed to bind to the immobilized antigen followed by horseradish

peroxidase-conjugated rabbit anti-human Ig (Cooper Biomedical Inc., Malvern,

Pennsylvania). The substrate, hydrogen peroxide with 5-amino salicylic acid was

degraded by the enzyme and the colorimetric change was measured at 492 nm.



Results



Fluorescence Properties of R, Labeled Virus

Figure 2-2 shows the excitation and emission spectra of octadecyl rhodamine

B chloride (R8) incorporated into virus membranes when relieved of self-quenching

with Triton X-100 (1% v/v final concentration). The excitation spectrum exhibits a

maximum peak at 560 nm. The peak emission wavelength displayed a maximum at

585 nm. The emission wavelength has been shown to be dependent upon the

environment of the probe (Hoekstra et al., 1984) with variance between 569 and 590 in

different solvents. Figure 2-3 demonstrates the stability of the quenching of the

fluorophore within the virus membrane and subsequent relief of quenching upon

addition of Triton X-100 (1% v/v).


















750



600

w
0
z 450
LU
CO

O 300
-j


150





540 560 580 600 620

WAVELENGTH (nm)


Figure 2-2. Excitation ( -0- ) and emission ( ---) spectra of R,a-containing
virions relieved of self-quenching with Triton X-100 (infinite dilution). Relative
fluorescence expressed in arbitrary units (a.u.).
















150









S 100
0
z
0
w
UJ



50












0 4 8 12 16 20 24 28 32


TIME (MINUTES)

Figure 2-3. Stability of self-quenching of R,,-labeled virions maintained at 37C and
relief of self-quenching upon addition of Triton X-100 (infinite dilution) after 30
minutes. Relative fluorescence expressed in arbitrary units (a.u.).










Effect of R,l on the Attachment of Virus

When adapting the fluorescence assay of Hoekstra and colleagues for use with

EBV, our first question was whether labeling of the virus with the fluorescent molecule

would qualitatively or quantitatively affect virus binding. To answer this question we

labeled virus metabolically with 3HTdR, divided the virus into three aliquots, left one

untreated, labeled one with an ethanolic solution of R,, and mock-labeled the third

aliquot with ethanol alone. The labeled and mock-labeled preparations of virus were

chromatographed on Sephadex G-75. A virus binding assay was done and the

amount of radioactivity bound to receptor positive and negative cells was measured.

Approximately half the bindable virus was lost during the labeling and mock-labeling

procedures. However, if the amount of radioactivity bound was expressed as a

percentage of the amount added, it could be seen that the labeling had no effect on

the ability of the virus to bind to receptor positive cells (Table 2-1). There was no

increase in nonspecific binding to receptor negative P3HR1-CI5 cells.

The specificity of binding was further confirmed by showing that preincubation

of virus with antibody 72A1 inhibited its ability to bind to receptor positive cells (Table

2-2). Two additional antibodies that have anti-CR2 activities were used in this

experiment. Preincubation of cells with one, OKB7, which normally blocks virus

binding (Nemerow et al., 1985a), inhibited labeled virus binding; preincubation of cells

with HB5, a monoclonal antibody to CR2 that does not block the virus binding sites,

appropriately failed to inhibit binding of labeled virus (Table 2-2).

Effect of R,. on Infectivity of Virus

Although the incorporation of R,, into the virus membrane did not alter the

binding properties of the virus, it remained possible that the probe interfered with a










Table 2-1. Effect of labeling with R,8 on the ability of [3H] EBV to bind to receptor
positive and negative cells.

Total acid % acid precipitable
Virus Virus precipitable
treatment dilution counts bound to: counts bound to:a
Rajib Cl5C Raji CI5

None neat 13284 543 28.9 1.2

1/2 6346 -d 27.6

1/4 2704 217 23.5 1.8

1/8 1408 24.5

Mock- neat 5816 113 29.4 0.6
labeled
1/2 2663 26.8

1/4 1366 38 27.5 0.7

1/8 758 30.6

R,1- neat 6962 248 25.5 1.0
labeled
1/2 3707 27.1

1/4 1831 104 26.8 1.5

1/8 1016 29.7


radioactivity bound/radioactivity added X 100
"receptor positive cells
Receptor negative cells
anot done










Table 2-2. Effect of monoclonal anti-EBV and anti-CR2 antibodies on the ability
of R,,-labeled ['H] EBV to bind to receptor positive cells.


R,,-labeled virus Mock-labeled virus
Antibody
(ug)
(ug) Total cpm % cpm Total cpm % cpm
bound bound bound bound
none 1904 23.5 2789 23.9

72A1 (10)' 123 1.5 231 1.9

OKB7 (5) 198 2.4 180 1.5

HB5 (5) 1390 17.1 2308 19.8


'amount of antibody used, expressed in micrograms
radioactivity bound/radioactivity added X 100










subsequent event in virus replication. Since the labeled virus was being used for

studying events post binding it was necessary to examine the infectivity of labeled

virus. A comparison was made of the ability of labeled and mock-labeled MCUV5

virus to induce immunoglobulin synthesis in cultures of T cell-depleted peripheral

leukocytes (Table 2-3). There was no indication that incorporation of probe into virus

had any detrimental effect on its biologic activity.

Changes in Fluorescence after Interaction of R'8-Labeled Virus with Lvmohoblastoid
cells
Figure 2-4 demonstrates the changes in fluorescence emission observed as

virus bound to Raji cells at 4C was warmed in the cuvette of the spectrofluorometer.

The fluorescence increased gradually over approximately 28-32 minutes, after which

time a plateau was reached. At this time, Triton X-100 (1% v/v) was added to relieve

any residual self-quenching of the fluorophore and thus providing a rough

approximation of the percentage of bound virus that fused. In Figure 2-4, 56% of the

maximal fluorescence was reached, this value proved reproducible for this particular

batch of labeled virus. The maximal value obtained with any batch of virus was 75%.

Parallel analysis of the receptor negative Daudi cell line confirmed that R18-labeled

virus failed to bind to these cells (Figure 2-4). This result also showed that there was

no significant diffusion of residual free or incorporated probe from the virus

preparation into cell membranes during the 1 hour incubation at 40C.

Further data indicating that relief of self-quenching was measuring a membrane

fusion event and not simple diffusion of probe from closely approximated membranes

were obtained using fixed Raji cells and the Molt 4 cell line (Figure 2-5). The increase

in fluorescence emission measured when virus was bound to Raji cells at 4C and

then warmed to 370C was almost completely eliminated if the cells were fixed with











Table 2-3. Effect of labeling with R,1 on the ability of MCUV5 virus to induce
immunoglobulin synthesis by fresh T-depleted human leukocytes.


Virus Immunoglobulin conc. ng/ml with:
dilution
R,,-labeled Mock-labeled
virus virus

1/5 24,754 22,366

1/10 45,720 23,836

1/20 38,609 23,639

1/40 39,902 27,404

1/80 42,326 30,105

1/160 33,921 21,093

1/320 18,168 15,999

1/640 3,970 not done
none 1,138













100




RAJI



75 DAUDI




Cu
w
z
w
W so








25











0 4 8 12 16 20 24 28 32

TIME (MINUTES)


Figure 2-4. Relief of self-quenching of R,,-labeled virus bound to receptor positive
Raji cells and receptor negative Daudi cells. At 32 minutes Triton X-100 was added to
measure maximum relief of self-quenching of bound probe (infinite dilution). Relative
fluorescence expressed in arbitrary units (a.u.).

















70

RAJI
60
RAJI FIXED

2 50 --- MOLT 4


40

z
I 30

O
i 20


10





0 4 8 12 16 20 24 28 32

TIME (MINUTES)


Figure 2-5. Comparison of relief of self-quenching of R,,-labeled P3HR1-CI13 virus
bound to Raji cells, fixed Raji cells, or Molt 4 cells. Increase in fluorescence is
expressed as a percent of the maximum release obtained with each cell line after
addition of Triton X-100 (infinite dilution). The average maximum fluorescence for
each cell line was: Raji, 100a.u.; fixed Raji, 97 a.u.; Molt 4, 75 a.u.. Vertical lines
indicate the standard deviation of the mean of experiments with the same batch of
labeled virus.








41

paraformaldehyde prior to binding to virus. When Molt 4 cells were substituted in the

assay for Raji, there was no significant relief of self-quenching of the bound probe,

which is compatible with the reported inability of virus to fuse with Molt 4 cell

membranes (Menezes, 1977). The fluorescence maxima obtained after addition of

Triton was slightly less for Molt 4 cells than Raji, which is in agreement with published

observations showing that Molt 4 cells express fewer receptors than Raji (Stoco et al.,

1988).

Chances in Fluorescence after Interaction of R_.-Labeled Virus with Normal B Cells

Two independent studies have demonstrated that although EBV fuses with the

plasmalemma of lymphoblastoid cells, it is endocytosed into normal B cells before

any fusion of virus and cell membranes occurs (Nemerow and Cooper, 1984a; Tedder

et al., 1986). However, if fusion was occurring within an endocytic vesicle, it seemed

possible that the event might still be detectable with the RI-labeled virus.

Experiments were done initially with B cells isolated from fresh tonsil tissue.

Tonsil tissue was obtainable on a sporadic basis from the surgical pathology

department and large numbers of cells could be obtained from a single piece of

tissue. Considerably less virus bound to normal B cells than to Raji cells. However,

even though the increase in fluorescence measured with R,,-labeled virus bound to

normal B cells was smaller than that measured with lymphoblastoid cells, a

measurable signal was obtained. The increase in fluorescence expressed as a

percentage of the maximum obtainable after addition of Triton was less than that seen

in experiments with lymphoblastoid cells (Figure 2-6).

In order to rule out the interference of monocyte engulfment of virus in the

determination of maximum relief of fluorescence, experiments were done using cells



















25



S 20


15


w
o 10



U. 5




0 4 8 12 16 20 24 28

TIME (MINUTES)

Figure 2-6. Relief of self-quenching of R,,-labeled P3HR1-C113 virus bound to tonsil
derived T-depleted leukocytes expressed as a percent of the maximum release
obtained after addition of Triton X-100 (infinite dilution).










that had been depleted of monocytes. Table 2-4 shows the extent of monocyte

depletion as determined by cell counts and nonspecific esterase stain pre and post

depletion. Figure 2-7 shows the increase in fluorescence of tonsil derived B cells pre

and post monocyte depletion by adherence to plastic. The cell preparations treated

with iron filings could not be used in the fluorometer due to scatter interference from

residual filings in the preparation. The maximum increase in fluorescence achieved

with tonsil derived B cells was 20-23% and depletion of monocytes from the cells used

did not affect this measurement.

Fusion experiments were also done using T depleted peripheral leukocytes.

Human peripheral leukocytes could be obtained with greater regularity than tonsil

tissue. Figure 2-8 shows data obtained using T depleted peripheral leukocytes. As

seen with the tonsillar B cells, less virus bound peripheral B cells than Raji cells, but

the maximum increase in fluorescence was higher than the level obtained with tonsil

derived cells. In this experiment the maximum increase was 55%, in other

experiments using different batches of labeled virus and different cells, values ranging

from 28-56% were achieved.

Changes in Fluorescence of R,.-labeled Virus with EBV-lmmortalized B Cells

Human B cells were infected with EBV and were immortalized. These cells,

designated BAT, have growth characteristics of a continuous cell line, but since they

are recently immortalized, they may be biologically more similar to B cells than the

lymphoblastoid cell lines, such as Raji, which has been in culture for many years. Raji

cells have been reported to have alterations in the cytoskeleton (Bachvaroff et al.,

1980). Figure 2-9 demonstrates how these cells function in the fluorescence

dequenching assay. Utilizing these cells reduces the need to obtain fresh human










Table 2-4. Monocyte depletion of T-depleted human leukocytes by adherence to
plastic.



Cell cell number % cells staining
treatment esterase positive1


none 3.0 X 108 45%


adherence 1.8 X 108 8%



















25

----- PRE
S 20
POST


15

z
0 10
Co

L. 5





0 4 8 12 16 20 24 28 32

TIME (MINUTES)

Figure 2-7. Comparison of relief of self-quenching of R,,-labeled P3HR1-CI13 virus
bound to tonsil derived B cells pre and post monocyte depletion by adherance to
plastic. Increase in fluorescence is expressed as a percent of the maximum release
obtained after addition of Triton X-100 (infinite dilution).


















70


60


50 o

40
LUt
z
u 30


O 20


10



0 4 8 12 16 20 24 28 32 36

TIME (MINUTES)

Figure 2-8. Relief of self-quenching of R,,-labeled MCUV5 virus bound to fresh T-
depleted peripheral leukocytes expressed as a percent of the maximum release
obtained after addition of Triton X-100 (infinite dilution).




















70


60


I 50
X


I 40
040
z
w 30


o 20

LL




0 4 8 12 16 20 24 28 32

TIME (MINUTES)


Figure 2-9. Relief of self-quenching of R8,-labeled MCUV5 virus bound to BAT cells
expressed as a percent of the maximum release obtained after addition of Triton X-
100 (infinite dilution).










peripheral blood for each experiment. In future experiments virus entry into BAT cells

will be studied in parallel and compared to entry into fresh normal B cells.



Discussion

The fluorescent amphiphile octadecyl rhodamine B chloride (R,) has been

used by several groups to study interactions of virus with biological membranes and

liposomes (Blumenthal et al., 1987; Gilbert et al., 1990; Hoekstra et al., 1984, 1985;

Lapidot et al., 1987; Morris et al., 1989; Sinangil et al., 1988; Stegmann et al., 1986;

Wunderli-Allenspach and Ott, 1990). The results from these papers indicate that

fluorescence dequenching reflects the occurrence of virus membrane fusion and when

discussing the results of the experiments in this dissertation, fluorescence

dequenching and membrane fusion will be considered interchangeable terms. The

behavior of R,,-labeled EBV, as demonstrated by relief of self-quenching of virus

bound to Raji cells, and the absence of fluorescence of virus bound to fixed Raji cells

or Molt 4 cells, provides strong corroborative support for this conclusion. Fixed cells

are resistant to virus membrane fusion (Gilbert et al., 1990; Lapidot et al., 1987) and

Molt 4 cells are reported to bind but not internalize virus (Menezes et al., 1977).

The R,1 labeling procedure did not affect the binding specificity or the amount

of EBV that bound to lymphoblastoid cells. This is in agreement with the effect of

labeling on attachment of Sendai virus (Hoekstra et al., 1985). Labeling of VSV with

R,1 has been reported to enhance virus binding by twofold, possibly because the

probe is positively charged and increases the net charge of the virus (Blumenthal et

al., 1987). Labeled virus retained its infectivity as indicated by its ability to induce

immunoglobulin synthesis by cultured T-depleted peripheral leukocytes.










The early events in infection of normal B cells and lymphoblastoid cells have

been examined previously by electron microscopy (Nemerow and Cooper, 1984a;

Seigneurin et al., 1977). These studies reported that EBV enters lymphoblastoid cells

by direct fusion with the outer cell membrane and that virus is endocytosed into thin-

walled non-clathrin coated vesicles in the normal B cell before it fuses with the cell

membrane. Both pathways were reported to initiate within two to five minutes at 37C.

The kinetics of fusion with Raji cells, normal lymphocytes, and recently immortalized

BAT cells were very similar, all exhibiting a measurable change within two minutes of

warming in the cuvette of the spectrofluorometer. A one to two minute lag time,

corresponding to the time required for initial entry of ligands, toxins, and virions into

an acidic compartment after receptor mediated endocytosis (Bridges et al., 1982) has

been reported for relief of self-quenching of R,,-labeled vesicular stomatitis virus

bound to Vero cells (Blumenthal et al., 1987).

Since EBV appears capable of fusing with the plasma membrane at the cell

surface, or after endocytosis, this may mean that either virus can enter normal B cells

by both routes, or that fusion with an endocytic vesicle wall occurs rapidly after

uptake, perhaps even before virus is exposed to low pH. It has been shown that

rotavirus enters cells by direct cell membrane penetration (Kaljot et al., 1988) even

though earlier electron microscopy studies had revealed presence of rotavirus

particles in coated pits and a variety of vesicles, signifying entry by endocytosis (Petrie

et al., 1981; Quann and Doane, 1983).

Experiments using lysosomotropic agents, inhibitors of endocytosis, and pH

sensitive fluorescent probes may help answer the question of whether EBV is capable

of fusing at both the plasma membrane and the endocytic vesicle of lymphocytes. In








50

1984, Nemerow and Cooper demonstrated a 96% reduction in infectivity by EBV of B

cells treated with 1mM chloroquine and a 20% reduction in infectivity of cells treated

with 10mM NH4CI. Infectivity was assessed by stimulation of host cell DNA synthesis

as measured by incorporation of ['H] thymidine after 4 to 6 days in culture. From

their studies they concluded that a reduction in pH was necessary for virus entry

because of inhibition by these agents. The fluorescence dequenching assay allowed

for analysis of the effects of these reagents on EBV fusion and the results are

presented and discussed in the following chapter.














CHAPTER 3
EFFECTS OF LYSOSOMOTROPIC AGENTS AND pH ON FUSION
OF EPSTEIN-BARR VIRUS WITH LYMPHOCYTES



Introduction

To initiate an infection, all enveloped animal viruses must fuse with a cellular

membrane and this fusion can be divided into two general classes, low pH dependent

and pH independent. It is generally considered that viruses that are low pH

dependent fuse from within acidic vesicles whereas viruses that are low pH

independent can fuse directly with the plasma membrane, but may fuse from

endosomes as well. Although fusion of EBV with lymphoblastoid cell lines occurs at

the plasma membrane and therefore presumably does not require exposure to low

pH, virus has been reported to fuse with normal B cells after endocytosis and certain

lysosomotropic agents have been shown to inhibit virus infectivity (Nemerow and

Cooper, 1984a). The possibility that penetration of Epstein-Barr virus nucleocapsids

into the cytosol might involve an acid-catalyzed fusion reaction in the endosomal

compartment was further investigated in this work since our assay measures

membrane fusion in isolation of other events in the virus life cycle that might be

affected by drugs. Exposure to pH values between 5.0 and 7.0 has dramatic effects

on many of the molecules brought into the cell by endocytosis. Many ligands

dissociate from their receptors at pH values below 7.

Some viruses undergo significant changes in conformation when exposed to

acidic pH (White, 1990). The fusion glycoprotein of influenza virus, the hemagglutinin








52

(HA) undergoes an irreversible conformational change upon exposure to mildly acidic

pH within acidic organelles after endocytosis. If virus is bound to the cell surface and

the extracellular pH is briefly lowered to pH 5.0, fusion of the virus can occur at the

plasma membrane. If the virus alone is exposed to acidic pH the conformation

occurs prematurely and the virus is unable to fuse. Treatment of cells with

lysosomotropic agents inhibited influenza infectivity. The unprotonated form of these

lipophilic amines crosses cell membranes but the protonated form does this far less

efficiently. When the uncharged form enters acidic compartments it becomes

protonated, thereby raising the pH and inhibiting its own escape across the

membranes of the vacuoles.

Vesicular stomatitis virus (VSV) is another example of a virus that fuses from

within an acidic compartment after endocytosis (Dahlberg, 1974; Dales, 1973; Dickson

et al., 1982; Matlin et al., 1982). The fusion activity can be shown to take place on the

plasma membrane if cells with VSV attached to their surfaces are placed in pH 5.9

medium (Matlin et al., 1982; Blumenthal et al., 1987). Lysosomotropic agents were

also shown to inhibit fusion from with an endocytic vesicle, but had no effect on fusion

at the plasma membrane at pH 5.9 (Blumenthal et al., 1987).

The work described here sought to determine whether EBV fusion is a truly pH

dependent event and where fusion takes place in lymphoblastoid cell lines, freshly

isolated human B cells and recently transformed human B cells.

Materials and Methods

Membrane Fusion Assay

Virus that has been labeled with R,8 at self-quenching concentration was added

to 2 X 106 cells and incubated for 1 hour on ice in the dark. Cells were washed four








53
times with ice-cold Dulbecco's saline at pH 7.4 and suspended in 400ul of Dulbecco's

at pH 7.4 (unless otherwise indicated) when transferred to the microcuvette of a

spectrofluorometer (SLM SPF 500C, SLM Instruments Co., Urbana, Illinois) equipped

with a magnetic stirrer and circulating water bath set at 37C. Fluorescence

dequenching was monitored continuously at an excitation wavelength of 560nm and

an emission wavelength of 585nm. At the end of the assay, Triton X-100 (1% v/v,

final concentration) was added to allow the measurement of fluorescence that would

be obtained upon infinite dilution of the fluorophore.

Cells

The lymphoblastoid cell lines Raji (Pulvertaft, R.J., 1964) and BAT, which are

both EBV genome-positive human B-cell lines expressing the virus receptor CR2

(CD21); Molt 4 (Minowda et al., 1972), an EBV genome negative human T cell line that

expresses CR2, but cannot internalize virus (Menezes et al., 1977) and P3HR1-CI5

(Heston et al., 1982), an EBV genome-positive human B-cell line which does not

express CR2 were grown at 370C and diluted at least biweekly in RPMI 1640

supplemented with heat-inactivated fetal calf serum, 1001U of penicillin and 100ug of

streptomycin per ml. Fresh human T cell-depleted leukocytes were isolated as

described previously from peripheral blood and used directly in assays.

Treatment of Cells with Lvsosomotropic Agents

Ammonium chloride (NH4CI), chloroquine, and methylamine were purchased

from Sigma and stock solutions were made in phosphate buffered saline of 100mM,

100mM, and 50mM respectively, from which dilutions were made in RPMI 1640 for

incubation with cells. Cells were incubated in one milliliter of media containing the

lysosomotropic agent for 35 minutes at 370C to neutralize acidic intracellular










compartments and control cells were incubated in medium only. At the end of the

incubation the cells were pelleted by centrifugation and the supernatant was removed.

Cells were resuspended in 100ul of media and incubated with virus for 1 hour on ice.

Determination of Intracellular pH

To determine intracellular pH, cells were incubated with a mixture of fluorescein

isothiocyanate (FITC) and tetramethylrhodamine (TRITC)-labeled dextrans (70,000 mw)

(Molecular Probes Inc., Junction City, Oregon) for 35 minutes at 370C to allow uptake

of the labeled dextrans into the cells. Cells were washed free of unassociated dextran

and analyzed in the spectrofluorometer by measuring the TRITC fluorescence at an

excitation wavelength of 560nm and an emission wavelength of 580nm followed by

measuring FITC emission at 522nm at excitation wavelengths from 450nm to 518nm.

Monensin (lug/ml) was then added to equilibrate extracellular (test pH) and

endosomal pH and another fluorescence measurement of the FITC was taken from

450nm to 518nm. If the intensity of the fluorescence rises at this step, the average pH

of the intracellular compartments is below the test pH. If the intensity falls, the

average pH was above the test pH.

Incorporation of 5-(N-octadecanovl)aminofluorescein into Virus Membranes

The membrane probe 5-(N-octadecanoyl)aminofluorescein (AF) is a fluorescent

amphiphile containing a long hydrocarbon chain which allow it to insert readily into

biological membranes (Figure 3-1). AF manifests the same property of concentration-

dependent quenching of fluorescence as R,1, in addition to sensitivity to changes in

pH similar to the FITC-dextran used for determination of intracellular pH. A stock

solution of 50mg/ml of AF (Molecular Probes, Inc., Junction City, Oregon) was

prepared in dimethylformamide and stored at -200C. The probe was incorporated into









O U -OH


o kCOOH
-
NHC-(CH2-CH3
II n
(n= 16)


Figure 3-1. Structural formula of 5-(N-octadecanoyl)aminofluorescein (AF).










virus membranes by modification of the method used to incorporate R, into virus

membranes. Briefly, 2ul of the stock AF was added to 250ul of the MCUV5 strain of

EBV that had been collected from culture supernatant and concentrated 250-fold.

Virus and AF were vortexed immediately after addition of the fluorescent probe and

incubated at room temperature in the dark for 1 hour. Virus and non-incorporated AF

were separated by chromatography on Sephadex G-75 (Sigma Chemical Co., St.

Louis, Missouri) recovering the AF-labeled virus in the void volume. Labeled virus was

aliquoted and stored at -700C.

Fluorescence measurements were made at an excitation wavelength of 496nm

and an emission wavelength of 522nm using a SLM SPF500c spectrofluorometer

equipped with a thermostatically-controlled cuvette chamber and magnetic stirrer

(SLM Aminco, Urbana, Illinois).



Results

Effect of Lowering Extracellular pH on Fusion

Previous studies with viruses that are known to be dependent on the low pH of

the endosome in order to fuse have shown that they are also able to fuse at the

plasma membrane if the pH of the extracellular medium if briefly lowered (Blumenthal

et al., 1987; Marsh et al., 1983a; White et al., 1980). Experiments were therefore done

to see if the rate or extent of fusion of EBV would be affected if the pH of the

extracellular media was decreased in order to drive low pH-dependent fusion to occur

at the plasma membrane. The results in Figures 3-2, 3-3, and 3-4 indicate that

altering the extracellular pH from 7.4 to 5.5 did not effect virus fusion with Raji, BAT, or

fresh T-depleted leukocytes.


















70


7 60

x 50


wL 40
400
z
w 30
LU
n-
o 20
LL E pH 7.2
10 -- pH 5.5


0 1 I1-- [ I I -- 1 I \ -
0 4 8 12 16 20 24 28 32

TIME (MINUTES)


Figure 3-2. Relief of self-quenching of R,,-labeled MCUV5 virus bound to Raji cells
at pH 7.2 or pH 5.5. Virus was bound to cells at pH 7.2, cells were washed to remove
unbound virus and cells were resuspended in pH 7.2 or pH 5.5 Dulbecco's saline.
Increase in fluorescence is expressed as a percent of the maximum release obtained
after addition of Triton X-100 (infinite dilution).


















70


60




40


30




10 pH 7.2
I pH 5.5
0-
0 4 8 12 16 20 24 28 32

TIME (MINUTES)

Figure 3-3. Relief of self-quenching of R,,-labeled MCUV5 virus bound to BAT cells
at pH 7.2 or pH 5.5. Virus was bound to cells at pH 7.2, cells were washed to remove
unbound virus and cells were resuspended in pH 7.2 or pH 5.5 Dulbecco's saline.
Increase in fluorescence is expressed as a percent of the maximum release obtained
after addition of Triton X-100 (infinite dilution).



















70


60


X 50

40






2 pH 7.2
10
20
o 20


---- pH 5.5


0 4 8 12 16 20 24 28 32 36

TIME (MINUTES)

Figure 3-4. Relief of self-quenching of R,,-labeled MCUV5 virus bound to T-depleted
leukocytes at pH 7.2 or pH 5.5. Virus was bound to cells at pH 7.2, cells were washed
to remove unbound virus and cells were resuspended in pH 7.2 or pH 5.5 Dulbecco's
saline. Increase in fluorescence is expressed as a percent of the maximum release
obtained after addition of Triton X-100 (infinite dilution).










Effect of Lvsosomotrooic Agents on Virus Fusion

Although fusion of EBV with lymphoblastoid cell lines occurs at the plasma

membrane and therefore presumably does not require exposure to low pH, virus has

been reported to fuse with normal B cells after endocytosis and certain

lysosomotropic agents capable of altering the pH of intracellular compartments have

been shown to inhibit virus infectivity (Nemerow and Cooper, 1984a). These agents

have been used in many virus systems to determine the mechanism by which virus

enters cells (Andersen and Nexo, 1983; Blumenthal et al., 1987; Cassel et al., 1984;

Gilbert et al., 1990; Gollins and Porterfield, 1986; Stein et al., 1987).

The effects of ammonium chloride (NH4CI), methylamine and chloroquine on

three cell types, Raji, BAT, and fresh T-depleted human leukocytes were studied in the

fluorescence dequenching assay. Figures 3-5, 3-6 and 3-7 demonstrate that 20mM

NH4CI did not have any effect on fusion of virus with any of the three cell types. Three

concentrations of chloroquine were tested with Raji cells and did not effect fusion

(Figure 3-8). In contrast, chloroquine BAT cells and fresh T-depleted leukocytes

exhibited dose-dependent inhibition of fluorescence dequenching shown in Figures 3-

9 and 3-10. Chloroquine inhibited fusion of virus with BAT cells by 34% at 1mM and

by 30% at 0.5mM. For peripheral B cells, the inhibition was 60% at 1mM, 50% at

0.5mM, and 24% at 0.2mM. The third agent used, methylamine, which in addition to

elevating the endosomal pH also is an inhibitor of transglutaminase which has been

suggested to be involved in receptor-mediated endocytosis (Davies et al, 1980), did

not inhibit relief of self-quenching with any of the three cell types (Figure 3-11), thus

paralleling the data for the NH4CI-treated cells. In confirmation that these









61

70

60

x 50-

40

Lu 30 -
0)

O 20
U. 20mM
10 I CONTROL


A 0
0 4 8 12 16 20 24 28 32
TIME (MINUTES)

70

60

50 -

C 40


0m
z 30-
0



10 ----- CONTROL
,-, 20 f -- -- -- -- -- -- -- --



0 4 8 12 16 20 24 28 32
TIME (MINUTES)

Figure 3-5. Effect of preincubation of Raji cells with ammonium chloride (NHCI) or
RPMI on relief of self-quenching of R,,-Iabeled MCUV5 virus bound to cells. Panel A,
20mM NHCI; panel B, 10mM NHCI. Increase in fluorescence is expressed as a
percent of the maximum release obtained after addition of Triton-X-100 (infinite
dilution).



















70


I 60

< 50


w 40




0 20

----0---- NH4CI
10
-j


CONTROL


0 4 8 12 16 20 24 28 32 36

TIME (MINUTES)


Figure 3-6. Effect of preincubation of BAT cells with 20mM ammonium chloride
(NH,CI) or RPMI on relief of self-quenching of R,,-labeled MCUV5 virus bound to
cells. Increase in fluorescence is expressed as a percent of the maximum release
obtained after addition of Triton-X-100 (infinite dilution).

















70

S 60




~ 40


0
( 30


S--Ei--- NH4CI
10 CONTROL

0-
0 4 8 12 16 20 24 28

TIME (MINUTES)

Figure 3-7. Effect of preincubation of T-depleted leukocytes with 20mM ammonium
chloride (NH4CI) or RPMI on relief of self-quenching of R,,-labeled MCUV5 virus
bound to cells. Increase in fluorescence is expressed as a percent of the maximum
release obtained after addition of Triton-X-100 (infinite dilution).









64

70
x 60
50
L 40
z 30
S20
c _- 1mM
0 10
3 10--- CONTROL
LL 0 I-'-- -- -- ---I-- ---- I
0 4 8 12 16 20 24 28 32
A TIME (MINUTES)


SC70O
60
50





0 .0.5 mM













10 ----- CONTROL
o0
0 4 8 12 16 20 24 28 32
B TIME (MINUTES)
70 -
60


40 4
Wz 30

-w 20.
10 CONTROL

0 4 8 12 16 20 24 28 32
B TIME (MINUTES)

Figure 3-8. Effect of preincubation of Raji cells with chloroquine or RPMI on relief of
self-quenching of R,,-labeled MCUV5 virus bound to cells. Panel A, 1mM; panel B,
0.5mM; panel C, 0.2mM. Increase in fluorescence is expressed as a percent of the
maximum release obtained after addition of Triton-X-100 (infinite dilution).









65

70


60


I 50


40











0 4 8 12 16 20 24 28 32 36
TIME (MINUTES)
z 30






0







60 ---------------------- --



50


40.,


30
X 0 -2


























O.5mM
10 -





CONTROL
0 4 8 12 16 20 24 28 32

TIME (MINUTES)
70


60





m 40
z
Lu
o 30

O 20


10



0 4 8 12 16 20 24 28 32

TIME (MINUTES)

Figure 3-9. Effect of preincubation of BAT cells with chloroquine or RPMI on relief of
self-quenching of RF-labeled MCUV5 virus bound to cells. Panel A, 1mM; panel B.
0.5mM. Increase in fluorescence is expressed as a percent of the maximum release
obtained after addition of Triton-X-100 (infinite dilution).









66









70
-- CONTROL
60 1mM

S "~- 0.5mM
X 50 0.ZmM

40

z
W 30
U,
O 20






0 4 8 12 16 20 24 28 32
TIME (MINUTES)

Figure 3-10. Effect of preincubation of T-depleted leukocytes with chloroquine or
RPMI on relief of self-quenching of R,,-labeled MCUV5 virus bound to cells. Increase
in fluorescence is expressed as a percent of the maximum release obtained after
addition of Triton-X-100 (infinite dilution).










60
S-CONTROL
x 50 METHYLAMINE
0 40

W 30
0
z
W 20
o
I 10
0

0 4 8 12 16 20 24 28 32
A TIME (MINUTES)
S60
0 CONTROL
< 5 METHYLAMINE
2 40

o 30
z
0 20

0 10
o io
U- 0
0 4 8 12 16 20 24 28 32
TIME (MINUTES)



< METHYLAMINE
2 40

0 30

U 20




0 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 3-11. Effect of preincubation of Raji cells (panel A). BAT cells (panel B), and
T-depleted leukocytes (panel C) with 5mM methylamine or RPMI on relief of self-
quenching of R,8-labeled MCUV5 virus bound to cells. Increase in fluorescence is
expressed as a percent of the maximum release obtained after addition of Triton-X-
100 (infinite dilution).










agents were indeed increasing the intracellular pH, the resulting pH after treatment of

the cells was determined.

Determination of oH of Intracellular Compartments After Treatment with
Lvsosomotrooic Agents

The pH in endocytic compartments can be measured using fluorescein-labeled

ligands such as dextran (Ohkuma and Poole, 1978; Tyoko and Maxfield, 1982;

Yoshimura and Ohnishi, 1984.). The fluorescence intensity of fluorescein decreases

dramatically between pH 7.0 and pH 5.0. Thus, changes in fluorescence intensity can

be used as an assay for changes in pH. The ratio of fluorescence intensities at the

wavelengths of 450nm and 496nm can be used to determine a standard curve from

which actual pH values can be extrapolated (Geisow, M.J., 1984). In various cell

types, lysosomes have pH values between 4.6 and 5.2, and endocytic vesicles have

pH values between 5.0 and 5.5 (Maxfield and Yamashiro, 1987; Ohkuma and Poole,

1978; Tyoko and Maxfield, 1982; Tyoko et al., 1983; Yamashiro and Maxfield, 1984).

For accurate pH determinations using the fluorescent conjugates it was

necessary either to ensure sufficient pinocytosis to produce a reliable signal at 450nm

or to collapse the intracellular pH gradients using monensin. Monensin is a carboxylic

ionophore which is able to promote exchange of protons for univalent cations and

thereby abolishes transmembrane proton gradients (Pressman, 1976; Tartakoff, 1977).

After addition of monensin to cells, the fluorescein emission will resemble that

expected at the external pH. By altering the external pH, a calibration curve can be

obtained of intracellular fluorescein isothiocyanate (FITC)-dextran. In addition to the

fluorescein conjugate, a rhodamine conjugate is also included to ensure sufficient

uptake of the ligands into the cells. The fluorescence of the rhodamine is insensitive

to changes in pH and was used as an internal reference for the amount of conjugate










uptake. Figure 3-12 demonstrates the change in the fluorescence intensity of FITC-

dextran in buffer at various pH values. This standard curve can be used in

conjunction with the differences in fluorescence seen upon addition of monensin to

cells to determination the intracellular pH. After addition of monensin to cells, the

fluorescein emission resembles that expected at the external pH. By altering the

external pH, the pH of the intracellular FITC-dextran was obtained.

Figure 3-13 shows the fluorescence emission at 522nm of BAT cells that have

endocytosed FITC and TRITC-labeled dextran. The value of the fluorescence of the

TRITC was recorded as a control value for dextran uptake and compared between

samples. Fluorescence measurements were made in pH 7.4 medium before and after

addition of monensin. In the initial scan, the FITC emission was very low, indicating

that the fluorescence was quenched due to acidic pH, after addition of monensin

there was a great change in the fluorescence with a peak at 496nm. The TRITC

fluorescence value was 1370 arbitrary units (a.u.). Cells treated with 20mM NH4CI

were incubated with the labeled dextrans and analyzed for fluorescence before and

after addition of monensin. Figure 3-14 (panel A) shows the results when the

extracellular medium was pH 7.4. The fluorescence before addition of monensin was

much higher than the fluorescence of the untreated cells, indicating that the pH had

been elevated. Addition of monensin produced a slightly higher emission pattern,

indicating that the internal pH was not at pH 7.4, but that it was higher than the

control cells. The TRITC fluorescence was 1405 a.u.. The same assay was performed

except with an extracellular medium of pH 7.0 (Figure 3-14, panel B). In this case the

fluorescence measurements were essentially equal before and after addition of


















1000



800


LJ
0
Z 600



S400



200



0

5.0 5.4 5.8 6.2 6.6 7.0 7.4 7.8 8.2

pH

Figure 3-12. Relative intensity of fluorescein isothiocyanate-dextran (FITC-dextran)
fluorescence as a function of changes in pH. FITC-dextran was excitated at a
wavelength of 496nm and emission was measured at 522nm.


















2000




1500




S 1000
LU

O

LL 500




0 *

450 460 470 480 490 500 510 520

WAVELENGTH (nm)

Figure 3-13. Excitation spectra at pH 7.4 of BAT cells containing FITC-dextran before
(-g-) and after (-4-) addition of monensin. Measurements were taken at a fixed
emission wavelength of 522nm and fluorescence is expressed in arbitrary units (a.u.).
















1600



i 1200

uJ
0
z
LU
0 800


-O
LL AAn


450 460 470 480 490 500 510 520


WAVELENGTH (nm)


1200





w 800


C,
z
0,


3 400
U-


450 460 470 480 490 500 510 520

B WAVELENGTH(nm)

Figure 3-14. Excitation spectra at pH 7.4 (panel A) and pH 7.0 (panel B) of NHCI
treated BAT cells containing FITC-dextran before ( -0- ) and after ( ) addition
of monensin. Measurements were taken at a fixed emission wavelength of 522nm and
fluorescence is expressed in arbitrary units (a.u.).










monensin indicating that the intracellular pH was neutralized to pH 7.0 by the NH4CI.

The TRITC fluorescence was 1390 a.u..

Cells that were treated with 1mM chloroquine were tested in the same manner

as the NH4CI treated cells. Figure 3-15 illustrates the results of chloroquine treated

cells at external test pH values of pH 7.4. The chloroquine appeared to have elevated

the intracellular pH to pH 7.4. since the fluorescence before and after monensin were

superimposable. The TRITC fluorescence value was 1540 a.u. for the control cells

and 890 a.u. for the chloroquine treated cells. The amount of TRITC fluorescence and

FITC fluorescence after addition of monensin were lower for the chloroquine treated

cells than the control cells reflecting that there was less uptake of the dextran into the

chloroquine treated cells than the control cells. There are reports in the literature of

many effects of chloroquine on cells besides elevating the endosomal pH, such as

rendering membranes very resistant to mechanical stress; also that chloroquine

reduces uptake of some ligands into cells (Matsuzawa and Hostetler, 1980; Wibo and

Poole, 1974).

Cells treated with 5mM methylamine were also tested for the ability of this

agent to neutralize intracellular compartments. The results with this agent paralleled

the results with NH4CI treated cells, the methylamine elevates the intracellular pH to

7.0 (Figure 3-16). The values for the TRITC fluorescence were 1480 a.u. for the

control and 1465 a.u. for the methylamine treated cells.

Fluorescence Dequenching of AF-labeled EBV

Virus that was labeled with AF was shown to be infectious by the same

methods used for R,1-labeled EBV (Table 3-1). Figure 3-17 demonstrates the

fluorescence properties of virus labeled with AF as a function of changes in pH. The


















3000


2400

w
z 1800


O 1200


600




450 460 470 480 490 500 510 520

WAVELENGTH (nm)
----- CONTROL

S CONTROL-M

---'-- CHLOROOUINE

0 CHLOROQUINE-M


Figure 3-15. Excitation spectra at pH 7.4 of chloroquine treated and untreated
(control) BAT cells containing FITC-dextran before and after addition of monensin (M).
Measurements were taken at a fixed emission wavelength of 522nm and fluorescence
is expressed in arbitrary units (a.u.).


















2000



1500



z
w 1000-


0
M-4
L. 500



0
450 460 470 480 490 500 510 520

WAVELENGTH (nm)


--B- CONTROL

---- CONTROL-M

-a--- METHYLAMINE

--- -- METHYLAMINE-M


Figure 3-16. Excitation spectra at pH 7.0 of methylamine treated and untreated
(control) BAT cells containing FITC-dextran before and after addition of monensin (M).
Measurements were taken at a fixed emission wavelength of 522nm and fluorescence
is expressed in arbitrary units (a.u.).










Table 3-1. Effect of labeling with AF on the ability of MCUV5 virus to induce
immunoglobulin synthesis by fresh T-depleted human leukocytes.


Virus Immunoglobulin conc. ng/ml with:
dilution
AF-labeled Mock-labeled
virus virus

1/20 14,259 18,265

1/40 28,425 24,579

1/80 32,675 31,469

1/160 34,996 37,904

1/320 28,490 30,246

1/640 21,245 24,170

1/1280 12,248 10,960

none 1,438



























---- pH 6.0

- pH 6.5


-0-


pH 7.0

pH 7.4


508 516 524 532 540


WAVELENGTH (nm)


Figure 3-17. Fluorescence properties of virus labeled with AF at pH 6.0 to pH 7.4.
Measurements were taken at an excitation wavelength of 496nm. Fluorescence
intensity is expressed in arbitrary units (a.u.).








78

fluorescence is very sensitive to changes below pH 7.0 and is essentially undetectable

below pH 6.0.

The first cell types to be tested with AF-labeled EBV were Raji and Molt-4 cells.

Virus fuses with the Raji cell at the plasma membrane, therefore the fluorescence

should not be subject to a low pH environment. The Molt-4 cells are able to bind

virus but the virus does not penetrate the cell membrane. The data in Figure 3-18

show fluorescence dequenching up to 45% of the total bound to Raji cells but only

5.2% relief of quenching of virus bound to Molt-4 cells. Raji cells were also tested

using media with a pH of 5.5. The fluorescence remained quenched over a time

course of 32 minutes and the fluorescence when Triton was added the fluorescence

was also quenched (Figure 3-19). The fluorescence could be unquenched by addition

of 1.OM sodium phosphate to the cuvette, thus allowing determination of the amount

of fluorescence that bound to the cells.

The fluorescence dequenching of virus bound to BAT cells was very different

from that seen with the Raji cells. Figure 3-20 shows a plot of the amount of

dequenching of virus bound to cells expressed as a percentage of the value upon

addition of Triton. Also shown on the same graph is the percent dequenching of Re-

labeled EBV bound to the same population of cells. This difference reflects the

inability to measure the fluorescence of the AF-labeled virus that was in an acidic

environment.

In order to confirm this hypothesis, BAT cells were treated with 20mM NH4CI

as done in previous experiments in order to neutralize the acidic vesicles and then

these cells were used in fusion assays with AF-labeled EBV. Figure 3-21 illustrates the

results of this experiment. Virus fusion could be measured in the NH4CI treated cells

















70

--- -- RAJI
60
--- MOLT-4
S 50


40

30
z

u 20
0
Z)
LL 10



0 4 8 12 16 20 24 28 32

TIME (MINUTES)


Figure 3-18. Relief of self-quenching of AF-labeled MCUV5 virus bound to Raji and
Molt 4 cells. Increase in fluorescence is expressed as a percent of the maximum
release obtained after addition of Triton X-100 (infinite dilution).

















70

60 PH 7.2
-- pH 5.5
50


40

z 30

0 20
10


0 -

0 4 8 12 16 20 24 28 32
TIME (MINUTES)


Figure 3-19. Relief of self-quenching of AF-labeled MCUV5 virus bound to Raji cells
at pH 7.2 or pH 5.5. Virus was bound to cells at pH 7.2, cells were washed to remove
unbound virus and cells were resuspended in pH 7.2 or pH 5.5 Dulbecco's saline.
Increase in fluorescence is expressed as a percent of the maximum release obtained
after addition of Triton X-100 (infinite dilution).






















S 60 AR
2 R18
< 50


w 40

30
z
o 30
w
S20 -


10



0 4 8 12 16 20 24 28 32

TIME (MINUTES)


Figure 3-20. Relief of self-quenching of AF-labeled or R,,-labeled MCUV5 virus
bound to BAT cells at pH 7.2. Increase in fluorescence is expressed as a percent of
the maximum release obtained after addition of Triton X-100 (infinite dilution).




















60 -. .
S NH4CI
50

S40
0
z
LE 30


o 20


10



0 4 8 12 16 20 24 28 32

TIME (MINUTES)


Figure 3-21. Effect of preincubation of BAT cells with ammonium chloride (NH4CI) or
RPMI on relief of self-quenching of AF-labeled MCUV5 virus bound to cells at pH 7.2.
Increase in fluorescence is expressed as a percent of the maximum release obtained
after addition of Triton X-100 (infinite dilution).








83

to an extent comparable to that found with Re8-labeled virus, whereas in the untreated

cells the virus fusion was marginally detectable due to the pH-dependent quenching of

the fluorophore. These data not only show that most of the virus fused from within an

acidic compartment, but that virus was not dependent upon acidic pH in order to fuse

and thus enter the cell to continue the infectious cycle.



Discussion

It is well established that enveloped viruses enter their host cells by membrane

fusion, either at the plasma membrane or from within an endocytic vesicle. In many

instances, virus entry from within an endocytic vesicle is catalyzed by the acidic

environment of the endosome (Blumenthal et al., 1987; White, 1990) and this acidic

environment is a requirement for successful virus entry into the cytoplasm of the cell.

In order to assess which conditions are necessary for entry of Epstein-Barr virus into

lymphocytes, the effects of lysosomotropic agents and low pH treatment were

examined on fusion between virus and cellular membranes.

Altering the extracellular medium to pH 5.5 did not result in any increase in the

rate or extent of fusion of virus with the lymphoblastoid cells line Raji, the recently

EBV-transformed B cell line BAT, or with fresh peripheral T-depleted leukocytes.

Viruses that are catalyzed by acidification fuse rapidly and efficiently once in the

endosome and this environment can be imitated at the cell surface by lowering the pH

of the extracellular medium. If viruses in this category, such as influenza, Semliki

Forest, and West Nile virus are acidified before binding to their target membranes,

their fusion activity is irreversibly inactivated, presumably due to premature triggering

of the acid-activated conformational change in the viral fusion protein necessary for

fusion to occur.










Lysosomotropic agents raise the pH of endosomes and they have been shown

to inhibit the infectivity of all enveloped viruses tested that display low pH-dependent

fusion (Marsh and Helenius, 1989). The three agents tested in this work, chloroquine,

NH4CI and methylamine failed to inhibit fusion of virus with lymphoblastoid cells. For

the lymphoblastoid cells tested, Raji, virus has been reported to gain entry via fusion

at the plasma membrane (Nemerow and Cooper, 1984a), so it is not surprising that

these agents had no effect on virus fusion. Methylamine and NH4CI did not inhibit

virus fusion with BAT cells or fresh peripheral T-depleted leukocytes.

In contrast, chloroquine inhibited fusion of virus with BAT cells by 34% at 1mM

and by 30% at 0.5mM. For peripheral B cells, the inhibition was 60% at 1mM, 50% at

0.5mM, and 24% at 0.2mM. These results raise the questions of whether chloroquine

is effecting other biological processes besides raising the endosomal pH and also

whether methylamine and NH4CI are effectively neutralizing the acidity of the endocytic

compartments. The intracellular pH of BAT cells was determined to be raised to pH

7.0 by treatment with 20mM NHCI or 5mM methylamine and to pH 7.4 by treatment

with chloroquine. These data rule out the possibility that these agents were not

effectively raising the intracellular pH. Electron microscopy studies of internalization of

EBV into chloroquine-treated B lymphocytes showed that formation of endocytic

vacuoles proceeded normally in comparison to untreated cells but that in chloroquine-

treated B cells, intact virions remained in the vacuoles and very few nucleocapsids

were observed in the cytoplasm (Nemerow and Cooper, 1984a). Virus entry into

methylamine or NH4CI treated cells was not evaluated by electron microscopy by

these investigators, presumably because these agents had minimal effect on virus

infectivity as determined by stimulation of DNA synthesis, whereas chloroquine










reduced infectivity by 96%. Treatment of cells with methylamine or NH4CI is

considered an acceptable way of establishing whether a virus is dependent on low pH

for fusion. The results with chloroquine treated cells indicate that further studies are

necessary to determine the mechanism by which this agent inhibits virus entry into B

lymphocytes. Modifications of the endocytic pathway will be investigated in addition

to investigating other effects of the drug besides elevating endosomal pH.

Further confirmation that fusion of EBV with B lymphocytes is a pH-

Independent event was obtained from fusion assays of virus labeled with the pH-

sensitive probe 5-(N-octadecanoyl)aminofluorescein (AF). Fusion could be measured

in equal amounts with AF or R,,-labeled EBV and Raji cells because the virus fused at

the plasma membrane with this cell type. Molt cells, which bound virus but did not

internalize virus, were negative for fluorescence dequenching of the bound AF-labeled

virus indicating that the probe did not exchange between membranes in a non-specific

manner. Fusion of AF-labeled virus could not be measured with BAT cells unless the

cells were treated with NH4CI, suggesting that the virus was fusing from an acidic

compartment in which the fluorescence of AF was quenched. This was overcome by

raising the endosomal pH with NH4CI. Thus, although the EBV fusion occurred within

an intracellular compartment at low pH, the fusion was not dependent on acidic pH in

order to occur.













CHAPTER 4
MODIFICATION OF THE ENDOCYTIC PATHWAY TO DETERMINE
THE MECHANISM OF ACTION OF CHLOROQUINE ON VIRUS FUSION



Introduction

Since EBV enters B cells by endocytosis, one might assume that interference

with this process would limit virus infectivity. This has been presumed to be at least

partially responsible for the reduction in infectivity by calmodulin antagonists

(Nemerow and Cooper, 1984b). It has been shown that inhibitors of oxidative

phosphorylation and glycolysis can affect uptake of ligands into cells. Sodium azide,

which inhibits oxidative phosphorylation, has been shown to inhibit partially the uptake

of rebound Semliki Forest virus (Marsh and Helenius, 1980) and VSV (Blumenthal et

al., 1987). It is possible that the inhibition of EBV fusion by chloroquine is due to a

modification of the endocytic pathway that is inhibiting uptake of the virus into vesicles

or altering the membrane so that virus is not able to fuse with the endosomal

membrane. In addition to its pH-elevating property, chloroquine has other effects on

lysosomal functions and other cellular processes (de Duve et al., 1974; Seglen, 1983).

Chloroquine reduced uptake of asialo-fetuin (ASF) into cells when the concentration

exceeded 0.1mM, and at concentrations above 1.0mM, chloroquine almost completely

inhibited both uptake and degradation of ASF (Berg and Tolleshaug, 1980). Protease

inhibition is another effect of chloroquine (Wibo, M. and B. Poole, 1974), in particular,

inhibition of the enzyme cathepsin B ibidd) and phospholipases are also effected










(Matsuzawa and Hostetler, 1980). Chloroquine is also reported to alter membrane

fluidity (Berg and Tolleshaug, 1980).

In order to investigate further the action of chloroquine on fusion of virus with B

lymphocytes, fusion studies were done with cells treated with sodium azide, leupeptin,

and chlorpromazine.


Materials and Methods

Membrane Fusion Assay

Epstein-Barr virus that had been labeled with the fluorophore octadecyl

rhodamine B chloride (R,) at self-quenching concentration was incubated with 2 X 10'

cells and incubated for 1 hour on ice in the dark. When membrane fusion occurs

there is dilution of the fluorophore in the membranes which relieves the self-quenching

of the fluorescence. Cells were washed of unbound virus and the fluorescence

emission was monitored continuously using a spectrofluorometer at an excitation

wavelength of 560nm and an emission wavelength of 585nm. At the end of the assay

Triton X-100 was added to allow the measurement of fluorescence that would be

obtained upon infinite dilution of the fluorophore.

Cells

The lymphoblastoid cell line Raji (Pulvertaft, 1964) and the recently EBV-

transformed cell line BAT, which both express the virus receptor CR2, were grown at

37C and diluted at least biweekly in RPMI 1640 supplemented with fetal calf serum

and antibiotics. Fresh human T-depleted leukocytes were isolated from peripheral

blood by flotation on LSM followed by rosetting with sheep erythrocytes and




Full Text
145
normal infectious process. Our data showing inhibition of relief of self-quenching of
Relabeled virus by the F-2-1 antibody and the failure of the E1D1 antibody to
influence fusion is evidence for involvement of gp85 in fusion of virus with the cell
membrane, possibly leading to virus penetration. These results have been further
investigated by comparing the ability of virosomes, which are liposomes with virus
proteins incorporated, to bind and fuse to receptor positive cells (Haddad and Hutt-
Fletcher, 1989). Virosomes were labeled with R,6, and were shown to behave in a
manner similar to that of labeled virus. Monoclonal antibodies that inhibited binding of
fusion of virus inhibited binding and fusion of virosomes, and virus competed with
virosomes for attachment to cells. Virosomes made from virus proteins depleted of
gp85 by immunoaffinity chromatography remained capable of binding to receptor
positive cells but failed to fuse. These results are compatible with the hypothesis that
gp85 is actively involved in the fusion of EBV with B lymphocytes and suggest that the
ability of the antibody F-2-1 to neutralize infectivity of EBV represents a direct effect on
the function of the envelope glycoprotein gp85 as a fusion protein. Virus entry
into epithelial cells is apparently different from that with B cells. The antibody F-2-1
and the antibody E1D1 were not able to inhibit virus binding or fusion with epithelial
cells. This contrast between epithelial cell fusion and B lymphocyte fusion was not the
only difference found between these two cell types. The antibody 72A1, which
recognizes the major viral envelope proteins gp350 and gp220, completely inhibits
virus binding to B lymphocytes, but was unable to inhibit more than 50% of the virus
bound to epithelial cells. However, the remaining virus that was bound to the
epithelial cells was unable to fuse as indicated by the fluorescence dequenching of
the bound virus.


158
Kaljot, K.T., R.D. Shaw, D.H. Rubin, and H.B. Greenberg. 1988. Infectious rotavirus
enters cells by direct cell membrane penetration, not by endocytosis. J. Virol.
62:1136-1144.
Keller, P.M., A.J. Davison, R.S. Lowe, M.W. Rlemon, and R.W. Ellis. 1987.
Identification and sequence of the gene encoding gplll, a major glycoprotein of
varicella-zoster virus. Virology 157:526-533.
Kieff, E., T. Dambaugh, M. Heller, W. King, A. Cheung, S. Fennewald, K. Henness,
and T. Heineman. 1982. The biology and chemistry of Epstein-Barr virus. J. Inf. Dis.
146:506-517.
Kiellan, M. and A. Helenlus. 1985. pH induced alterations in the fusogenic spike
protein of Semliki forest virus. J. Cell Biol. 101:2284-2291.
Kirchner, H., G. Tosato, M.R. Blase, S. Broder, and I. Magrath. 1979. Polyclonal
immunoglobulin secretion by human B lymphocytes exposed to EBV in vitro. J.
Immunol. 122:1310.
Klein, G. 1983. Specific chromosomal translocations and the genesis of B-cell derived
tumors in mice and men. Cell 32:311-315.
Klein G. 1989. Viral latency and transformation: the strategy of Epstein-Barr virus.
Cell 58:5-8.
Klein, G., M. Andersson-Anvret, and N. Foresby. 1978. Nasopharyngeal carcinoma.
Progr. Exp. Tumor Res. 21:100-116.
Klein, E., G. Klein, S. Nadkarni, H. Wigzell, and P. Clifford. 1968. Surface IgM-kappa
specificity on a Burkitt lymphoma cell in vivo and in derived culture lines. Cancer Res.
28:1300-1310.
Lapidot, M., O. Nussbaum, and A. Loyter. 1987. Fusion of membrane vesicles
bearing only the Influenza hemmaglutinln with erythrocytes, living cultured cells and
liposomes. J. Biol. Chem. 262:13736-13741.
Lazarowitz, S. and P.W. Choppin. 1975. Enhancement of the infectivity of influenza A
and B viruses by proteolytic cleavage of the hemagglutinin polypeptide. Virology
68:440-454.
Lemon, S.M., L.M. Hutt-Fletcher, J.E. Shaw, L.H. Li, and J.S. Pagano. 1977.
Replication of Epstein-Barr virus in epithelial cells during infectious mononucleosis.
Nature (London) 268:268-270.
Lenoir, G.M. 1987. Role of the virus chromosomal translocation and cellular
oncogenes in the aetiology of Burkitt's lymphoma, p.184-205. in: M. Epstein and B.
Achong (ed.), The Epstein-Barr virus: recent advances. John Wiley and Sons, New
York.


140
experiment. The inhibition by the antiviral antibody 72A1, assayed in parallel, was
comparable to the inhibition by 20ug of CR2.
Effect of Antibodies on Fusion of Virus Bound to Epithelial Cells
The antibody 72A1 failed to block virus binding to parabasal and basal
epithelial cells to the same extent as it could block binding to T-depleted lymphocytes
or Raji cells. This residual virus binding was evaluated for its ability to fuse with the
cell membrane as indicated by fluorescence dequenching of the Relabeled EBV.
Figure 6-3 shows that the virus that bound to the cells after incubation with 72A1 was
unable to fuse with the cell membrane.
The monoclonal antibody F-2-1 which recognizes another envelope
glycoprotein gp85 and inhibits infectivity of B lymphocytes without having any effect
on virus binding was also tested in the fusion assay with epithelial cells. In contrast to
what has been found with B cells, this antibody does not inhibit virus binding or fusion
with epithelial cells (Figure 6-4). Another antibody that recognizes gp85, but is not a
neutralizing antibody, E1D1, was also included in this study and this antibody had no
effect on virus binding and fusion with epithelial cells (Figure 6-5).


92
Effect of Chlorpromazine on Virus Fusion
Chlorpromazine is a phenothiazine that acts as a calmodulin antagonist by
binding to calmodulin. Phenothiazines have been shown to block EBV Infectlvlty of
human B cells (Nemerow and Cooper, 1984b). Chlorpromazine has been shown to
exhibit this activity at a dose of 20uM with no Impairment of virus binding. The
endocytosis of Immunoglobulin complexes, concavalln A and alpha^macroglobulln
are Inhibited by phenothiazines. The data In Figures 4-3, 4-4 and 4-5, show that
50uM chlorpromazine Inhibited fusion with T-depleted leukocytes from 48.8% to 15.6%
with no Inhibition on virus binding, fusion with BAT cells was Inhibited by 62%, but
fusion with Rajl cells was not Inhibited.
Effect of Leupeptin on Virus Fusion
Leupeptln is N-proplonyl- and N-acetylleucylleucyl-arginal In an approximately
3:1 ratio. This agent Is a strong Inhibitor of cysteine proteinases and inhibits the
lysosomal cathepsins B, H, L, N, S, and T as well as the nonlysosomal Ca2*-
dependent proteinase II. It also inhibits a number of serine proteinases, including
trypsin, plasmln, tissue kallikrein, and ribosomal serine proteinase. Leupeptin
dissolves easily in aqueous solutions; for maximal effect It should be used at
concentration of 100 ug/ml. Leupeptin has no effect on protein synthesis or ATP
levels. The results In Figures 4-6, 4-7, and 4-8 show that this enzyme Inhibitor had no
detrimental effect on virus fusion with cells.




21
membranes in order to quantitate fusion events. Electron spin labels have been used
extensively with virus systems (Maeda et al., 1975, 1981, Lyles and Landesberger,
1979) but the extent of fusion is difficult to quantitate and continuous monitoring of the
fusion event is technically challenging. Assays utilizing fluorescent probes are much
faster and easier to perform than assays using electron spin probes and easily permit
continuous monitoring of the fusion events. The assay presented and utilized
throughout this work relies upon the relief of fluorescence self-quenching of the
fluorophore octadecyl rhodamine B chloride.
Quenching of fluorescence intensity can occur by a variety of mechanisms.
These include collisional processes with specific quenching molecules, excitation
transfer to nonfluorescent species, and complex formation or aggregation that forms
nonfluorescent species, also known as concentration quenching. Quenching of
fluorescence by added substances or by impurities can occur by a collisional process.
Molecular oxygen is one of the most widely encountered quenchers. This is because
02 is a triplet species in its ground electronic state and is able to transfer unpaired
electrons to the fluorescent species which is in the singlet state. The fluorescence
quenching of octadecyl rhodamine B chloride (Rla) is due to complex formation and is
dependent upon the concentration of the fluorophore in the lipid-containing
membrane. The self-quenching is concentration dependent because of the formation
of excimers (excited dimers) when interactions of the excited-state species occurs.
Most excited fluorophores emit fluorescence from a singlet state. The formation of
dimers results in quenching since the doublet species is not fluorescent (Tinoco et al.,
1985). The efficiency of self-quenching is directly proportional to the ratio of R18 to
total lipid. When the fluorophore is incorporated into a lipid bilayer at concentration


149
Importance of Present Studies and Future Directions
These studies have determined the mechanism of virus entry into B
lymphocytes and epithelial cells. The contribution of two viral envelope glycoproteins,
gp350/220 and gp85, in the fusion process has been evaluated. Future experiments
with recombinant gp85, gp350, and gp220 proteins Incorporated into liposomes will
allow analyses of the functional roles of these proteins with the two target cell types.
It will be Important to determine if gp350 and gp220 have separate functions In
epithelial cell and B lymphocytes and whether gp85 is dlspenslble for fusion of EBV
with epithelial cells.


121
O 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 5-6. Effect of preincubation of basal cells with sodium azide (NaN3) or RPMI
on relief of self-quenching of R18-labeled MCUV5 virus bound to cells. Increase in
fluorescence expressed as a percent of the maximum release after addition of Triton
X-100 (infinite dilution).


22
up to 9 mol% with respect to total lipid, the efficiency of the self-quenching is
proportional to its surface density (Hoekstra et al., 1984). When fusion of a labeled
membrane with a nonlabeled membrane occurs, there Is a decrease in the surface
density of the fluorophore and this results In a proportional relief of the self-quenching.
Purpose of this Work
The overall objective of this dissertation is to understand how Epstein-Barr virus
enters Its two target cells, the B lymphocyte and the epithelial cell. A greater
understanding of the conditions for successful virus penetration into both epithelial
cells and lymphocytes, as well as the viral components necessary to mediate these
events, will help to understand the unique tropism of EBV for B lymphocytes and
epithelial cells. This work presents experiments undertaken to develop an assay for
measuring fusion of EBV with cell membranes and application of the assay to follow
fusion with lymphoblastoid cells, B lymphocytes, and epithelial cells.


28
dilution series starting at 1:10. Clonal outgrowths were selected and propagated in
RPMI 1640 with 10% fetal calf serum. A cell line Initiated In this manner, designated
BAT, was utilized for comparing virus fusion with Immortalized B cells, freshly isolated
human B cells and lymphoblastold cell lines derived from tumor tissues that had been
in culture for many years.
Virus Titration and Neutralization
Infectlvity of EBV was measured in terms of Its ability to induce human
peripheral B lymphocytes to secrete Immunoglobulin In culture (Kircher et al., 1979).
T-depleted leukocytes were Incubated with or without virus at 37C In 96-well round-
bottomed tissue culture plates at concentrations of 105 cells per well In 10Oul of RPMI
1640 supplemented with 10% heat-inactivated fetal calf serum, 100IU of penicillin per
milliliter, and 100ug of streptomycin per milliliter. After 6 days in culture, 10Oul of
medium were added to each well. On day 12, the culture supernatants were collected
and the Immunoglobulin concentrations were measured. The ability of antibody to
neutralize infectivity was determined by preincubating virus for 1 hour at room
temperature with an equal volume of normal rabbit antibody at 100ug per ml, or with
mixtures of rabbit antibody and test antibody adjusted so that the total amount of
Immunoglobulin remained constant at 100ug per ml. All antibodies were heated for
30 minutes at 56C to inactivate complement prior to Incubation with virus.
Incorporation of Octadecvl Rhodamine B Chloride (R^) into Virus Membranes
Octadecyl rhodamine B chloride (R,a), is a fluorescent amphiphlle that can be
readily Inserted Into biological membranes (Figure 2-1). A stock solution of 13
nmoles/ul of R18 (Molecular Probes, Inc., Junction City, Oregon) was prepared in
chloroform/methanol (1:1) and stored at -20C. The probe was incorporated into virus


103
45/100% interfaces. Each of these populations of cells was collected separately and
extensively washed before use in experiments.
Cell Lines
The human B lymphoblastoid cell line Raji was grown at 37C and diluted at
least biweekly In RPMI 1640 (Sigma) supplemented with 10% heat-inactivated fetal calf
serum, 100 IU of penicillin and 100ug of streptomycin per ml.
Virus Production
Virus-producing cells were Induced with 30ng of 12-0-tetradecanoylphorbol-13-
acetate per ml, and after 7 days, virus was collected from the spent culture medium.
The cells were centrifuged at 4,000 X g for 10 minutes to remove cells; 100ug of
bacitracin per ml was added to the clarified supernatant, and the virus was pelleted by
centrifugation at 20,000 X g for 90 minutes. Pellets were suspended In 1/250 of the
original volume of medium containing I00ug of bacitracin per ml, redarlfied by
centrifugation three to four times at 400 X g, and filtered through a 0.45um pore filter
(Acrodisc; Gelman Sciences, Inc., Ann Arbor, Mich.).
Incorporation of R10 and AF into Virus Membranes
A stock solution of 13nmol of R18 per ul (Molecular Probes, Inc., Junction City,
Oregon) was prepared in chloroform-methanol (1:1,v/v) and stored at -20C. The
probe was Incorporated into virus membranes by a modification of the method of
Hoekstra and colleagues (Hoekstra et al., 1984). Three microliters of the stock probe
were dried under nitrogen and solubilized In ethanol, and 15ul of this solution
containing 15nmol of R18 was added to 250ul of virus with vortexing. The probe and
virus were Incubated at room temperature in the dark for 1 hour, after which the virus
and unincorporated R]S were separated by chromatography on Sephadex G-75


LIST OF ABBREVATIONS
AF
5-(N-octadecanoyl)aminofIuorescein
AIDS
acquired ¡mmunodeficiency syndrome
ASF
aslalofetuin
a.u.
arbitrary units
ATP
adenosine triphosphate
BL
Burkitts lymphoma
CMV
cytomegalovirus
CR2
complement receptor 2
DMEM
Dulbeccos modified eagles medium
DNA
deoxyribonucleic acid
EBNA
Epstein-Barr virus nuclear antigen
EBV
Epstein-Barr virus
FACS
fluorescent activated cell sorter
FITC
fluorescein Isothlocyanate
HA
hemagglutinin
HIV
human Immunodeficiency virus
HN
hemagglutinin-neuramidinase
HSV
herpes simplex virus
ig
Immunoglobulin
I.M.
infectious mononucleosis
LCL
lymphoblastoid cell line
LSM
lymphocyte separation medium
NaN3
sodium azide
NH4CI
ammonium chloride
NPC
nasopharyngeal carcinoma
OHL
oral hairy leukoplakia
SCR
short concensus repeat
SFV
Semllkl Forest virus
TPA
12-O-tetradecanoyl phorbol-13-acetate
vi


54
compartments and control cells were Incubated In medium only. At the end of the
Incubation the cells were pelleted by centrifugation and the supernatant was removed.
Cells were resuspended In 10Oul of media and Incubated with virus for 1 hour on ice.
Determination of Intracellular oH
To determine intracellular pH, cells were incubated with a mixture of fluorescein
Isothiocyanate (FITC) and tetramethylrhodamine (TRITC)-labeled dextrans (70,000 mw)
(Molecular Probes Inc., Junction City, Oregon) for 35 minutes at 37C to allow uptake
of the labeled dextrans into the cells. Cells were washed free of unassociated dextran
and analyzed In the spectrofluorometer by measuring the TRITC fluorescence at an
excitation wavelength of 560nm and an emission wavelength of 580nm followed by
measuring FITC emission at 522nm at excitation wavelengths from 450nm to 518nm.
Monensin (1ug/ml) was then added to equilibrate extracellular (test pH) and
endosomal pH and another fluorescence measurement of the FITC was taken from
450nm to 518nm. If the intensity of the fluorescence rises at this step, the average pH
of the intracellular compartments is below the test pH. If the intensity falls, the
average pH was above the test pH.
Incorporation of 5-fN-octadecanovllaminofluorescein into Virus Membranes
The membrane probe 5-(N-octadecanoyl)aminofluorescein (AF) is a fluorescent
amphlphile containing a long hydrocarbon chain which allow it to insert readily Into
biological membranes (Figure 3-1). AF manifests the same property of concentration-
dependent quenching of fluorescence as Ft18, in addition to sensitivity to changes in
pH similar to the FITC-dextran used for determination of intracellular pH. A stock
solution of 50mg/ml of AF (Molecular Probes, Inc., Junction City, Oregon) was
prepared in dimethylformamide and stored at -20C. The probe was incorporated into


4
immune system is so important for keeping the abnormal cell proliferation in control,
lymphoproliferative disorders can occur in the immunocompromised patient during
primary infection or thereafter due to failure to control the persisting latent infection.
Particularly at risk are immunosuppressed organ transplant recipients and those with
acquired immunodefiency syndrome (AIDS) (Cleary et al., 1986; Fauci, 1988; Hanto et
al., 1985; Purtilo, 1985).
Nasooharvnoeal Carcinoma
Epstein-Barr virus has also been associated with another human cancer,
nasopharyngeal carcinoma (NPC) (Anderson-Anvret et al., 1979; de-The, 1982). The
association was initially based on the finding of high antibody titers to EBV in all NPC
patients examined (de-The and Zeng, 1987; Henle and Henle, 1976). The antibody
levels for viral capsid antigen were unusually high, only paralleled by BL sera, which
increased the likelihood of involvement of EBV with the carcinoma. In 1976, EBV DNA
was found in all the undifferentiated carcinomas of the nasopharynx studied (Henle
and Henle, 1976). Subsequently, EBV DNA has been consistently found in all
undifferentiated carcinomas of the nasopharynx and has also been detected in
differentiated forms of the carcinoma as well (de-The and Zeng, 1987; Raab-Traub et
al., 1987). Nasopharyngeal carcinoma occurs throughout the world, but occurs with a
much higher incidence in populations of southern Asia. The high frequency of NPC in
the Kwantung Providence of southern China (de-The and Zeng, 1987) suggests that
other factors, perhaps genetic or environmental, are acting with EBV in the
development of the cancer (Henderson et al., 1976; Klein et al., 1978). Despite
considerable effort to identify carcinogenic substances and cultural patterns which
might operate as cofactors, no firm identification of such a factor has yet been made.


73
monensin indicating that the intracellular pH was neutralized to pH 7.0 by the NH4CI.
The TRUC fluorescence was 1390 a.u..
Cells that were treated with 1 mM chloroquine were tested in the same manner
as the NH4CI treated cells. Figure 3-15 illustrates the results of chloroquine treated
cells at external test pH values of pH 7.4. The chloroquine appeared to have elevated
the intracellular pH to pH 7.4. since the fluorescence before and after monensin were
superimposable. The TRITC fluorescence value was 1540 a.u. for the control cells
and 890 a.u. for the chloroquine treated cells. The amount of TRITC fluorescence and
FITC fluorescence after addition of monensin were lower for the chloroquine treated
cells than the control cells reflecting that there was less uptake of the dextran into the
chloroquine treated cells than the control cells. There are reports in the literature of
many effects of chloroquine on cells besides elevating the endosomal pH, such as
rendering membranes very resistant to mechanical stress; also that chloroquine
reduces uptake of some ligands into cells (Matsuzawa and Hostetler, 1980; Wibo and
Poole, 1974).
Cells treated with 5mM methylamine were also tested for the ability of this
agent to neutralize intracellular compartments. The results with this agent paralleled
the results with NH4CI treated cells, the methylamine elevates the intracellular pH to
7.0 (Figure 3-16). The values for the TRITC fluorescence were 1480 a.u. for the
control and 1465 a.u. for the methylamine treated cells.
Fluorescence Deauenchina of AF-labeled EBV
Virus that was labeled with AF was shown to be infectious by the same
methods used for Relabeled EBV (Table 3-1). Figure 3-17 demonstrates the
fluorescence properties of virus labeled with AF as a function of changes in pH. The


I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Lindsey(^i. Hutt-Fletcher, Chair
Professor of Immunology and
Medical Microbiology
I certify that I have read this study and that in my opinion it conforms to
acceptable standards of scholarly presentation and is fully adequate, In scppe and
quality, as a dissertation for the degree of Doctor of Philqsophy.
William W. Hauswiri
Professor of Immunology and
Medical Microbiology
I certify that I have read this study and that in my opinion It conforms to
acceptable standards of scholarly presentation and is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
Sue Anne Moyer
Professor of Immunology and
Medical Microbiology
I certify that I have read this study and that in my opinion It conforms to
acceptable standards of scholarly presentation and Is fully adequate, in scope and
quality, as a dissertation for the degree of Doctor of Philosophy.
John R. Dankert
Assistant Professor of Veterinary
Medicine


ENTRY OF EPSTEIN-BARR VIRUS INTO LYMPHOCYTES AND EPITHELIAL CELLS
By
NANCIMAE MILLER
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT
OF THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1991


Figure page
5-7. Effect of preincubation of basal cells with NH4CI or RPMI on relief of self
quenching of AF-labeled MCUV5 virus bound to cells 122
6-1. Effect of preincubation with 100ug of monoclonal antibodies on relief of
self-quenching of R18-labeled P3HR1-CI13 virus bound to Rajl cells 133
6-2. Effect of preincubation of virus with monoclonal antibodies on relief of
self-quenching of Relabeled MCUV5 virus bound to BAT cells 136
6-3. Effect of preincubation with 100ug of normal mouse immunoglobulin or
antibody 72A1 on relief of self-quenching of Relabeled MCUV5 virus bound
to basal epithelial cells 141
6-4. Effect of preincubation with 100ug of normal mouse Immunoglobulin or
antibody F-2-1 on relief of self-quenching of Relabeled MCUV5 virus bound
to basal epithelial cells 142
6-5. Effect of preincubation with 100ug of normal mouse immunoglobulin or
antibody E1D1 on relief of self-quenching of Relabeled MCUV5 virus bound
to basal epithelial cells 143
x


130
Table 6-1. Effect of antibodies F-2-1, E1D1,72A1, and E8D2 on the ability of MCUV5
virus to induce immunoglobulin synthesis by fresh T-depleted human leukocytes.
Monoclonal
antibody
Monoclonalmorma
I
Immunoglobulin cone,
(ng/ml) with;
%
rabbit antibody
(ng per culture)
antibody
alone
antibody
+ virus
inhibition
F-2-1
1000:0
426
5958
62
300:700
256
9396
41
100:900
268
15628
1
30:970
447
17862
0
10:990
329
17696
0
E1D1
1000:0
151
14998
5
300:700
120
15963
0
100:900
224
17583
0
30:970
132
17887
0
10:990
144
15656
1
72A1
1000:0
208
686
96
300:700
318
2429
85
100:900
404
7604
52
30:970
232
10303
35
10:990
325
16632
0


98
Discussion
The purpose of these studies was to elucidate the mechanism by which
chloroquine inhibits virus fusion with fresh B lymphocytes and BAT cells, but not Raji
cells. It had been established from the work presented in chapter 3 that this inhibition
was not due to the effect that chloroquine has on the endosomal pH because other
agents that elevate endosomal pH, such as methylamlne and NH4CI, had no effect on
fusion with any cell type. Two other possible effects of the drug were considered.
First, since chloroquine only had effects on cells Into which virus was endocytosed
(see above and chapter 5) and the amount of dextran taken up into cells treated with
chloroquine was less than that taken Into untreated cells or cells treated with the other
two lysosomotropic agents, it seemed possible that endocytosis was required for
fusion with some cell types. Second, chloroquine has been reported to have activity
as an inhibitor of cathepsln B and also some phospholipases (Wibo and Poole, 1974).
Proteolytic cleavage Is a prerequisite for function of several viral fusion proteins (White
et al, 1983). Although this process usually occurs during maturation of virus It is
possible that activation of fusion via proteolysis could occur following binding of virus
to the host cell (Hattori et al., 1989) and be required for fusion with the normal B cell.
The first of these possibilities was examined by testing the effects of two other
inhibitors of endocytosis on virus fusion.
Sodium azide (NaNJ inhibits oxidative phosphorylation in cells which affects
their endocytic capacity; fusion of VSV via the endocytic pathway was inhibited by
treatment of the cells with NaN3 (Blumenthal et al., 1987). Fusion of EBV with Raji
cells was not Inhibited by sodium azide, which would be expected because the virus
fuses at the plasma membrane, but both BAT cells and T-depleted leukocytes treated


Table 5-2. Morphological distribution of epithelial cells in fractions from Percoll
gradient.
108
gradient0
fraction
%
of
total
distribution of cells (%)
basal
parabasal
squamous
1 -media/30%
9.2
6.2
18.4
75.4
interface
2.3
1.2
3.5
6.2
2-30/40%
15.8
17.3
66.2
16.5
interface
8.1
3.4
7.9
5.9
3-40/45%
26.5
23.5
71.5
5.0
interface
5.2
5.6
6.9
3.0
4-45/100%
47.9
71.2
28.8
0
interface
10.3
7.6
7.4
30%. 40%, 45%, 100% Percoll


2
time, M. A. Epstein was in search of a human cancer caused by a virus and became
interested in the tumor that Dr. Burkitt described. EBV fulfilled many requirements
used to define an oncogenic virus. Virus was present in all tumor cells but not normal
tissue from the same patient, patients had extremely high antibody titers to EBV, EBV
could Immortalize human B lymphocytes in vitro, and EBV was capable of inducing
tumors in subhuman primates. However, there could be no simple causal relationship
between EBV and BL because the virus was found to infect humans worldwide at a
frequency of 90-100% (Evans, 1984). The virus was considered likely to play the role
of a cofactor in development of African BL, but additional cellular changes were
assumed to occur to create malignant BL cells. The presence of phenotypic
differences between EBV-genome containing BL cells and EBV-immortalized
nonmalignant cells provided support for this theory. All cell lines derived from BL
contained chromosomal translocations (Klein, 1983). The characteristic translocation
found in BL is a translocation of chromosome 8 with chromosome 14, but can also
involve 2 or 22 (Miyoshi et al., 1981). The c-myc oncogene has been localized to
chromosome 8 in humans. The translocations involve the juxtaposition of c-myc with
the Immunoglobulin heavy chain gene cluster on chromosome 14, the kappa light-
chain genes on chromosome 2 and the lambda light-chain genes on chromosome 22
(Lenoir, 1987). It Is likely that the c-myc oncogene plays a role in the development of
BL, but that the translocation is not Induced by EBV. Rather, the appearance of
malignant cell clones that have altered c-myc may be facilitated when EBV causes
unrestricted proliferation of B cells in cooperation with immunosuppression from
persistent malaria infections that are holoendemic in central Africa.


89
O 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 4-1. Effect of preincubation of Raji cells with sodium azide (NaN3) or RPMI on
relief of self-quenching of Relabeled MCUV5 virus. Increase in fluorescence is
expressed as a percent of the maximum release obtained after addition of Triton X-
100 (infinite dilution).


66
O 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 3-10. Effect of preincubation of T-depleted leukocytes with chloroquine or
RPMI on relief of self-quenching of Relabeled MCUV5 virus bound to cells. Increase
In fluorescence is expressed as a percent of the maximum release obtained after
addition of Triton-X-100 (Infinite dilution).


114
O 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 5-1. Relief of seif-quenching of Relabeled MCUV5 virus bound to parabasal
and basal epithelial cells. Increase In fluorescence is expressed as a percent of the
maximum release obtained after addition of Triton X-100 (infinite dilution).


34
Effect of R on the Attachment of Virus
When adapting the fluorescence assay of Hoekstra and colleagues for use with
EBV, our first question was whether labeling of the virus with the fluorescent molecule
would qualitatively or quantitatively affect virus binding. To answer this question we
labeled virus metabollcally with 3HTdR, divided the virus Into three aliquots, left one
untreated, labeled one with an ethanolic solution of R18 and mock-labeled the third
aliquot with ethanol alone. The labeled and mock-labeled preparations of virus were
chromatographed on Sephadex G-75. A virus binding assay was done and the
amount of radioactivity bound to receptor positive and negative cells was measured.
Approximately half the blndable virus was lost during the labeling and mock-labeling
procedures. However, If the amount of radioactivity bound was expressed as a
percentage of the amount added, it could be seen that the labeling had no effect on
the ability of the virus to bind to receptor positive cells (Table 2-1). There was no
increase in nonspecific binding to receptor negative P3HR1-CI5 cells.
The specificity of binding was further confirmed by showing that preincubation
of virus with antibody 72A1 inhibited Its ability to bind to receptor positive cells (Table
2-2). Two additional antibodies that have anti-CR2 activities were used In this
experiment. Preincubation of cells with one, OKB7, which normally blocks virus
binding (Nemerow et al., 1985a), inhibited labeled virus binding; preincubation of cells
with HB5, a monoclonal antibody to CR2 that does not block the virus binding sites,
appropriately failed to inhibit binding of labeled virus (Table 2-2).
Effect of Rln on Infectivitv of Virus
Although the incorporation of R18 Into the virus membrane did not alter the
binding properties of the virus, it remained possible that the probe interfered with a


LIST OF FIGURES
Figure Page
2-1. Structural formula of octadecyl rhodamlne B chloride (R1S) 29
2-2. Excitation and emission spectra of Ri8-contalning virions relieved of self
quenching with Triton X-100 (Infinite dilution) 32
2-3. Stability of self-quenching of Relabeled virions 33
2-4. Relief of self-quenching of Relabeled virus bound to receptor positive
Raji cells and receptor negative Daudl cells 39
2-5. Comparison of relief of self-quenching of Relabeled P3FIR1-CI13 virus
bound to Raji cells, fixed Raji cells, or Molt 4 cells 40
2-6. Relief of self-quenching of Relabeled P3HR1-CI13 virus bound to tonsil
derived T-depleted leukocytes 42
2-7. Comparison of relief of self-quenching of Relabeled P3HR1-CI13 virus
bound to tonsil derived B cells pre and post monocyte depletion by
adherance to plastic 45
2-8. Relief of self-quenching of Relabeled MCUV5 virus bound to fresh
T-depleted peripheral leukocytes 46
2-9. Relief of self-quenching of Relabeled MCUV5 virus bount to BAT cells. 47
3-1. Structural formula of 5-(N-octadecanoyl)aminofluoresceln (AF) 55
3-2. Relief of self-quenching of Relabeled MCUV5 virus bound to Raji cells
at pH 7.2 or pH 5.5 57
3-3. Relief of self-quenching of Relabeled MCUV5 virus bound to BAT cells
at pH 7.2 or pH 5.5 58
3-4. Relief of self-quenching of Relabeled MCUV5 virus bound to T-depleted
leukocytes at pH 7.2 or pH 5.5 59
3-5. Effect of preincubation of Raji cells with ammonium chloride or RPMI on
relief of self-quenching of Relabeled MCUV5 virus bound to cells 61
vii


FLUORESCENCE (% MAXIMUM)
81
TIME (MINUTES)
Figure 3-20. Relief of self-quenching of AF-labeled or Relabeled MCUV5 virus
bound to BAT cells at pH 7.2. Increase in fluorescence is expressed as a percent of
the maximum release obtained after addition of Triton X-100 (infinite dilution).


131
E8D2
1000:0
199
15250
4
300:700
316
14821
6
100:900
352
16487
0
30:970
124
17775
0
10:990
140
17379
0
None
0:1000
263
15840
0


74
a CHLOROQUINE
O CHLOROQUINE-M
Figure 3-15. Excitation spectra at pH 7.4 of chloroquine treated and untreated
(control) BAT cells containing FITC-dextran before and after addition of monensin (M).
Measurements were taken at a fixed emission wavelength of 522nm and fluorescence
is expressed in arbitrary units (a.u.).


53
times with ice-cold Dulbeccos saline at pH 7.4 and suspended in 400ul of Dulbeccos
at pH 7.4 (unless otherwise indicated) when transferred to the microcuvette of a
spectrofluorometer (SLM SPF 500C, SLM Instruments Co., Urbana, Illinois) equipped
with a magnetic stirrer and circulating water bath set at 37C. Fluorescence
dequenching was monitored continuously at an excitation wavelength of 560nm and
an emission wavelength of 585nm. At the end of the assay, Triton X-100 (1% v/v,
final concentration) was added to allow the measurement of fluorescence that would
be obtained upon infinite dilution of the fluorophore.
Cells
The lymphoblastoid cell lines Raji (Pulvertaft, R.J., 1964) and BAT, which are
both EBV genome-positive human B-cell lines expressing the virus receptor CR2
(CD21); Molt 4 (Minowda et al., 1972), an EBV genome negative human T cell line that
expresses CR2, but cannot internalize virus (Menezes et al., 1977) and P3HR1-CI5
(Heston et al., 1982), an EBV genome-positive human B-cell line which does not
express CR2 were grown at 37C and diluted at least biweekly in RPMI 1640
supplemented with heat-inactivated fetal calf serum, 100IU of penicillin and lOOug of
streptomycin per ml. Fresh human T cell-depleted leukocytes were isolated as
described previously from peripheral blood and used directly in assays.
Treatment of Cells with Lysosomotropic Agents
Ammonium chloride (NH4CI), chloroquine, and methylamine were purchased
from Sigma and stock solutions were made in phosphate buffered saline of 100mM,
100mM, and 50mM respectively, from which dilutions were made in RPMI 1640 for
incubation with cells. Cells were incubated in one milliliter of media containing the
lysosomotropic agent for 35 minutes at 37C to neutralize acidic intracellular


132
Table 6-2. Effect of antibodies F-2-1,72A1, and E8D2 on the ability of [3H] EBV to
bind to receptor positive cells.
Antibody
concentration
(ug/mi)
Total acid precipitable counts in presence of:
72A1
F-2-1
E8D2
600
177
5831
4891
400
162
5163
5080
200
187
5899
5832
100
220
5044
5379
50
417
5549
5682
25
1183
5038
5297
12.5
2296
4760
5494
None
4764
Counts bound to receptor negative cell = 110


141
TIME (MINUTES)
Figure 6-3. Effect of preincubation with 100ug of normal mouse Immunoglobulin (A)
or antibody 72A1 (B) on relief of self-quenching of Relabeled MCUV5 virus bound to
basal epithelial cells. Increase in fluorescence Is expressed in arbitrary units (a.u.).
Arrow indicates point at which Triton X-100 was added to measure maximum relief of
self-quenching of bound virus (infinite dilution).


Table 6-4. Effect of antibody or unlabeled virus on binding of Relabeled virus.
Relative amounts of virus bound to:
Treatment
Parabasal
cells
Basal
cells
Raji
none
100 (285)a
100 (535)
100 (1076)
unlabeled
virus
14 (41)
10 (54)
3 (35)
antibody
72A1
48 (137)
54 (290)
7 (73)
'Figures in parentheses Indicate maximum fluorescence in arbitrary units.


85
reduced infectivity by 96%. Treatment of cells with methylamlne or NH4CI is
considered an acceptable way of establishing whether a virus is dependent on low pH
for fusion. The results with chloroquine treated cells indicate that further studies are
necessary to determine the mechanism by which this agent Inhibits virus entry Into B
lymphocytes. Modifications of the endocytlc pathway will be Investigated In addition
to investigating other effects of the drug besides elevating endosomal pH.
Further confirmation that fusion of EBV with B lymphocytes Is a pH-
Independent event was obtained from fusion assays of virus labeled with the pH-
sensitlve probe 5-(N-octadecanoyl)amlnofluorescein (AF). Fusion could be measured
In equal amounts with AF or Relabeled EBV and Raji cells because the virus fused at
the plasma membrane with this cell type. Molt cells, which bound virus but did not
internalize virus, were negative for fluorescence dequenching of the bound AF-labeled
virus indicating that the probe did not exchange between membranes in a non-specific
manner. Fusion of AF-labeled virus could not be measured with BAT cells unless the
cells were treated with NHCI, suggesting that the virus was fusing from an acidic
compartment in which the fluorescence of AF was quenched. This was overcome by
raising the endosomal pH with NH4CI. Thus, although the EBV fusion occurred within
an intracellular compartment at low pH, the fusion was not dependent on acidic pH in
order to occur.


FLUORESCENCE (a.u.)
33
Figure 2-3. Stability of self-quenching of Relabeled virions maintained at 37C and
relief of self-quenching upon addition of Triton X-100 (infinite dilution) after 30
minutes. Relative fluorescence expressed in arbitrary units (a.u.).


24
fusion between membranes but also provides an opportunity to analyze the kinetics of
fusion between membranes, which can be useful for comparing the kinetics of virus
fusion with different cell types.
Materials and Methods
Lvmphoblastoid Cell Lines
Cell lines were grown at 37C and diluted at least biweekly in RPMI 1640
(Sigma Chemical Co., St. Louis, Missouri) supplemented with heat-inactivated fetal
calf serum (5-10%, depending on cell type), 100 IU of penicillin and 100ug of
streptomycin per milliliter. The cell lines used include four human EBV genome
positive B lymphoblastoid cell lines, Raji (Pulvertaft, 1964), Daudi (Klein et al., 1968),
P3HR1-CI13 and P3HR1-CI5 (Heston et al., 1982). Raji is a latently infected virus
nonproducing cell line expressing CR2. Daudi Is a genome positive nonproducing cell
line that currently, in our laboratory, does not express CR2. P3HR1-CI13 is a
superinducible virus producing cell line and P3HR1-CI5 is a genome positive cell line
derived from the same parent line as P3HR1-CI13, but currently in our laboratory does
not produce virus. Also used were MCUV5, an EBV producing marmoset cell line and
Molt 4 (Minowda et al., 1972), an EBV genome negative human T cell line that
expresses CR2, but cannot internalize virus (Menezes et al., 1977).
Virus Production and Radiolabeling
A small percentage of the P3HR-CI13 cells spontaneously produce low levels
of virus, but this amount can be increased after induction with 30ng of 12-0-
tetradecanoyl phorbol-13-acetate (TPA) per milliliter (Sigma). The virus obtained from


CHAPTER 5
ISOLATION AND IDENTIFICATION OF EPITHELIAL CELLS EXPRESSING
A RECEPTOR FOR EPSTEIN-BARR VIRUS AND STUDIES OF VIRUS ENTRY
INTO THESE CELLS
Introduction
The ability of EBV to Infect lymphocytes Is initiated by attachment of virus to
the cell membrane glycoprotein CR2, which also binds the C3d fragment of
complement (Fingeroth et al., 1984; Nemerow et al., 1985b). CR2 Is a 145-kllodalton
B cell membrane glycoprotein (Weis et al., 1984) whose precise function is not known,
but appears to be Immunoregulatory (Cooper et al., 1988). The B cell CR2 Is lost with
activation and differentiation to the plasma cell. CR2 has been assigned to the CD21
antigen cluster of B cell differentiation (Relnherz et al., 1986).
Epithelial cells have been shown to express a receptor for virus attachment In a
differentiation-dependent manner. The receptor has been detected on the surface of
non-keratinized squamous cells and deeper layers (Slxbey et al., 1987; Young et al.,
1986). The receptor expression appears limited to the less differentiated cells,
although there Is some confusion In the literature about Its precise distribution.
Epithelia In certain anatomical sites can support EBV Infection, most dramatically
demonstrated In oral hairy leucoplakia and nasopharyngeal carcinoma, but the
functional significance of a CR2-like molecule on epithelial cells remains yet
undetermined. Epithelial Involvement in both acute Infection and the chronic carrier
101


161
Miyoshi, !., K. Hamasaki, K. Miyamoto, K. Nagase, K. Narahara, K. Kitajima, I. Kimure,
and J. Sato. 1981. Chromosomal translocations in Burkitts lymphoma. N. Engl. J.
Med. 304:734.
Morgan, D.G, J.C. Niederman, G. Miller, H.W. Smith, and J.M. Dowaliby. 1979. Site of
Epstein-Barr virus replication in the oropharynx. Lancet 2:1154-1157.
Morris, S.J., D.P. Sarkar, J.M. White, and R. Blumenthal. 1989. Kinetics of pH-
dependent fusion between 3T3 fibroblasts expressing influenza hemagglutinin and red
blood cells. J. Biol. Chem. 264:3972-3978.
Nadler, L.M., P. Stashenko, R. Hardy, A. vanAgthoven, C. Terhost, and S.
Schlossman. 1981. Characterization of a human B cell-specific antigen (B2) distinct
from B1. J. Immunol. 126:1941-1947.
Nemerow, G.R. and N.R. Cooper. 1984a. Early events in the infection of human B
lymphocytes by EBV: the internalization process. Virology 132:186-198.
Nemerow, G.R. and N.R. Cooper. 1984b. Infection of B lymphocytes by a human
herpesvirus, Epstein-Barr virus, is blocked by calmodulin antagonists. Proc. Natl.
Acad. Sci. USA 81:4955-4959.
Nemerow, G.R., R.A. Houghten, M.D. Moore, and N.R. Cooper. 1989. Identification of
an epitope in the major envelope protein of Epstein-Barr virus that mediates viral
binding to the B lymphocyte EBV receptor (CR2). Cell 56:369-377.
Nemerow, G.R., M.E. McNaughton, and N.R. Cooper. 1985a. Binding of monoclonal
antibody to the Epstein-Barr virus CR2 receptor induces activation and differentiation
of human B lymphocytes. J. Immunol. 135:3068-3073.
Nemerow, G.R., C. Mold, V.K. Schwend, V. Tollefson, and N.R. Cooper. 1987.
Identification of gp350 as the viral glycoprotein mediating attachment of Epstein-Barr
virus to the EBV/C3d receptor of B cells: sequence homology of gp350 and C3
complement fragment C3d. J. Virol. £1:1416-1420.
Nemerow, G.R., J.J. Mullen III, P.W. Dickson, and N.R. Cooper. 1990. Soluble
recombinant CR2 (CD21) inhibits Epstein-Barr infection. J. Virol. 64:1348-1352.
Nemerow, G.R., R. Wolfert, M. McNaughton and N.R, Cooper. 1985b. Identification
and characterization of the Epstein-Barr virus receptor on human B lymphocytes and
its relationship to the C3d complement receptor CR2. J. Virol. 55:347-351.
Niederman, J.C., A.S. Evans, L. Subrahmanyan, and R.C. McCollum. 1970.
Prevalence, incidence and persistence of EB virus antibody in young adults. New
Engl. J. Med. 282:361-365.


5
Oral Hairy Leucoplakia
Epsteln-Barr virus is also associated with oral hairy leucoplakia (OHL), a
proliferative lesion of the lateral tongue epithelium found in persons infected with HIV
(Greenspan et al., 1984). The presence of OHL indicates that patients are severely
immunocompromised and has proven to be a valuable prognosticator of the onset of
AIDS (Greenspan et al., 1987; Schiodt et al., 1987). Studies of OHL lesions reveal
EBV particles within the nucleus, cytoplasm and the intercellular spaces of epithelial
cells (Sclubba et al., 1989). In the basal layers, the BZLF1 gene is expressed, which
activates the switch from latency into replication. In the upper third of the epithelium,
structural proteins and viral envelope components are found (Ibid). There is
temporary regression of the OHL lesions when the patients are treated with acyclovir,
but the lesions recur weeks or months after cessation of acyclovir therapy, indicating
that EBV plays an active role in development of the lesions (Resnlck et al., 1988).
Description of EBV
Classification and Morphology
Epstein-Barr virus is classified as human herpesvirus 4 and as a member of the
gamma herpesvirus subfamily (Roizman, 1982). Morphologically, EBV is
indistinguishable from other members of the herpes family. The diameter of the
mature virus particle is about 150 to 180 nm. The virus envelope is acquired as the
virus buds through the nuclear membrane. The envelope consists of at least five
proteins that are encoded by the virus, four of which are glycosylated. The lipid
component of the envelope Is derived from host cell membrane in which cellular
proteins have been replaced by those encoded by the virus (Spear, 1980). Within the


19
(Noble et al., 1983). Virus lacking gB (Cal et al., 1988) or gD (Johnson and Ligas,
1988) attaches but does not penetrate. The glycoprotein gH is present in the viral
envelope at concentrations at least 10-fold lower that gD (Richman et al., 1986).
Despite this fact, antibodies against gH have neutralizing activity comparable to that of
antibodies against gD (Minson et al., 1986). A monoclonal antibody to gH has also
been shown to exhibit anti-fusion activity (Ibid). Thus three glycoproteins, gB, gD, and
gH, are likely either to induce or influence the fusion process which occurs In a pH-
Independent manner at the surface of the cell. There Is no evidence to suggest that
they act as a single functional heteropolymer. Homodimers of gB extracted from
virions or infected cells are not associated with other glycoproteins (Claesson-Welsh
and Spear, 1986), and gB and gD have been shown to form morphologically distinct
structures In the virion envelope (Stannard et al., 1987).
Entry of Epstein-Barr Virus
Infection of B lymphocytes and epithelial cells with EBV Is initiated by
attachment of virus to a 145-kilodalton cell membrane glycoprotein, CR2, which also
serves as the receptor for the C3d fragment of the complement cascade (Cooper et
al., 1990; Flngeroth et al., 1984; Nemerow et al., 1985b; Sixbey et al., 1987).
Expression of the CR2 molecule on both cell types is linked to cell differentiation.
CR2 expression on human B lymphocytes is lost at the plasma cell stage of
differentiation (Tedder et al., 1984). Immunofluorescent studies have demonstrated
expression of CR2 on epithelia in a differentiation-linked manner as it is on B
lymphocytes (Sixbey et al., 1987; Young et al., 1986, 1989). Binding of EBV to CR2 is
mediated by attachment of at least one virus membrane glycoprotein, gp350


104
(Sigma). Labeled virus was stored at -70C. Labeled virus was still infectious and
retained binding specificity of unlabeled virus.
A stock solution of 50mg/ml of AF (Molecular Probes, Inc., Junction City,
Oregon) was prepared in dimethylformamide and stored at -20C. The probe was
incorporated into the virus membrane and stored in the same manner as the R,s
probe.
Fluorescence Measurement
R18 labeled virus (or AF-labeled virus) was added to pellets of 2 X 106 cells and
incubated for 1 hour on ice in the dark. Cells were washed four times with ice-cold
Dulbeccos saline, suspended in 400ul, and transferred to the microcuvette of the
spectrofluorometer (SLM SPF500C) equipped with a magnetic stirrer and circulating
water bath. For the Relabeled virus, fluorescence was monitored continuously at an
excitation wavelength of 560nm and an emission wavelength of 585nm, AF-labeled
virus was measured at an excitation wavelength of 496nm and an emission
wavelength of 522nm. At the end of the assay, Triton X-100 (1% v/v, final
concentration) was added to allow the measurement of fluorescence that would be
obtained upon infinite dilution of the fluorophore.
Cells were also analyzed for virus binding and fusion by direct fluorescence
microscopy with Relabeled virus and evaluated for receptor expression by indirect
immunofluorescence microscopy. Cells were incubated with Relabeled virus for 35
minutes at 37C, washed of excess virus and mounted onto a slide for analysis. For
indirect immunofluorescence, cells were incubated with monoclonal antibody, washed
three times and incubated with FITC-conjugated sheep anti-mouse immunoglobulin G
and washed again before analysis.


Table 5-5. Microscopic analysis of virus binding and fusion with epithelial cells.
Treatment
parabasal8
basal8
unfixed
fixed
unfixed
fixed
ebv-r18
20 + 5
1+2
266
31
HB5
27+7
24 + 3
8+4
7 + 5
Results expressed as percent positive staining cells.
Antibody binding was visualized with FITC-labeled anti-mouse antibody.


146
The specificity of virus binding to epithelial ceils was evaluated by the ability of
unlabeled virus to compete for binding of Relabeled material. Unlabeled virus
markedly reduced the amount of virus binding, whereas the antibody 72A1 was
unable to inhibit all virus binding. Virus binding in the presence of soluble receptor
was the next approach investigated and this reagent was also unable to completely
inhibit virus attachment of virus to epithelial cells. The residual virus binding to
epithelial cells is apparently non-functional in terms of the ability of the virus to fuse
with the cell membrane as measured by the fluorescence dequenching assay. This
binding may be irrelevant to the infectious cycle in this cell type but it could also be
an indication that there is more than one type of binding interaction that is necessary
for virus fusion to occur with the epithelial cell and that the antibody 72A1 is capable
of preventing one of the interactions to an extent that fusion is inhibited.
The ability of a peptide corresponding to the amino terminus of gp350/220 to
block virus binding to B cells indicates that this region is primarily responsible for virus
attachment to B cells via CR2 (Nemerow et al., 1989). This peptide has a homology
with the C3d protein, suggesting that a single domain may be used for binding of
both ligands. This does not eliminate the possibility that other regions of gp350/220
also mediate or contribute to receptor interaction. It is once again relevant to point
out that the anti-CR2 monoclonal antibody OKB7 was able to block binding of EBV
and C3d with CR2 on B cells, but that OKB7 did not react with epithelial cells.
Considering the complexity of the EBV envelope and the specific tropism that
the virus maintains, it is conceivable that certain envelope proteins may have functions
that are unique to one type of host cell.


14
and although they found no evidence of an endocytic entry pathway, they did not rule
out the possibility that virus could enter by both pathways in a pH-independent
manner. Analysis of cells expressing a mutant form of CD4 that had Impaired ability
to undergo endocytosis revealed that HIV Infection did not require endocytosis of its
receptor, CD4 (Maddon et al., 1988).
For EBV, studies utilizing electron microscopy and immunoelectron microscopy
have reported direct fusion at the plasma membrane of EBV with the lymphoblastold
cell line, Raji (Nemerow and Cooper, 1984a; Selgneurin et al., 1977). Virus
nucleocapslds were found in the cytoplasm directly beneath the cellular plasma
membrane, while virus was never found to be bound to the clathrin-coated areas of
the plasma membrane, nor observed in endocytic vesicles. The same studies using
normal B lymphocytes revealed transfer of membrane bound virus Into vesicles.
These vesicles were distinct In size and appearance from clathrin-coated vesicles.
After 30 minutes at 37C very few virus particles remained In the vesicles.
Membrane Fusion Proteins
A virus envelope has a relatively simple protein composition that has three
main functions: facilitation of assembly and egress of virus particles, protection of the
genome during the extracellular transport of virus, and delivery of nucleocapsids Into
host cells. The following viruses have proteins well characterized for ability to mediate
viral and cell fusion: Sendai, Semlikl Forest, Influenza, and vesicular stomatitis virus.
Fusion proteins identified to date are glycoproteins which span the bilayer and have
the bulk of their mass exposed externally. The transmembrane anchor region of the
glycoprotein is frequently composed of hydrophobic residues that favor alpha helix
formation.


82
Figure 3-21. Effect of preincubation of BAT cells with ammonium chloride (NH4CI) or
RPMI on relief of self-quenching of AF-labeled MCUV5 virus bound to cells at pH 7.2.
Increase in fluorescence is expressed as a percent of the maximum release obtained
after addition of Triton X-100 (Infinite dilution).


Table 3-1. Effect of labeling with AF on the ability of MCUV5 virus to induce
Immunoglobulin synthesis by fresh T-depleted human leukocytes.
Virus
dilution
Immunoglobulin cone, ng/ml with:
AF-labeled
virus
Mock-labeled
virus
1/20
14,259
18,265
1/40
28,425
24,579
1/80
32,675
31,469
1/160
34,996
37,904
1/320
28,490
30,246
1/640
21,245
24,170
1/1280
12,248
10,960
none
1,438


78
fluorescence Is very sensitive to changes below pH 7.0 and is essentially undetectable
below pH 6.0.
The first cell types to be tested with AF-labeled EBV were Raji and Molt-4 cells.
Virus fuses with the Raji cell at the plasma membrane, therefore the fluorescence
should not be subject to a low pH environment. The Molt-4 cells are able to bind
virus but the virus does not penetrate the cell membrane. The data in Figure 3-18
show fluorescence dequenching up to 45% of the total bound to Raji cells but only
5.2% relief of quenching of virus bound to Molt-4 cells. Raji cells were also tested
using media with a pH of 5.5. The fluorescence remained quenched over a time
course of 32 minutes and the fluorescence when Triton was added the fluorescence
was also quenched (Figure 3-19). The fluorescence could be unquenched by addition
of 1.0M sodium phosphate to the cuvette, thus allowing determination of the amount
of fluorescence that bound to the cells.
The fluorescence dequenching of virus bound to BAT cells was very different
from that seen with the Raji cells. Figure 3-20 shows a plot of the amount of
dequenching of virus bound to cells expressed as a percentage of the value upon
addition of Triton. Also shown on the same graph is the percent dequenching of Re
labeled EBV bound to the same population of cells. This difference reflects the
Inability to measure the fluorescence of the AF-labeled virus that was in an acidic
environment.
In order to confirm this hypothesis, BAT cells were treated with 20mM NH4CI
as done in previous experiments in order to neutralize the acidic vesicles and then
these cells were used In fusion assays with AF-labeled EBV. Figure 3-21 illustrates the
results of this experiment. Virus fusion could be measured In the NH4CI treated cells


Rose, J.K., M.J. Welch, B.M. Sefton, F.S. Esch, and N.C. Ling. 1980. Vesicular
stomatitis virus glycoprotein is anchored In the viral membrane by a hydrophobic
domain near the COOH terminus. Proc. Natl. Acad. Sci. USA 77:3884-3888.
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Rowe, M., D.T. Rowe, C.D. Gregory, L.S. Young, P.J. Farrell, H. Rupani, and A.S.
Rickinson. 1987. Differences in B cell growth phenotype reflect novel patterns of
Epstein-Barr virus latent gene expression in Burkitt's lymphoma cells. EMBO J.
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isolates extends to the EBNA-3 family of nuclear proteins. J. Virol. 63:1031-1039.
Ruigrok, R.W., A. Aiken, L.J. Calder, S.R. Martin, and J.J. Skehel. 1988. Studies on
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Salisbury, J.L., J.S. Condeelis, and P. Satlr. 1980. Evidence for the involvement of
calmodulin in endocytosis. Ann. N.Y. Acad. Sci. 356:429-432.
Sarmiento, M., M. Kaffey, and P.G. Spear. 1979. Temperature-sensitive mutant of
herpes simplex-1 gB adsorbs but does not penetrate. J. Virol. 29:1149-1158.
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64:716-720.
Sclubba, J., J. Brandsma, M. Schwartz, and N. Barrezueta. 1989. Hairy leucoplakia:
and AIDS-associated opportunistic infection. Oral Surg. Oral. Med. Oral. Pathol.
67:404-410.
Sculley, T.B., D.G. Sculley, J.H. Pope, G.W. Bornkamm, G.M. Lenoir, and A.B.
Rickinson. 1988. Epstein-Barr virus nuclear antigens 1 and 2 in Burkltt lymphoma cell
lines containing either A- or 'B'-type virus. Intervirology 29:77-85.


155
Gething, M.J., R.W. Doms, D. York, and J. White. 1986. Studies on the mechanism of
membrane fusion: site specific mutagenesis of the hemagglutinin of influenza virus. J.
Cell Biol. 102:11-23.
Gething, M.J., J.M. White, and M.D. Waterfield. 1978. Purification of the fusion protein
of Sendai virus: analysis of the NH2-terminal sequence generated during precursor
activation. Proc. Natl. Acad. Sci. USA 75:2737-2740.
Gilbert, J.M., D. Mason, and J.M. White. 1990. Fusion of Rous sarcoma virus with
host cells does not require exposure to low pH. J. Virol. 64:5106-5113.
Given, D., D. Yee, K. Griem, and E. Kieff. 1979. DNA of EBV V. Direct repeats of the
ends of EBV DNA. J. Virol. 30:852-867.
Golden, H.D., R.S. Chang, W. Prescott, E. Simpson, and T.Y. Cooper. 1973.
Leukocyte-transforming agent: prolonged excretion by patients with mononucleosis
and excretion by normal individuals. J. Infect. Dis. 127:471-473.
Goldstein, J.L., R.R. Anderson, and M.S. Brown. 1979. Coated pits, coated vesicles,
and receptor-mediated endocytosis. Nature (London) 279:679-685.
Gollins, S.W. and J.S. Porterfield. 1986. The uncoating and infectivity of the Flavivirus
West Nile on interaction with cells; effects of pH and ammonium chloride. J. Gen.
Virol. 6Z: 1941-1950.
Gompels, U. and A. Minson. 1986. The properties and sequence of glycoprotein H of
herpes simplex type 1. Virology 153:230-247.
Greenspan, D., J.S. Greenspan, M. Conant, V. Peterson, S. Silverman, and Y.
DeSouza. 1984. Oral hairy leucoplakia in male homosexuals: evidence of association
with both papillomavirus and a herpes-group virus. Lancet 2:831-834.
Greenspan, D., J.S. Greenspan, and N.G. Hearst. 1987. Relation of oral hairy
leucoplakia to infection with the human immunodeficiency virus and the risk of
developing AIDS. J. Infect. Dis. 155:475-481.
Greenspan, J.S., D. Greenspan, E.T. Lennette, D.l. Abrams, M.A. Conant, V. Peterson,
and U.K. Freese. 1985. Replication of Epstein-Barr virus within the epithelial cells of
oral "hairy" leukoplakia, an AIDS-associated lesion. New Engl. J. Med. 313:1564-1571.
Grogan, E., G. Miller, W. Henle, M. Rabson, D. Shedd, and J.C. Neiderman. 1981.
Expression of Epstein-Barr virus viral early antigen in monolayer tissue cultures after
transfection with viral DNA and DNA fragments. J. Virol. 40:861-869.
Gussander, E. and A. Adams. 1984. E.M. evidence for replication of circular EBV
genome in latently infected Raji cells. J. Virol. 52:549-556.


84
Lysosomotropic agents raise the pH of endosomes and they have been shown
to inhibit the infectivity of all enveloped viruses tested that display low pH-dependent
fusion (Marsh and Helenius, 1989). The three agents tested in this work, chloroquine,
NH4CI and methylamine failed to inhibit fusion of virus with lymphoblastoid cells. For
the lymphoblastoid cells tested, Raji, virus has been reported to gain entry via fusion
at the plasma membrane (Nemerow and Cooper, 1984a), so it is not surprising that
these agents had no effect on virus fusion. Methylamine and NH4CI did not inhibit
virus fusion with BAT cells or fresh peripheral T-depleted leukocytes.
In contrast, chloroquine inhibited fusion of virus with BAT cells by 34% at 1mM
and by 30% at 0.5mM. For peripheral B cells, the inhibition was 60% at 1mM, 50% at
0.5mM, and 24% at 0.2mM. These results raise the questions of whether chloroquine
is effecting other biological processes besides raising the endosomal pH and also
whether methylamine and NH4CI are effectively neutralizing the acidity of the endocytic
compartments. The intracellular pH of BAT cells was determined to be raised to pH
7.0 by treatment with 20mM NH4CI or 5mM methylamine and to pH 7.4 by treatment
with chloroquine. These data rule out the possibility that these agents were not
effectively raising the intracellular pH. Electron microscopy studies of internalization of
EBV into chloroquine-treated B lymphocytes showed that formation of endocytic
vacuoles proceeded normally in comparison to untreated cells but that in chloroquine-
treated B cells, intact virions remained in the vacuoles and very few nucleocapsids
were observed in the cytoplasm (Nemerow and Cooper, 1984a). Virus entry into
methylamine or NH4CI treated cells was not evaluated by electron microscopy by
these investigators, presumably because these agents had minimal effect on virus
infectivity as determined by stimulation of DNA synthesis, whereas chloroquine


50
1984, Nemerow and Cooper demonstrated a 96% reduction in infectivity by EBV of B
cells treated with 1mM chloroquine and a 20% reduction in infectivity of cells treated
with 10mM NH4CI. Infectivity was assessed by stimulation of host cell DNA synthesis
as measured by incorporation of [3H] thymidine after 4 to 6 days in culture. From
their studies they concluded that a reduction in pH was necessary for virus entry
because of inhibition by these agents. The fluorescence dequenching assay allowed
for analysis of the effects of these reagents on EBV fusion and the results are
presented and discussed in the following chapter.


6
envelope is a nucleocapsid exhibiting isosahedral symmetry which contains 162
capsomeres arranged in hexagonal and pentameric array. An amorphous tegument
fills the cavity between the nucleocapsid and the envelope. Inside the nucleocapsid is
the core virus particle consisting of core proteins and a large double-stranded
deoxyribonucleic acid (DNA) genome of approximately 172,000 base pairs in length
(Kieff et al., 1982). The viral mRNAs are translated in the cytoplasm and many of the
translational products then return to the nucleus where the nucleocapsid is
assembled.
Tropism and Latency
An unique feature of the gamma herpesviruses is their limited host range. All
members of this group infect lymphoblastoid cells in vivo and in vitro. The only
human member of the group is EBV and was originally identified as having tropism for
human B lymphocytes. Other members of the group include Mareks disease virus of
chickens and Herpes teles and Herpes saimiri virus of new world monkeys. These
viruses infect T cells (Fleckenstein and Desrosiers, 1982; Nonoyama, 1982). The host
range of EBV in vitro is restricted to B lymphocytes of humans and new world
primates. EBV establishes latency in these cells and immortalizes them. Latently
infected lymphocytes, but not those fully permissive for virus replication, have been
demonstrated in vivo. The infected cells retain the complete viral genome and
express a restricted set of viral genes necessary to maintain latency (Hayward and
Kieff, 1976; Pritchett et al., 1975).
Three types of latently infected cells have been extensively studied,
lymphoblastoid cell lines (LCLs), Burkitt-lymphoma cells (BL) and nasopharyngeal
cells (NPC). All three types express Epstein-Barr nuclear antigen 1 (EBNA-1), which is


55
COOH
NHC^CH^Hs
II n
(n=16)
Figure 3-1. Structural formula of 5-(N-octadecanoyl)amlnofluoresceln (AF).


CHAPTER 1
INTRODUCTION
Discovery of Epstein-Barr Virus
Epstein-Barr virus was discovered by electron microscopy during investigations
undertaken with lymphoblastoid cells cultured from a biopsy of an African Burkltts
lymphoma In the early 1960s (Epstein and Barr, 1964). The virus was Identified
morphologically as a member of the herpesviridae, but extensive vlrologic
Investigations proved It to be distinct from any previously known herpesvirus; It could
not be transmitted to host cells known to be susceptible to herpes-simplex virus
(HSV), cytomegalovirus (CMV), or varicella-zoster virus (VZV) and was given the name
Epstein-Barr virus (Epstein et al., 1965). The uniqueness of EBV was confirmed
serologically when antisera to known herpesviruses failed to react in
immunofluorescence tests with cells carrying the virus (Henle and Henle, 1966).
Seroepidemioiogic studies established the worldwide distribution of the virus in normal
healthy people.
Clinical Manifestations
Burkitts Lymphoma
In the 1950s Dr. Dennis Burkitt became Interested In a childrens tumor in
Africa that was not only the most common children's tumor in Africa but also more
common than all other childrens tumors added together (Burkitt, 1987). Also at that
1


120
=>
x
<
LLI
O
cn
LU
CC
O
Z)
0 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 5-5. Effect of preincubation of basal cells with methylamine, NH4CI, or RPMI
on relief of self-quenching of Relabeled MCUV5 virus bound to cells. Increase in
fluorescence expressed as a percent of the maximum release after addition of Triton
X-100 (infinite dilution).


25
the MCUV5 cell line will transform fresh human B cells and Induce them to secrete
Immunoglobulin (Gerber and Lucas, 1972), whereas the P3HR1-CI13 virus Is a non
transforming lytic strain of virus. Virus was obtained from producer cells by harvesting
the virus from culture supernatant 7 days after Induction with TPA. Cell culture
supernatant was cleared of cells by centrifugation at 4,000 x g for 10 minutes.
Bacitracin (Sigma) was added to the clarified supernatant (100 ug/ml) to reduce virus
aggregation, and the virus was pelleted by centrifugation at 20,000 x g for 90 minutes.
The virus pellets were resuspended In 1 /250 original volume of medium containing
100ug per ml bacitracin, reclarlfled of cell debris by centrifugation three to four times
at 400 x g, and filtered through a ,45um-pore filter (Acrodlsc, Gelman Sciences, Inc.,
Ann Arbor, Michigan).
P3HR1-CI13 virus was intrinsically labeled with (3H) thymidine (3HTdR;
Amersham Corp., Arlington Heights, Illinois) by feeding cells with medium containing
100uM hypoxanthine and 0.4uM aminopterin (Sigma), inducing them with TPA when
they reached confluency (day 0) in the presence of 2 uCi of 3HTdR (specific activity 5
Ci/mmol) per ml, adding an additional 2uCi of 3HTdR (specific activity 52 Ci/mmol)
on day 3, and harvesting the virus on day 7 in the same manner as described above.
All virus stocks were stored at -70C.
Monoclonal Antibodies
Monoclonal antibodies were purified from hybridoma culture supernatants by
chromatography on protein A-Sepharose (Genzyme, Boston, Massachusetts). The
antibody 72A1 (Hoffman et al., 1980) is an lgG1 antibody that recognizes the viral
glycoprotein gp350/220. Two monoclonal antibodies that react with CR2 were used,


16
prerequisite for infectivity (Lazarowitz and Choppin, 1975; White et al., 1983). The
cleavage generates a new N-terminus on HA2 which is hydrophobic and highly
conserved in different influenza strains and has partial homology with the N-terminus
of F,. Synthetic peptides analogous to the N-terminus sequence of HAj inhibit
infectivity by influenza viruses (Gething et al., 1986; Richardson et al., 1980). The HA
molecule in its neutral form is a trimer and the hydrophobic fusion peptide in each
monomer is unexposed until the low pH of the endocytic vesicle causes partial
dissociation of the HA trimer, thus exposing the fusion peptide which can insert into
the target bilayer (Dorns et al., 1985; Schlegel et al., 1982) and initiate endosomal
membrane fusion. Collective research findings suggest that the pH induced
conformation does not involve any changes in secondary structure and that the stem
region of the spike remains trimeric. However, elements of the spike change their
relative positions with the globular heads dissociating from one another by bending
about a hinge region. This movement of the three proteins composing the spike is
thought to release the terminal fusion peptide from the molecular interior (Dorns et al.,
1990; Dorns and Helenius, 1988; Harter et al., 1989; Ruigrok et al., 1988; Stegmann et
al., 1987, 1989; Wharton, 1987; Wharton et al., 1988; White et al., 1983; White and
Wilson, 1987; Wiley and Skehel, 1987). The HA is the only membrane fusion protein
for which a crystal structure is known (White, 1990).
The envelope spike of Semliki Forest virus (SFV), a togavirus, consists of a
complex of three glycopeptides, E1, E2, and E3. E1 and E2 are transmembrane
glycoproteins; E3 is noncovalently associated with E2 and is external to the bilayer.
This virus does not fuse with the plasma membrane at physiologic pH (Helenius et al.,
1980a). Virions are endocytosed and a fall in pH within the endocytic vesicle activates


162
Niederman, J.C., R.W. McCollum, G. Henle, and W, Henle. 1968. Infectious
mononucleosis: Clinical manifestations in relation to Epstein-Barr virus antibodies. J.
Am. Med. Assoc. 203:205-209.
Niederman, J.C., G. Miller, H.A. Pearson, J.S. Pagano, and J.M. Dowallby. 1976.
Infectious mononucleosis: Epstein-Barr virus shedding in saliva and the oropharynx.
N. Engl. J. Med. 294:1355-1359.
Noble, A.G., G. Lee, and P.G. Spear. 1983. Anti-gD monoclonal antibodies inhibit cell
fusion induced by herpes simplex virus type 1. Virology 129:218-224.
Nonoyama, M. 1982. The molecular biology of Marek's disease herpesvirus, p. 333.
in: B. Roizman (ed.), The herpesviruses, vol. 1. Plenum Press, New York.
Oba, D.E. and L.M. Hutt-Fletcher. 1988. Induction of antibodies to the Epstein-Barr
virus glycoprotein gp85 with a synthetic peptide corresponding to a sequence in the
BXLF2 open reading frame. J. Virol. 62:1108-1114.
Ohkuma, S. and B. Poole. 1978. Fluorescence probe measurement of the
intralysosomal pH in living cells and the perturbation of pH by various agents. Proc.
Natl. Acad. Sci. USA 75:3327-3331.
Pearse, B.M.F. 1975. Clathrin: an unique protein associated with intracellular transfer
of membrane by coated vesicles. Proc. Natl. Acad. Sci. USA 73:1255-1259.
Pellegrino, M.A., S. Ferrone, M.P. Dietrich, and R.A. Reisfeld. 1975. Enhancement of
sRBC human lymphocyte rosette formation by the sulfhydryl compound 2-aminoethyl
isothiouronium. Clin. Immunol. Immunopath. 3:224-333.
Pellet, P.E., K.G. Kousoulas, L. Pereira, and B. Roizman. 1985. The anatomy of the
herpes simplex virus gB gene: primary sequence and predicted protein structure of
the wild type and of monclonal antibody resistants mutants. J. Virol. 53:243-253.
Petrie, B.L., D.Y. Graham, and M.K. Estes. 1981. Identification of rotavirus particle
types. Intervirology 16:20-28.
Pressman, B.C. 1976. Biological applications of ionophores. Annu. Rev. Biochem.
45:501-530.
Prichett, R.F., S.D. Hayward, and E. Kieff. 1975. DNA of Epstein-Barr virus I.
Comparative studies of the DNA of Epstein-Barr virus of virus from HR-1 and B95-8
cells: size, structure and relatedness. J. Virol. 15:556-569.
Pulvertaft, R.J. 1964. Cytology of Burkitts tumor (African lymphoma). Lancet 1:238-
240.
Purtillo, D.T. 1985. Association of Epstein-Barr virus and lymphoproliferative diseases
in immune deficient persons, p. 3-18. In: P. Levine, D. Ablashi, G. Pearson, and S.


42
Figure 2-6. Relief of self-quenching of Relabeled P3HR1-CI13 virus bound to tonsil
derived T-depleted leukocytes expressed as a percent of the maximum release
obtained after addition of Triton X-100 (infinite dilution).


65
O 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 3-9. Effect of preincubation of BAT cells with chloroquine or RPMI on relief of
self-quenching of Relabeled MCUV5 virus bound to cells. Panel A, 1mM; panel B,
0.5mM. Increase in fluorescence is expressed as a percent of the maximum release
obtained after addition of Triton-X-100 (infinite dilution).


124
recognized by all the monoclonal reagents selected for reactivity with the B cell CR2.
Polymorphic variations In the CR2 coding sequence, resulting both from alternative
splicing of exons In the DNA and from the existence of allelic differences could result
In generation of distinct forms of the protein In different cell types. More surprising,
however, was the finding that the monoclonal antibody OKB7, which inhibits EBV
binding with the B cell, (Carel et al., 1990; Nemerow et al., 1985a) did not react with
the epithelial cells which bound virus. OKB7 has been mapped to the terminal SCR at
a site very close to, though not Identical to the viral binding site (Lowell et al., 1989).
The goal of this work was to investigate virus binding and entry Into epithelial
cells in order to compare it to lymphocytes. When cells from the basal cell population
and the parabasal cell population were evaluated for their ability to bind and
internalize virus as measured by the fluorescence dequenching assay, there was not a
strict correlation between virus binding and reactivity of the cells with the HB5
antibody. Cells from the basal cell population bound 50 per cent more virus than cells
from the parabasal population despite the lack of reactivity of basal cells with the HB5
antibody. The amount of fluorescence dequenching of the virus bound to the two
populations was similar, reflecting an equal ability of functional virus to enter these
cells.
The basal cell population represents approximately 50 per cent of the total
number of cells Isolated from the epidermal pieces of tissue and this population was
able to bind and internalize virus. The entry of virus Into these cells was pursued
further In studies utilizing lysosomotropic agents that raise the intracellular pH. The
three agents tested In this work, chloroqulne, NH4CI, and methylamine had no
inhibitory effects on virus binding and fusion with basal epithelial cells. These results


Abstract of Dissertation Presented to the Graduate School
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy
ENTRY OF EPSTEIN-BARR VIRUS INTO LYMPHOCYTES AND EPITHELIAL CELLS
By
Nancimae Miller
May 1991
Chairman: Lindsey Hutt-Fletcher
Major Department: Immunology and Medical Microbiology
Epsteln-Barr virus (EBV) is a human herpesvirus which causes Infectious
mononucleosis and is associated with two cancers, Burkltts lymphoma and
nasopharyngeal carcinoma. To understand the biologic activity of EBV, it is crucial to
understand how EBV infects cells, and what viral components are Important to this
process. Epstein-Barr virus infects two cell types, B lymphocytes and epithelial cells.
To examine the early events in virus Infection, binding and fusion, we have adapted an
assay that measures membrane fusion. Virus membranes were labeled with
concentrations of octadecylrhodamine (R18) or 5-(N-octadecanoyl)aminofluorescein
(AF) at which fluorescence Is self-quenched. The fluorescence of AF is also sensitive
to changes in pH. Fusion and mixing of virus and cell membranes was measured In
terms of relief of self-quenching and was monitored kinetlcally.
The assay was used to compare virus fusion with lymphoblastoid cell lines,
lymphocytes recently transformed with EBV, normal B lymphocytes and epithelial
xii


43
that had been depleted of monocytes. Table 2-4 shows the extent of monocyte
depletion as determined by cell counts and nonspecific esterase stain pre and post
depletion. Figure 2-7 shows the increase in fluorescence of tonsil derived B cells pre
and post monocyte depletion by adherence to plastic. The cell preparations treated
with iron filings could not be used in the fluorometer due to scatter interference from
residual filings in the preparation. The maximum increase in fluorescence achieved
with tonsil derived B cells was 20-23% and depletion of monocytes from the cells used
did not affect this measurement.
Fusion experiments were also done using T depleted peripheral leukocytes.
Human peripheral leukocytes could be obtained with greater regularity than tonsil
tissue. Figure 2-8 shows data obtained using T depleted peripheral leukocytes. As
seen with the tonsillar B cells, less virus bound peripheral B cells than Raji cells, but
the maximum increase in fluorescence was higher than the level obtained with tonsil
derived cells. In this experiment the maximum increase was 55%, in other
experiments using different batches of labeled virus and different cells, values ranging
from 28-56% were achieved.
Changes in Fluorescence of Relabeled Virus with EBV-lmmortalized B Cells
Human B cells were Infected with EBV and were immortalized. These cells,
designated BAT, have growth characteristics of a continuous cell line, but since they
are recently immortalized, they may be biologically more similar to B cells than the
lymphoblastoid cell lines, such as Raji, which has been in culture for many years. Raji
cells have been reported to have alterations in the cytoskeleton (Bachvaroff et al.,
1980). Figure 2-9 demonstrates how these cells function in the fluorescence
dequenching assay. Utilizing these cells reduces the need to obtain fresh human


11
and Hutt-Fletcher, 1988). Although no function has been conclusively ascribed to this
molecule, antibodies to it can neutralize virus infectivlty (Strnad et al., 1982), thus
Implying that it may play a role in the initiation of ceil infection. The function of
gp78/55 has not been determined; neither a monoclonal antibody nor a polyclonal
sera to the recombinant molecule neutralized the ability of virus to transform cells.
Preliminary studies with recombinant vaccinia virus expressing the gene
product from the BDLF3 open reading frame have immunoprecipitated a protein of
90kd using serum from a patient with chronic mononucleosis (LC. Davenport and
L.M. Hutt-Fletcher, personal communication).
Entry of Enveloped Viruses into Animal Cells
The earliest events in the virus replication cycle are attachment, penetration,
and uncoatlng. The initial event, virus attachment to specific cell receptors, is a major
determinant of cellular troplsm and pathogenesis of viruses. Virus membrane proteins
protruding from the virus envelope mediate virus attachment to host cells. These
membrane proteins have other functions in addition to cell recognition and
attachment, namely fusion, penetration, and possibly, direction of egress of the virus.
Enveloped viruses enter cells by fusing with cellular membranes (Lonberg-Holm and
Philipson, 1974; White et al 1983; White, 1990). Since fusion Is an energetically
unfavorable process, viruses utilize specific proteins to fuse with host cells and
introduce their genetic material into the host cell (White, 1990). Two pathways of
entry are commonly utilized and viral fusion reactions fall Into two classes, low pH-
dependent and pH-independent. Some viruses, such as Sendai (Scheid and
Choppln, 1976), deposit their nucleocapsids directly into the cytoplasm by fusing with


93
0 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 4-4. Effect of preincubation of BAT cells with chlorpromazine or RPMI on relief
of self-quenching of Relabeled MCUV5 virus. Increase In fluorescence is expressed
as a percent of the maximum release obtained after addition of Triton X-100 (infinite
dilution).


CHAPTER 3
EFFECTS OF LYSOSOMOTROPIC AGENTS AND pH ON FUSION
OF EPSTEIN-BARR VIRUS WITH LYMPHOCYTES
Introduction
To Initiate an Infection, all enveloped animal viruses must fuse with a cellular
membrane and this fusion can be divided Into two general classes, low pH dependent
and pH independent. It is generally considered that viruses that are low pH
dependent fuse from within acidic vesicles whereas viruses that are low pH
independent can fuse directly with the plasma membrane, but may fuse from
endosomes as well. Although fusion of EBV with lymphoblastoid cell lines occurs at
the plasma membrane and therefore presumably does not require exposure to low
pH, virus has been reported to fuse with normal B cells after endocytosis and certain
lysosomotropic agents have been shown to inhibit virus Infectivity (Nemerow and
Cooper, 1984a). The possibility that penetration of Epstein-Barr virus nudeocapsids
into the cytosol might Involve an acid-catalyzed fusion reaction in the endosomal
compartment was further investigated in this work since our assay measures
membrane fusion In Isolation of other events in the virus life cycle that might be
affected by drugs. Exposure to pH values between 5.0 and 7.0 has dramatic effects
on many of the molecules brought into the cell by endocytosis. Many ligands
dissociate from their receptors at pH values below 7.
Some viruses undergo significant changes in conformation when exposed to
acidic pH (White, 1990). The fusion glycoprotein of influenza virus, the hemagglutinin
51


CHAPTER 4
MODIFICATION OF THE ENDOCYTIC PATHWAY TO DETERMINE
THE MECHANISM OF ACTION OF CHLOROQUINE ON VIRUS FUSION
Introduction
Since EBV enters B cells by endocytosls, one might assume that Interference
with this process would limit virus infectivity. This has been presumed to be at least
partially responsible for the reduction in Infectivity by calmodulin antagonists
(Nemerow and Cooper, 1984b). It has been shown that inhibitors of oxidative
phosphorylation and glycolysis can affect uptake of ligands into cells. Sodium azide,
which inhibits oxidative phosphorylation, has been shown to inhibit partially the uptake
of prebound Semliki Forest virus (Marsh and Helenius, 1980) and VSV (Blumenthal et
al., 1987). It Is possible that the Inhibition of EBV fusion by chloroquine is due to a
modification of the endocytic pathway that is inhibiting uptake of the virus into vesicles
or altering the membrane so that virus is not able to fuse with the endosomal
membrane. In addition to Its pH-elevating property, chloroquine has other effects on
lysosomal functions and other cellular processes (de Duve et al., 1974; Seglen, 1983).
Chloroquine reduced uptake of asialo-fetuln (ASF) Into cells when the concentration
exceeded 0.1mM, and at concentrations above 1.0mM, chloroquine almost completely
inhibited both uptake and degradation of ASF (Berg and Tolleshaug, 1980). Protease
inhibition is another effect of chloroquine (Wibo, M. and B. Poole, 1974), In particular,
inhibition of the enzyme cathepsln B (ibid) and phospholipases are also effected
86


49
The early events in Infection of normal B cells and lymphoblastold cells have
been examined previously by electron microscopy (Nemerow and Cooper, 1984a;
Selgneurin et al., 1977). These studies reported that EBV enters lymphoblastold cells
by direct fusion with the outer cell membrane and that virus Is endocytosed into thin-
walled non-dathrin coated vesicles In the normal B cell before It fuses with the cell
membrane. Both pathways were reported to initiate within two to five minutes at 37C.
The kinetics of fusion with Raji cells, normal lymphocytes, and recently immortalized
BAT cells were very similar, all exhibiting a measurable change within two minutes of
warming in the cuvette of the spectrofluorometer. A one to two minute lag time,
corresponding to the time required for initial entry of ligands, toxins, and virions into
an acidic compartment after receptor mediated endocytosis (Bridges et al., 1982) has
been reported for relief of self-quenching of Relabeled vesicular stomatitis virus
bound to Vero cells (Blumenthal et al., 1987).
Since EBV appears capable of fusing with the plasma membrane at the cell
surface, or after endocytosis, this may mean that either virus can enter normal B cells
by both routes, or that fusion with an endocytic vesicle wall occurs rapidly after
uptake, perhaps even before virus Is exposed to low pH. It has been shown that
rotavirus enters cells by direct cell membrane penetration (Kaljot et al., 1988) even
though earlier electron microscopy studies had revealed presence of rotavirus
particles in coated pits and a variety of vesicles, signifying entry by endocytosis (Petrie
et al., 1981; Quann and Doane, 1983).
Experiments using lysosomotropic agents, inhibitors of endocytosis, and pH
sensitive fluorescent probes may help answer the question of whether EBV is capable
of fusing at both the plasma membrane and the endocytic vesicle of lymphocytes. In


58
Z)
1
X
<
2
L
O
cr
o
3
0 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 3-3. Relief of self-quenching of Relabeled MCUV5 virus bound to BAT cells
at pH 7.2 or pH 5.5. Virus was bound to cells at pH 7.2, cells were washed to remove
unbound virus and cells were resuspended in pH 7.2 or pH 5.5 Dulbecco's saline.
Increase in fluorescence is expressed as a percent of the maximum release obtained
after addition of Triton X-100 (infinite dilution).


Choppin, P.W. and R.W. Compans. 1975a. Replication of paramyxoviruses, p. 94-
178. In: H. Fraenkel-Conrat and R. Wagner (ed.), Comphrensive virology. Plenum
Press, New York.
Choppin, P.W. and R.W. Compans. 1975b. The structure of Influenza virus, p. 15-51.
in: E. Kilbourne (ed.), The Influenza viruses and influenza. Academic Press, New
York.
Choppin, P.W. and A. Scheid. 1980. The role of glycoproteins in adsorption and
pathogenicity of viruses. Reviews of Infectious Diseases. 2:40-61.
Claesson-Welsh, L. and P.G. Spear. 1976. Oligomerization of herpes simplex virus
glycoprotein B. J. Virol. 60:803-806.
Clague, M.J., C. Schoch, L. Zech, and R. Blumenthal. 1990. Gating kinetics of pH-
activated membrane fusion of vesicular stomatitis virus with cells: stopped-flow
measurements by dequenching of octadecylrhodamine fluorescence. Biochemistry
29:1303-1308.
Cleary, M., R. Dorfman, and J. Sklar. 1986. Failure In immunologic control of the virus
infection: post transplant lymphomas, p. 163-181. In: M. Epstein and B. Achong (ed.),
The Epstein-Barr virus: recent advances. John Wiley and Sons, New York.
Cooper, N.R., B.M. Bradt, J.S. Rhim, and G.R. Nemerow. 1990. CR2 complement
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128
protein p105. An antibody that recognizes the major envelope glycoprotein
gp350/220, 72A1 (Hoffman et al., 1980) was also used.
Membrane Fusion Assay
Epstein-Barr virus that has been labeled with R,8 at self-quenching
concentration was added to 2 X 106 cells and incubated for 1 hour on ice. Cells were
washed four times with ice cold Dulbeccos saline and suspended in 400ul of
Dulbeccos when transferred to the microcuvette of a spectrofluorometer.
Fluorescence dequenching was monitored continuously at an excitation wavelength of
560nm and an emission wavelength of 585nm. At the end of the assay, Triton X-100
(1% v/v, final concentration) was added to allow the measurement of fluorescence
that would be obtained upon infinite dilution of the fluorophore.
Soluble CR2
Binding inhibition assays were done using a soluble form of the first two short
consensus repeats (SCR) of the CR2 protein. This reagent was a gift from Glen
Nemerow (Research Institute of Scripps Clinic, La Jolla, California). Raji cells and
basal epithelial cells from newborn foreskin tissue were evaluated for their ability to
bind Relabeled EBV in the presence of soluble CR2. Virus that had been
preincubated with the anti-viral antibody 72A1 was also included in the experiments.
Cells were either incubated with 72A1 and virus or CR2 and virus for 1 hour on ice
follow by three washes to remove unbound material and the cell-associated
fluorescence was measured by lysing the cells and virus with 1% Triton X-100.


109
from the very large keratinized squamous cells. Parabasal cells were found at both
the 45/45% and the 35/40% interfaces. There is no significant morphological
difference between cells seen at these two interfaces except that more basal cells
were found at the 40/45% interface. The migration of the parabasal cells into two
fractions may reflect differences in the extent of the keratinization of these cells. The
two fractions were handled as two different populations when evaluating virus binding
and receptor expression. The terminally differentiated squamous cells were found at
the media/30% interface. These ceils were very large in comparison to the other cell
types and a majority of the non-viable cells in the total population were of this type.
Reactivity of Epithelial Cells with Anti-CR2 Antibodies
Epithelial cells that were separated by size on discontinuous Percoll gradients
into four populations were analyzed for expression of a CR2-llke molecule using the
anti-CR2 antibody HB5 (Weis et al., 1984). Expression of this receptor has been
demonstrated on cells separated in this manner from cultures epithelial cell explants
(Sixbey et al., 1987). Table 5-3 Illustrates the results of this experiment. The large
keratinized squamous cells which were found at the media/30% interface exhibited
very little staining with this antibody. Cells that were found at the 30/40% and the
40/45% Interfaces exhibited the highest amount of reactivity. The small basal cells
found at the 45/100% interface exhibited more staining than the squamous cells but
not as much as the suprabasal cells.
On subsequent analysis only parabasal and basal cells were analyzed due to
lack of reactivity of squamous cells with HB5 and also because it was more likely that
the less keratinized cells would actually bind and internalize virus. Table 5-4 illustrates
the results of binding assays of epithelial cells with additional anti-CR2


160
Matlin, S., H. Reggio, A. Helenius, and K. Simons. 1981. Infectious entry pathway of
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phospholipase C by chloroquine and 4,4'-bis(diethylaminoethoxy) alpha,beta-
diethyldiphenylethane. J. Biol. Chem. 255:5190-5194.
Maxfield, F.R. and D.J. Yamashlro. 1987. Endosme acidification and the pathways
of receptor-mediated emdocytosis. Adv. Exp. Med. Biol. 225:189-198.
McClure, M.O., M. Marsh, and R.A. Weiss. 1988. Human immunodeficiency virus
infection of CD4-bearing cells occurs by a pH-independent mechanism. EMBO
Z:513-518.
McGeoch, D.J., A. Dolan, S. Donald, and F.J. Rixon. 1985. Sequence determination
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substitutions that confer resistance to neutralization. J. Gen. Virol. 67:1001-1013.


47
Figure 2-9. Relief of self-quenching of Relabeled MCUV5 virus bound to BAT cells
expressed as a percent of the maximum release obtained after addition of Triton X-
100 (infinite dilution).


97
O 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 4-8. Effect of preincubation of T-depleted leukocytes with leupeptin or RPMI
on relief of self-quenching of Relabeled MCUV5 virus. Increase in fluorescence is
expressed as a percent of the maximum release obtained after addition of Triton X-
100 (infinite dilution).


168
White, J., A. Helenius, and J. Kartenbeck. 1982. Membrane fusion ability of influenza
virus. EMBO J. 1:217-222.
White, J., J. Kartenbeck, and A. Helenius. 1980. Fusion of Semliki Forest virus with
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viruses. Q. Rev. Biophys. 16:151-195.
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and vesicular stomatitis virus. J. Cell Biol. 89:674-679.
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conformational change: Low-pH activation of the influenza virus hemagglutinin. J. Cell
Biol. 105:2887-2896.
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chloroqulne by rat fibroblasts and the inhibition of cellular protein degradation and
cathepsln B. J. Cell Biol. 63:430-440.
Wiley, D.C. and J.J. Skehel. 1987. The structure and function of the hemagglutinin
membrane glycoprotein of influenza virus. Annu. Rev. Biochem. 56:365-394.
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parotid gland. J. Virol. 51:795-798.
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cells is binding to heparan sulfate. J. Virol. 63:52-58.
Wunderli-Allenspach, H. and S Ott. 1990. Kinetics of fusion and lipid transfer between
virus receptor containing lysosomes and influenza viruses as measured with the
octadecylrhodamine B chloride assay. Biochemistry 29:1990-1997.
Yao, Q.Y., A.B. Ricklnson, and M.A. Epstein. 1985. A re-examination of the Epstein-
Barr virus carrier state in healthy sreoposltive individuals. Int. J. Cancer 35:35-42.
Yamashlro, D.J. and F.R. Maxfield. 1984. Acidification of endocytic compartments
and the Intracellular pathways of ligands and receptors. J. Cell. Biochem. 26:231
Yoshimura, A. and S. Ohnishi. 1984. Uncoating of influenza virus in endosomes. J.
Virol. 51:497-504.
Young, L.S., J.W. Sixbey, D. Clark, and A.B. Rickinson. 1986. Epstein-Barr virus
receptors on human pharyngeal epithelia. Lancet 1:240-242.
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human epithelial cell surface protein sharing an epitope with the C3d/Epstein-Barr
virus receptor molecule of B lymphocytes. Int. J. Cancer. 43:786-


119
2
D
X
<
2
UJ
o
o
(/)
LU
cr
o
=)
0 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 5-4. Effect of preincubation of basal cells with chloroquine or RPMI on relief of
self-quenching of Relabeled MCUV5 virus bound to cells. Increase in fluorescence
expressed as a percent of the maximum release after addition of Triton X-100 (infinite
dilution).


136
O 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 6-2. Effect of preincubation of vims with monoclonal antibodies on relief of
self-quenching of R18-labeled MCUV5 virus bound to BAT cells. Relief of self-
quenching is expressed as a percent of the maximum release obtained after addition
of Triton X-100 (infinite dilution).


Table 2-3. Effect of labeling with R18 on the ability of MCUV5 virus to induce
Immunoglobulin synthesis by fresh T-depleted human leukocytes.
Virus
dilution
Immunoglobulin cone, ng/ml with:
Relabeled
virus
Mock-labe!ed
virus
1/5
24,754
22,366
1/10
45,720
23,836
1/20
38,609
23,639
1/40
39,902
27,404
1/80
42,326
30,105
1/160
33,921
21,093
1/320
18,168
15,999
1/640
3,970
not done
none
1,138


CHAPTER 6
EFFECTS OF MONOCLONAL ANTIBODIES TO VIRUS MEMBRANE PROTEINS
ON BINDING AND ENTRY OF EPSTEIN-BARR VIRUS INTO
LYMPHOCYTES AND EPITHELIAL CELLS
Introduction
Previous studies on the function of EBV membrane glycoproteins have focused
on the largest and most abundant molecules, gp350 and gp220, primarily because
antibodies that recognized these proteins were capable of neutralizing virus infectivity
by inhibiting virus from binding. Even though the majority of neutralizing activity in
normal sera can be accounted for by antibody that reacts with these molecules
(Thorley-Lawson and Poodry, 1982), the predominant anti-membrane reactivity in
neutralizing sera taken from patients during the acute phase of infectious
mononucleosis is directed against another envelope protein, gp85 (Qualtiere and
Pearson, 1979). The development of an assay to measure virus entry by measuring
membrane fusion was pursued in the interest of investigating the function of viral
membrane proteins in events occurring after virus binding.
Monoclonal antibodies are a valuable tool for identifying proteins and their
specificity has been widely used for virus function studies in many systems. The
studies presented in this chapter were in pursuit of greater understanding of which
viral surface membrane proteins are Important for virus fusion.
126


68
agents were indeed increasing the intracellular pH, the resulting pH after treatment of
the cells was determined.
Determination of pH of Intracellular Compartments After Treatment with
Lysosomotropic Agents
The pH in endocytic compartments can be measured using fluorescein-labeled
ligands such as dextran (Ohkuma and Poole, 1978; Tyoko and Maxfleld, 1982;
Yoshlmura and Ohnlshl, 1984.). The fluorescence Intensity of fluorescein decreases
dramatically between pH 7.0 and pH 5.0. Thus, changes in fluorescence intensity can
be used as an assay for changes In pH. The ratio of fluorescence Intensities at the
wavelengths of 450nm and 496nm can be used to determine a standard curve from
which actual pH values can be extrapolated (Gelsow, M.J., 1984). In various cell
types, lysosomes have pH values between 4.6 and 5.2, and endocytic vesicles have
pH values between 5.0 and 5.5 (Maxfleld and Yamashlro, 1987; Ohkuma and Poole,
1978; Tyoko and Maxfield, 1982; Tyoko et al., 1983; Yamashlro and Maxfleld, 1984).
For accurate pH determinations using the fluorescent conjugates it was
necessary either to ensure sufficient plnocytosis to produce a reliable signal at 450nm
or to collapse the Intracellular pH gradients using monensin. Monensin Is a carboxylic
Ionophore which is able to promote exchange of protons for univalent cations and
thereby abolishes transmembrane proton gradients (Pressman, 1976; Tartakoff, 1977).
After addition of monensin to cells, the fluorescein emission will resemble that
expected at the external pH. By altering the external pH, a calibration curve can be
obtained of Intracellular fluorescein Isothlocyanate (FITC)-dextran. In addition to the
fluorescein conjugate, a rhodamine conjugate is also Included to ensure sufficient
uptake of the ligands Into the cells. The fluorescence of the rhodamine is Insensitive
to changes In pH and was used as an Internal reference for the amount of conjugate


4 MODIFICATION OF THE ENDOCYTIC PATHWAY TO DETERMINE THE
MECHANISM OF ACTION OF CHLOROQUINE ON VIRUS FUSION . 86
Introduction 86
Materials and Methods 87
Results 88
Discussion 98
5 ISOLATION AND IDENTIFICATION OF EPITHELIAL CELLS EXPRESSING
A RECEPTOR FOR EPSTEIN-BARR VIRUS AND STUDIES OF VIRUS
ENTRY INTO THESE CELLS 101
Introduction 101
Materials and Methods 102
Results 106
Discussion 123
6 EFFECTS OF MONOCLONAL ANTIBODIES TO VIRUS MEMBRANE
PROTEINS ON BINDING AND ENTRY OF EPSTEIN-BARR VIRUS INTO
LYMPHOCYTES AND EPITHELIAL CELLS 126
Introduction 126
Materials and Methods 127
Results 129
Discussion 144
7 SUMMARY AND CONCLUSIONS 147
Recapitulation 147
Importance of Present Studies and Future Directions 149
REFERENCES 150
BIOGRAPHICAL SKETCH 169
v


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87
(Matsuzawa and Hostetler, 1980). Chloroquine is also reported to alter membrane
fluidity (Berg and Tolleshaug, 1980).
In order to Investigate further the action of chloroquine on fusion of virus with B
lymphocytes, fusion studies were done with cells treated with sodium azide, leupeptin,
and chlorpromazine.
Materials and Methods
Membrane Fusion Assay
Epstein-Barr virus that had been labeled with the fluorophore octadecyl
rhodamine B chloride (Ria) at self-quenching concentration was incubated with 2 X 106
cells and incubated for 1 hour on ice in the dark. When membrane fusion occurs
there is dilution of the fluorophore in the membranes which relieves the self-quenching
of the fluorescence. Cells were washed of unbound virus and the fluorescence
emission was monitored continuously using a spectrofluorometer at an excitation
wavelength of 560nm and an emission wavelength of 585nm. At the end of the assay
Triton X-100 was added to allow the measurement of fluorescence that would be
obtained upon infinite dilution of the fluorophore.
Cells
The lymphoblastoid cell line Raji (Pulvertaft, 1964) and the recently EBV-
transformed cell line BAT, which both express the virus receptor CR2, were grown at
37C and diluted at least biweekly in RPMI 1640 supplemented with fetal calf serum
and antibiotics. Fresh human T-depleted leukocytes were isolated from peripheral
blood by flotation on LSM followed by resetting with sheep erythrocytes and


142
O 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 6-4. Effect of preincubation with 100ug of normal mouse immunoglobulin or
antibody F-2-1 on relief of self-quenching of Rls-labeled MCUV5 virus bound to basal
epithelial cells. Increase in fluorescence is expressed as a percent of the maximum
release after addition of Triton x-100 (infinite dilution). The vertical lines indicate the
standard deviation of the mean of 4 experiments.


ACKNOWLEDGEMENTS
I greatly appreciate the support and guidance from all those who helped me in
completing this work. My committee members, Drs. Sue Anne Moyer, William
Hauswirth, and John Dankert have been extremely encouraging and helpful. None of
this would have been possible without the excellent guidance from the chairperson of
my committee, Dr. Lindsey Hutt-Fletcher. She has been my teacher, my mentor, and
my friend. A special thanks goes to Dr. Alfred Esser for his input into the project and
the use of his spectrofluorometer. The time spent on this project has not been spent
alone, my thanks go to all the members of the laboratory past and present, especially
to Susan, Linda, Lisa, and Doug. Thanks are extended to my parents, my sister,
Michelle, and my brother, John, for always being there for me. Special thanks to
Dave for supporting me throughout the writing of this dissertation.


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accompanied by alterations of the golgi complex. J. Exp. Med. 146:1332-1345.
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during human B cell differentiation: immunofluorescence analysis with the HB5
monoclonal antibody. J. Immunol. 133:678-683.
Tedder, T.F., V.S. Goldmacher, J.M. Lambert, and S.F. Schlossman. 1986. Epstein-
Barr virus binding induces internalization of the C3d receptor: a novel immunotoxin
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membrane antigen complex. J. Virol. 32:458-467.


18
this virus. The fusion activity has been shown to occur at the plasma membrane if
cells with VSV attached to their surfaces are placed in a low pH medium (Blumenthal
et al 1987; Matlin et al., 1982).
Herpesviruses are considerably more complex. The best studied, herpes
simplex virus (HSV), has an envelope that contains at least nine glycoproteins, five of
them have been characterized and sequenced (Bzik et al., 1984; Frink et al., 1983;
Gompels and Minson, 1986; McGeoch et al., 1985; Pellet et al., 1985; Watson et al.,
1982). Studies indicate that the receptor molecules recognized in one of the initial
binding events are heparan sulfate proteoglycans (WuDunn and Spear, 1989).
Recently, it was determined that glycoprotein gC is principally responsible for virus
adsorption to cells (Herald et al., 1991). Glycoprotein gC bound heparin and virions
devoid of gC exhibited significant impairment in adsorption and penetration. Three of
the glycoproteins, namely gB, gD, and gH, induce antibodies capable of neutralizing
HSV infectivity in the absence of complement and have been implicated in virus
penetration (Fuller and Spear, 1987; Gompels and Minson, 1986, Sarmiento et al.,
1979). Evidence implicating gB in penetration comes from studies of temperature
sensitive HSV-1 mutants that fail to process precursor gB molecules to mature forms
at nonpermissive temperature. The virions produced are noninfectious but can bind
to cells and the block to their infectivity can be overcome by treating virus-cell
complexes with the membrane fusing agent polyethylene glycol (Little et al., 1981;
Sarmiento et al., 1979). Neutralizing anti-gD monoclonal antibodies have been shown
to block HSV infection by preventing virus-cell fusion at the plasma membrane (Fuller
and Spear, 1987) and antibodies to this glycoprotein also block HSV-induced cell-cell
fusion, a process which may be analogous to the virus-cell fusion required for entry


70
5.0 5.4 5.8 6.2 6.6 7.0 7.4 7.8 8.2
pH
Figure 3-12. Relative intensity of fluorescein isothiocyanate-dextran (FITC-dextran)
fluorescence as a function of changes in pH. FITC-dextran was excitated at a
wavelength of 496nm and emission was measured at 522nm.


122
Figure 5-7. Effect of preincubation of basal cells with NH4CI or RPMI on relief of self-
quenching of AF-labeled MCUV5 virus bound to cells. Increase in fluorescence
expressed as a percent of the maximum release after addition of Triton X-100 (Infinite
dilution).


Bramhall, J. and B. Wisniewski. 1981. The role of lipids in virus-cell interactions,
p.143-153. In: K. Lonberg-Holm and L. Philipson (ed.). Virus receptors part 2.
Chapman and Hall, London.
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Bridges, K., J. Hartford, G. Ashwell, and R.D. Klausner. 1982. Fate of receptor and
ligand during endocytosls of asialoglycoproteins by isolated hepatocytes. Proc. Natl.
Acad. Sci. USA 79:350-354.
Brown, J.C., W.W. Newcomb, and S. Lawrenz-Smith. 1988. pH-dependent
accumulation of the vesicular stomatitis virus glycoprotein at the ends of intact virions.
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Burkitt, D.P. 1987. Discovering Burkitts lymphoma, p. xxi-xxxi. in: P.H. Levine, D.V.
Ablashi, M. Nonoyama, G.P. Pearson, and R. Glaser (ed.), Epstein-Barr virus and
human disease. Humana Press, Clifton, New Jersey.
Bzik, D.J., B.A. Fox, N.A. DeLuca, and S. Pearson. 1984. Nucleotide sequence
specifying the glycoprotein gB of herpes simplex virus type 1. Virology 133:301-314.
Cai, W., B. Gu, and S. Person. 1988. Role of glycoprotein gB of herpes simplex virus
type I in virus entry and cell fusion. J. Virol. 62:2596-2504.
Cain, C.C. and R.F. Murphy. 1988. A chioroquine-resistant Swiss 3T3 cell line with a
defect in late endocytic acidification. J. Cell Biol. 106:269-277.
Cameron, K.R., T. Stamminger, M. Craxton, W. Bodemer, R.W. Honess, and B.
Fleckenstein. 1987. The 160,000 Mr virion protein encoded at the right end of the
herpesvirus saimiri genome is homologous to the 140,000-Mr membrane antigen
encoded at the left end of the EBV genome. J. Virol. 61:2063-2070.
Carel, J., B.L. Myones, B. Frazier, and V.H. Holers. 1990. Structural requirements for
C3d,g/Epstein-Barr virus receptor (CR2/CD21) ligand binding, internalization, and viral
infection. J. Biol. Chem. 265:12293-12299.
Cassel, S., J. Edwards, and D.T. Brown. 1984. Effects of lysosomotropic weak bases
on infection of BHK-21 cells by Sindbis virus. J. Virol. 52:875-864.
Chang, R.S., J.P. Lewis, and C.F. Abildgaard. 1973. Prevalence of oropharyngeal
excretors of leukocyte-transforming agents among a human population. N. Engl. J.
Med. 289:1325-1329.
Cheung, W.Y. 1980. Calmodulin plays a pivotal role in cellular regulation. Science
207:19-27.
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inhibition of T cell mitogenesis by calmodulin antagonists. J. Immunol. 131:2291-
2295.


118
agents have been widely used to determine if virus fusion is dependent on low pH in
order to occur. They were used to determine that fusion of EBV with lymphocytes is
not catalyzed by low pH. Figures 5-4 and 5-5 show that chloroquine, NH4CI, and
methylamine did not have any inhibitory effects on virus fusion with basal epithelial
cells.
Effects of Sodium Azide on Virus Fusion
Sodium azide, which inhibits endocytosis in cells, was able to inhibit virus
fusion with B lymphocytes, but not with the lymphoblastoid cell line Raji. Basal
epithelial cells were treated with 10mM sodium azide followed by incubation with virus.
Sodium azide had no effect on virus fusion with epithelial cells (Figure 5-6).
Fusion of AF-labeled Virus
Virus labeled with AF has proven to be a valuable method for determining
whether virus fusion is occurring in a neutral or acidic environment from previous
experiments with lymphoblastoid cell lines and fresh B cells. AF-labeled virus was
incubated with cells that had been treated with NH4CI to neutralize acidic
compartments and untreated cells. The results in Figure 5-7 show that fluorescence
dequenching could be measured with the AF-labeled virus regardless of whether or
not the cells were treated with NH4CI.


15
The envelope of Sendai virus, a paramyxovirus, has two proteins. The
hemagglutinin-neuramidase (HN) protein is responsible for attachment of the virus to
cell surface sialic acid residues. The fusion (F) protein initiates fusion at the plasma
membrane allowing virus penetration, virus-induced cell fusion and hemolysis (Hsu et
al., 1981; Scheid and Choppin, 1974; Soheid and Choppin, 1976). The F protein
consists of two sulfhydryl-linked glycopeptides (F, and F2) resulting from proteolytic
cleavage of an inactive precursor (F0) by a host cell enzyme (Hsu et al., 1982).
Viruses produced by cells that lack a suitable protease for F protein activation are
noninfectious (Hsu et al, 1982). F2 corresponds to the N-terminus of F0, and the
protein is anchored in the bilayer through F,. The N-terminus of F,, resulting after
cleavage of F has been found to be unusually hydrophobic (Gething et al., 1978) and
it was suggested that the hydrophobic terminal peptide might play a role in fusion.
Support for this role has been provided by experiments with synthetic peptides
corresponding to the hydrophobic amino-terminus of F, showing that such molecules
inhibit virus fusion (Richardson et al., 1980). The amino acid sequence in this region
is highly conserved among paramyxoviruses (Scheid et al., 1978).
Orthomyxoviruses also have two types of spike glycoproteins which have
neuraminidase, hemagglutination, and fusion activities. One of the glycoproteins is a
neuraminidase (NA) and the other, the hemagglutinin (HA), has the capability to bind
to cell surface sialic acid residues and to catalyze fusion (Choppin and Compans,
1975b; White et al., 1982). Unlike paramyxoviruses, orthomyxoviruses are
endocytosed and fuse with the endocytic vesicle. The HA consists of two disulphide
linked glycopeptide chains, HA, and HA2, resulting from proteolytic cleavage of a
precursor glycoprotein HA0. The cleavage is irrelevant to adsorption, but is a


Table 2-1. Effect of labeling with R18 on the ability of [3H] EBV to bind to receptor
positive and negative cells.
35
Virus
treatment
Virus
dilution
Total acid
precipltable
counts bound to:
% acid preclpitable
counts bound to:a
Rajl
CI5
Raji
CI5
None
neat
13284
543
28.9
1.2
1/2
6346
a
27.6
-
1/4
2704
217
23.5
1.8
1/8
1408
-
24.5
-
Mock-
neat
5816
113
29.4
0.6
labeled
1/2
2663
*
26.8
"
1/4
1366
38
27.5
0.7
1/8
758
-
30.6
-
neat
6962
248
25.5
1.0
labeled
1/2
3707
-
27.1
1/4
1831
104
26.8
1.5
1/8
1016
-
29.7
-
radioactivity bound/radloactivlty added X 100
receptor positive cells
receptor negative cells
not done


44
Table 2-4. Monocyte depletion of T-depleted human leukocytes by adherance to
plastic.
Cell
treatment
cell number
% cells staining
esterase positive1
none
3.0 X108
45%
adherance
1.8 X 10s
8%


cells. Entry of EBV Into all cell types occurred independent of exposure to low pH.
However, virus fusion with normal and recently transformed lymphocytes occurred
from within endocytic vesicles, whereas fusion with lymphoblastold and epithelial cells
occurred at the plasma membrane.
The contribution to fusion made by virus envelope proteins to fusion was
studied with monoclonal antibodies that neutralized virus infectivity. Antibody to
glycoprotein gp85 inhibited fusion with all cells except epithelial cells. Antibody to
glycoprotein gp350, responsible for virus attachment to CR2 on lymphocytes, only
partially Inhibited virus binding to epithelial cells and the remaining bound virus did not
fuse. Soluble CR2 Inhibited virus binding to lymphocytes but only partially inhibited
binding to epithelial cells.
These studies document clear differences between virus entry Into lymphocytes
and epithelial cells and suggest that the virus proteins involved In fusion with the two
cell types may be distinct.
xiii


7
one of the latently transcribed genes that Is necessary to maintain the eplsomal form
of the EBV DNA (Fahraesus et al., 1988; Rowe et al., 1987). Additional latently
transcribed genes EBNA-2, EBNA-3-6 and latent membrane protein (LMP) are
expressed in LCLs, but are down-regulated in BL and NPC cells (Klein, 1989; Rowe et
al., 1987).
For a long time It was generally accepted that EBV infected only B cells in vivo.
Recently, a second target for EBV, the undifferentiated epithelial cell, has been
identified (Greenspan et al., 1985; Lemon et al., 1977; Sixbey et al., 1984, 1987; Wolf
et al., 1984). The epithelial cell is permissive for replication and is thought to be the
source of virus that is shed in the oropharynx. Cultures of human epithelial cells have
been transfected (Grogan et al., 1981) and directly infected in vitro (Sixbey, 1983), but
the only cell currently available for studying the virus replication cycle in vitro is the
lymphocyte. Lymphocytes latently infected with EBV provide an unique system for
studying the biochemistry of herpes virus latency.
The ability of EBV to infect B lymphocytes is Initiated by attachment of virus to
the 145-kilodalton (kDa) cell membrane glycoprotein, CR2, which also binds the C3d
fragment of complement (Fingeroth et al., 1984; Nemerow et al., 1985b). Recently It
has been shown that epithelial cells also express a receptor for virus attachment but It
Is lost during differentiation of the epithelium (Sixbey et al., 1984, 1987; Young et al.,
1986). The B cell CR2 receptor Is also lost during differentiation to the plasma cell
(Tedder et al, 1984). Immunopreclpltation from the surface of epithelial cells with an
antl-CR2 antibody yielded a 200-kilodalton membrane protein (Young et al., 1989).
The CR2 receptor has been detected on three T-lymphoblastoid cell lines (Fingeroth et
al., 1988) and on a fraction of normal human peripheral blood T lymphocytes (Fischer


69
uptake. Figure 3-12 demonstrates the change in the fluorescence intensity of FITC-
dextran in buffer at various pH values. This standard curve can be used in
conjunction with the differences In fluorescence seen upon addition of monensin to
cells to determination the intracellular pH. After addition of monensin to cells, the
fluorescein emission resembles that expected at the external pH. By altering the
external pH, the pH of the Intracellular FITC-dextran was obtained.
Figure 3-13 shows the fluorescence emission at 522nm of BAT cells that have
endocytosed FITC and TRITC-labeled dextran. The value of the fluorescence of the
TRITC was recorded as a control value for dextran uptake and compared between
samples. Fluorescence measurements were made In pH 7.4 medium before and after
addition of monensin. In the initial scan, the FITC emission was very low, indicating
that the fluorescence was quenched due to acidic pH, after addition of monensin
there was a great change in the fluorescence with a peak at 496nm. The TRITC
fluorescence value was 1370 arbitrary units (a.u.). Cells treated with 20mM NH4CI
were incubated with the labeled dextrans and analyzed for fluorescence before and
after addition of monensin. Figure 3-14 (panel A) shows the results when the
extracellular medium was pH 7.4. The fluorescence before addition of monensin was
much higher than the fluorescence of the untreated cells, Indicating that the pH had
been elevated. Addition of monensin produced a slightly higher emission pattern,
Indicating that the Internal pH was not at pH 7.4, but that it was higher than the
control cells. The TRITC fluorescence was 1405 a.u.. The same assay was performed
except with an extracellular medium of pH 7.0 (Figure 3-14, panel B). In this case the
fluorescence measurements were essentially equal before and after addition of


This dissertation was submitted to the Graduate Faculty of the College of
Medicine and to the Graduate School and was accepted as partial fulfillment of the
requirements for the degree of Doctor of Philosophy.
May 1991
A.P4M
Dean, College of Medicine
Dean, Graduate School


94
TIME (MINUTES)
Figure 4-5. Effect of preincubation of Raji cells with chlorpromazine or RPMI on relief
of self-quenching of Ria-labeled MCUV5 virus. Increase in fluorescence is expressed
as a percent of the maximum release obtained after addition of Triton X-100 (infinite
dilution).


41
paraformaldehyde prior to binding to virus. When Molt 4 cells were substituted in the
assay for Rajl, there was no significant relief of self-quenching of the bound probe,
which is compatible with the reported inability of virus to fuse with Molt 4 cell
membranes (Menezes, 1977). The fluorescence maxima obtained after addition of
Triton was slightly less for Molt 4 cells than Raji, which is in agreement with published
observations showing that Molt 4 cells express fewer receptors than Raji (Stoco et al.,
1988).
Changes in Fluorescence after Interaction of R1f-Labeled Virus with Normal B Cells
Two independent studies have demonstrated that although EBV fuses with the
plasmalemma of lymphoblastoid cells, it is endocytosed into normal B cells before
any fusion of virus and cell membranes occurs (Nemerow and Cooper, 1984a; Tedder
et al., 1986). However, if fusion was occurring within an endocytic vesicle, it seemed
possible that the event might still be detectable with the Relabeled virus.
Experiments were done initially with B cells Isolated from fresh tonsil tissue.
Tonsil tissue was obtainable on a sporadic basis from the surgical pathology
department and large numbers of cells could be obtained from a single piece of
tissue. Considerably less virus bound to normal B cells than to Raji cells. However,
even though the Increase In fluorescence measured with Relabeled virus bound to
normal B cells was smaller than that measured with lymphoblastoid cells, a
measurable signal was obtained. The increase in fluorescence expressed as a
percentage of the maximum obtainable after addition of Triton was less than that seen
In experiments with lymphoblastoid cells (Figure 2-6).
In order to rule out the interference of monocyte engulfment of virus in the
determination of maximum relief of fluorescence, experiments were done using cells


115
deflected out of the main stream toward the deflection plate bearing an opposite
charge, and the charged droplet streams are collected, while the uncharged main
stream passes into an aspiration tube leading to the waste reservoir. The sorter also
permits formation of two sort streams, one positively charged and the other negatively
charged, which allows isolation of cells selected by two criteria (Shapiro, 1988).
To examine the virus receptor on the epithelial basal cell population further,
HB5 positive and negative cells were sorted in the fluorescence activated cell sorter.
FACS analysis seemed to be considerably more sensitive than our previous micro
scopic analysis and indicated that approximately 19% of basal cells were HB5 positive
(Figure 5-2). The HB5 negative population was recovered from the population and
then analyzed for its ability to bind and to fuse with Relabeled EBV. Figure 5-3
shows a spectrofluorometric analysis of virus bound to unsorted basal cells and two
sorts of basal cells from which the HB5 reactive population was removed. Removal of
the HB5 positive population reduced the amounts of virus binding by 85 to 90%. The
numbers of HB5 positive cells recovered were too small for analysis in the
spectrofluorometer, but microscopic analysis of these cells indicated that greater than
85% of these cells were able to bind and fuse with virus.
Effects of Lysosomotropic Agents on Virus Fusion
Lysosomotropic agents cross cell membranes easily in the unprotonated form
but the protonated form does this far less efficiently. When the uncharged form enters
acidic vesicles it become protonated, thereby raising the pH and Inhibiting its own
escape across the membranes of the vacuoles. Basal epithelial cells were treated
with the lysosomotropic agents chloroquine, ammonium chloride (NHCI), and
methylamine and evaluated for the ability to bind and fuse Relabeled virus. These


LIST OF TABLES
Table page
2-1. Effect of labeling with R18 on the ability of [3H] EBV to bind to receptor
positive and negative cells 35
2-2. Effect of monoclonal anti-EBV and anti-CR2 antibodies on the ability of
Relabeled [3H] EBV to bind to receptor positive cells 36
2-3. Effect of labeling with R1S on the ability of MCUV5 virus to induce
immunoglobulin synthesis by fresh T-depleted human leukocytes 38
2-4. Monocyte depletion of T-depleted human leukocytes by adherance
to plastic 44
3-1. Effect of labeling with AF on the ability of MCUV5 virus to induce
immunoglobulin synthesis by fresh T-depleted human leukocytes 76
5-1. Cell counts and viability of cells recovered from infant foreskin epidermis. 107
5-2. Morphological distribution of epithelial cells In fractions from Percoll
gradient 108
5-3. Reactivity of epithelial cells with the monoclonal anti-CR2 antibody HB5. 110
5-4. Reactivity of epithelial cells with anti-CR2 antibodies 110
5-5. Microscopic analysis of virus binding and fusion with epithelial cells. ... 112
6-1. Effect of antibodies F-2-1, E1D1, 72A1, and E8D2 on the ability of
MCUV5 virus to Induce immunoglobulin synthesis by fresh T-depleted
human leukocytes 130
6-2. Effect of antibodies F-2-1, 72A1, and E8D2 on the ability of [3H] EBV to
bind to receptor positive cells 132
6-3. Effect of antibody on the relief of self-quenching of Relabeled virus
added to T-depleted leukocytes 135
6-4. Effect of antibody or unlabeled virus on binding of R18-EBV 137
6-5. Effect of soluble CR2 or 72A1 on binding of Relabeled EBV 139
XI


72
B WAVELENGTH (nm)
Figure 3-14. Excitation spectra at pH 7.4 (panel A) and pH 7.0 (panel B) of NHCI
treated BAT cells containing FITC-dextran before ( a ) and after ( ) addition
of monensin. Measurements were taken at a fixed emission wavelength of 522nm and
fluorescence is expressed in arbitrary units (a.u.).


39
O 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 2-4. Relief of self-quenching of Relabeled virus bound to receptor positive
Raji cells and receptor negative Daudi cells. At 32 minutes Triton X-100 was added to
measure maximum relief of self-quenching of bound probe (infinite dilution). Relative
fluorescence expressed in arbitrary units (a.u.).


154
Fahraesus, R., L.F. Hu, I. Ergberg, J. Finke, M. Rowe, G. Klein, K. Falk, E, Nilsson, M.
Yadav, P. Busson, T. Tursz, and B. Kallin. 1988. Expression of Epstein-Barr virus-
encoded proteins In nasopharyngeal carcinoma. Int. J. Cancer 42:329-338.
Fauci, A.S. 1988. The human Immunodeficiency virus: Infectlvlty and mechanisms of
pathogenesis. Science 239:617-622.
Fingeroth, J.D., M.L. Clabby, and J.D. Strominger. 1988. Characterization of a T-
lymphocyte Epstein-Barr virus/C3d receptor (CD21). J. Virol. 62:1442-1447.
Fingeroth, J.D., J.J. Weiss, T.F. Tedder, J.L. Strominger, P.A. Biro, and D.T. Fearon.
1984. Epstein-Barr virus receptor of human lymphocytes is the C3d receptor CR2.
Proc. Natl. Acad. Sci. USA. 81:4510-4514.
Fischer, E., C. Delibrias, and M.D. Kazatchkine. 1991. Expression of CR2 (the
C3dg/EBV receptor, CD21) on normal human peripheral blood T lymphocytes. J.
Immunol. 146:865-869.
Fleckenstein, B. and R.C. Desroslers. 1982. Herpesvirus samiri and Herpesvirus
teles, p. 253. ¡n: B. Roizman (ed.), The Herpesviruses, vol.1. Plenum Press, New
York.
Frink, R.J., R. Elsenberg, G. Cohen, and E.K. Wagner. 1983. Detailed analysis of the
portion of the herpes simplex virus type 1 genome encoding glycoprotein C. J. Virol.
45:634
Fujisaku, A., J.B. Harley, M.B. Frank, B.A. Gruner, B. Frazier, and V.M. Holers. 1989.
Genomic organization and polymorphisms of the human C3d/Epstein-Barr virus
receptor. J. Biol. Chem. 264:2118-2125.
Fuller, A.O. and P.G. Spear. 1987. Anti-glycoprotein D antibodies that permit
adsorption but block Infection by herpes simplex virus 1 prevent virion-cell fusion at
the cell surface. Proc. Natl. Acad. Sci. USA. 84:5454-5458.
Garoff, H., A.M. Frischauf, K. Simon, H. Lehrach, and H. Delius. 1980. Nucleotide
sequence of cDNA coding for Semllki Forest virus membrane glycoproteins. Nature
(London) 288:236-241.
Gelsow, M.J. 1984. Fluorescein conjugates as indicators of subcellular pH. Exp. Cell
Res. 150:29-35.
Gelsow, M.J. and W.H. Evans. 1984. pH in the endosme. Exp. Cell Res. 150:36-46.
Gerber, P. and S.J. Lucas. 1972. Lymphocyte stimulation by Epstein-Barr virus. Cell
Immunol. 5:318-324.


Table 2-2. Effect of monoclonal antl-EBV and anti-CR2 antibodies on the ability
of Relabeled [3H] EBV to bind to receptor positive cells.
Antibody
(ug)
Relabeled virus
Mock-labeled virus
Total cpm
bound
% cpm
bound0
Total cpm
bound
% cpm
bound
none
1904
23.5
2789
23.9
72A1 (10)
123
1.5
231
1.9
OKB7 (5)
198
2.4
180
1.5
HB5 (5)
1390
17.1
2308
19.8
amount of antibody used, expressed In micrograms
radioactivity bound/radloactivlty added X 100


Haddad, R.S. and L.M. Hutt-Fletcher. 1989. Depletion of glycoprotein gp85 from
virosomes made with Epstein-Barr virus proteins abolishes their ability to fuse with
virus receptor-bearing cells. J. Virol. 63:4998-5005.
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Hampar, B., A. Tanaka, M. Nonoyama, and J.G. Derge. 1974. Replication of the
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29
Figure 2-1. Structural formula of octadecyl rhodamine B chloride (R,s).


133
Figure 6-1. Effect of preincubation with 100ug of monoclonal antibodies on relief of
self-quenching of R,s-labeled P3HR1-CI13 virus added to Raji cells. Relief of self
quenching expressed as a percent of the maximum release obtained after addition of
Triton X-100 (infinite dilution).


centrifugation over 60% Percoll. The non-rosetting cells were collected from the
gradient, extensively washed, and used directly in assays.
Preparation and Use of Cellular Inhibitors
88
Sodium azide, leupeptin, and chlorpromazine were purchased from Sigma
(Sigma Chemical Co., St. Louis, Missouri). A 5mM stock solution of chlorpromazine
was prepared in phosphate buffered saline (PBS) with 1.0% dimethylsulfoxide.
Leupeptin was prepared by reconstituting 10mg of solid in 0.5ml distilled water to
which 4.5ml of RPMI 1640 was added for a final concentration of 2mg/ml which was
allquoted and stored at -20C. Stock solutions of the other inhibitors were prepared in
PBS and all working dilutions were made in RPMI 1640. Cells were incubated with the
inhibitors for 40 minutes at 37, while control samples were Incubated In RPMI
medium alone. After incubation, cells were pelleted and the supernatant was removed
and the cells were incubated with virus as described previously.
Results
Effect of Sodium Azide on Virus Fusion
Sodium azide (NaNJ, which inhibits oxidative phosphorylation, thus blocking
the energy production required for endocytosis, had no effect on virus binding or
fusion with Raji cells at a concentration of 10mM (Figure 4-1). When BAT cells were
treated in the same manner, there was a 35% inhibition of fusion at 10mM (Figure 4-
2), and some inhibition of binding was seen at 50mM. Virus fusion with peripheral T-
depleted leukocytes was also partially inhibited with 10mM NaN3 (Figure 4-3).


129
Results
Effect of Antibodies on Virus Binding and Infectivitv of B Lymphocytes
Of the four monoclonal antibodies utilized in this work, two of them, 72A1 and
F-2-1, have been previously reported to neutralize virus infectivity in the absence of
complement (Hoffman et al., 1980; Strnad et al., 1982). Neutralization of virus
infectivity is measured by the ability of the antibody to prevent the transforming strain
of virus, MCUV5, from inducing immunoglobulin synthesis by human peripheral B
cells in culture. The data in Table 6-1 show the extent of neutralization of virus with
72A1 and F-2-1, and also the effect of two non-neutralizing antibodies, E1D1 and
E8D2. It was then important to compare the ability of the two neutralizing antibodies
to inhibit virus binding as this would be an effective means by which virus could be
neutralized. The non-neutralizing antibody E8D2 had no inhibitory effects on binding
(Table 6-2). In contrast, the ability of 72A1 to neutralize virus infectivity can be
accounted for by its ability to block virus binding. The observation that antibody F-2-1
failed to block virus attachment despite its ability to neutralize infectivity suggested
that it blocked replication at a point following virus attachment.
Effect of Antibodies on Fusion of Virus Bound to Raii cells
Preincubation of Relabeled P3HR1-CI13 virus with the nonneutralizing
antibodies E8D2 and E1D1 had no effect on the amount of R18-labeled virus that
bound to Raji cells, nor on the relief of self-quenching of the probe after cells were
warmed to 37C (Figure 6-1). In contrast, preincubation with antibody F-2-1 effectively
inhibited the relief of self-quenching, even though the antibody did not affect binding
of the labeled virus. These data suggested that the neutralization of virus


FLUORESCENCE (% MAXIMUM)
79
0 4 8 12 1 6 20 24 28 32
TIME (MINUTES)
Figure 3-18. Relief of self-quenching of AF-labeled MCUV5 virus bound to Raji and
Molt 4 cells. Increase In fluorescence is expressed as a percent of the maximum
release obtained after addition of Triton X-100 (infinite dilution).


135
Table 6-3. Effect of antibody on the relief of self-quenching of Relabeled virus added
to T-depleted leukocytes.
Antibody
Amount added
(ug)
Fluorescence
(% maximum)
F-2-1
100
6.1
50
4.7
25
5.6
12.5
6.1
6.25
9.4
3.13
13.3
1.56
24.3
E8D2
100
33.1
E1D1
100
29.7
None
0
31.1


134
infeotivity by F-2-1 might be due to interference with the ability of the virus to fuse with
the cell membrane.
Effect of Antibodies on Fusion on Virus Bound to T-deoleted Leukocytes and BAT
Cells
In order to rule out the possibility that the effects of the antibody were unique
to the P3HR1-CI13 strain of virus, the experiment was repeated with Relabeled
MCUV5 virus and T-depleted human leukocytes. Of the three antibodies tested, only
F-2-1 influenced the relief of self-quenching and the inhibition seen with F-2-1 was
dose dependent (Table 6-3).
Although it was unlikely that the effects of the antibodies would be different
from the EBV-transformed BAT cell line, these cells were also tested in assays with the
antibodies. Figure 6-2 shows the results of the antibodies with BAT cells. Once
again, the F-2-1 antibody inhibited fusion of bound virus with cells.
Specificity of Virus Binding to Epithelial Cells
Although previous work with the Relabeled EBV had indicated that the
labeling had no effect on the specificity of binding to lymphocytes, and virus binding
seemed to be confined to a subset of epithelial cells, there was still concern that the
mild trypsinization of the epidermal sheets shortly before use might make them more
non-specifically sticky for virus. To investigate this possibility, specificity was judged
by two criteria, namely the ability of unlabeled virus to compete for binding of R18
labeled material, and the ability of the antibody 72A1, which inhibits virus binding to B
cells, to inhibit R1S labeling of epithelial cells. The results are presented in Table 6-4
Raji cells were included as a comparative control. The figures in parentheses indicate


TABLE OF CONTENTS
page
DEDICATION ¡i
ACKNOWLEDGEMENTS ¡
LIST OF ABBREVATIONS vi
LIST OF FIGURES vil
LIST OF TABLES xi
ABSTRACT xii
CHAPTERS
1 INTRODUCTION 1
Discovery of Epsteln-Barr Virus 1
Clinical Manifestations 1
Description of EBV 5
Entry of Enveloped Viruses into Animal Cells 11
Measuring Fusion 20
Purpose of This Work 22
2 ESTABLISHMENT OF AN ASSAY TO MEASURE VIRUS FUSION .... 23
Introduction 23
Materials and Methods 24
Results 31
Discussion 48
3 EFFECTS OF LYSOSOMOTROPIC AGENTS AND pH ON FUSION OF
VIRUS WITH LYMPHOCYTES 51
Introduction 51
Materials and Methods 52
Results 56
Discussion 83
IV


DEDICATION
This dissertation Is dedicated to my parents, John and Nancy Miller, who have
always encouraged all my endeavors and have provided me with love and support
throughout all that I have done.


138
maximum fluorescence obtained with each cell type after addition of Triton X-100. For
comparison within cell type, this value was then given a score of 100. As expected,
more virus actually bound to Raji cells, which are known to have a high density of
receptors, than to any other cell type. As previously noted, almost twice as much
virus bound to basal cells than parabasal cells despite the decreased reactivity of HB5
and B2 antibody with this population assayed in parallel. Unlabeled virus markedly
reduced the amount of R18 label bound to each cell type, suggesting that little virus
bound non-specifically to cells. In contrast, however, there was a significant difference
between the ability of antibody to block attachment of virus to Raji cells and its ability
to block attachment to epithelial cells.
Virus Binding in Presence of Soluble CR2
The extracellular domain of the receptor CR2 is composed of 15-16 short
consensus repeat elements (SCR) that consist of 60-75 amino acids each. Analysis of
CR2 deletion mutants and chimeric receptors of CR2 and CR1 have determined that
the binding site of EBV and C3dg are within the first two amino-terminal SCRs (Lowell
et al., 1989). The first two amino terminal SCRs of CR2 were expressed in the
Baculovirus expression system by Nemerow et al. (1990). This protein is able to block
infection of B lymphocytes by blocking binding of virus. In order to investigate further
the difference in the ability to inhibit virus binding to epithelial cells with an anti-viral
antibody, virus binding in the presence of soluble virus receptor was evaluated, the
results are presented in Table 6-5. For Raji cells, 20ug of CR2 was able to inhibit virus
binding by 90%, which parallels the effectiveness of the 72A1 antibody for inhibiting
virus binding to these cells. The inhibition of virus binding to basal epithelial cells was
54% of the control in one experiment and 77% of the control in another


0 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 4-6. Effect of preincubation of Raji cells with leupeptln or RPMI on relief of
self-quenching of Relabeled MCUV5 virus. Increase in fluorescence is expressed
a percent of the maximum release obtained after addition of Triton X-100 (infinite
dilution).


144
Discussion
The initial observation that the monoclonal antibody F-2-1 was able to
neutralize virus infectivity of B lymphocytes without inhibition of virus binding,
suggested that the glycoprotein gp85 might be involved in events occurring after virus
binding. The data presented in this chapter suggest that gp85 is involved in fusion of
virus and B lymphocyte cell membranes. This antibody was tested with normal B
cells, recently EBV-transformed cells and lymphoblastoid cells that have been in
culture for many years. Fusion of virus with all three of these cell types was inhibited
by this antibody against gp85. The gene encoding gp85 has been mapped to the
BXLF2 open reading frame of EBV DNA (Heineman et al., 1988; Oba and Hutt-
Fletcher, 1988). The sequence encoded a relatively hydrophobic protein that has
homology with genes encoding herpes simplex hype (HSV) 1 glycoprotein gH,
varicella zoster (VZV) gplll and glycoprotein p86 of human cytomegalovirus (CMV)
(Cranage et al., 1988). Antibodies to each of these proteins neutralize virus in the
absence of complement and antibodies to both HSV gH and VZV gplll have been
shown to block cell fusion by syncytial virus strains (Gompels and Minson, 1986;
Keller et al., 1987). Thus EBV gp85 appears to be one of a family of homologous
herpesvirus glycoproteins whose role in infectivity may be one of membrane fusion.
Studies with purified recombinant gp350 and gp220 have implicated these two
proteins in the internalization process (Tanner et al., 1987). Polyacrolein beads 0.9
um in diameter coated with the recombinant proteins bound to the EBV receptor on
normal B cells and twenty-four hours later 10% of the beads were found to be
discharged free into the cytoplasm. It is possible however that the discharge of
gp350/220 coated beads into the cytoplasm of B cells does not completely reflect the


CHAPTER 2
ESTABLISHMENT OF AN ASSAY TO MEASURE VIRUS FUSION
Introduction
Membrane fusion is an effective process for delivering membrane-bound
contents from one cellular compartment to another. Viruses take advantage of this
important process and utilize membrane fusion for entry into cells. The mechanism of
fusion has become one of the most Intriguing questions in cell biology, and viruses
provide a natural experimental system for studying the fusion process. Fusion of two
lipid bilayers is an energetically unfavorable process, and the fact that viruses, which
have relatively simple membranes, use this process for entry Into cells makes them a
very Interesting model for studying membrane fusion.
Studies of membrane fusion have, In the past, been largely morphological and
descriptive due to lack of techniques for measuring and analyzing the fusion process
in isolation. Many assays used involved radioisotopes (Haywood and Boyer, 1982;
White et al., 1983) and electron spin probes (Maeda et al., 1975, 1981; Lyles and
Landesberger, 1979) or involved use of Indirect techniques such as hemolysis and
infectivlty (White et al., 1983). Of all these techniques only electron spin resonance
permits the continuous monitoring of the fusion process, which is desirable In a fusion
assay. More recently, the assay described in this work has been widely adopted for
measurement of membrane fusion in isolation from other events in the virus life cycle
that precede or follow it. This method not only allows for continuous monitoring of
23


139
Table 6-5. Effect of soluble CR2 or 72A1 on binding of Relabeled virus.
Treatment
amount of R1S-EBV bound3
% Inhibition of binding0
Rajl
Basal
Raji
Basal
control
1090
858
0
0
CR2
118
398
90.2
54.6
72 A1
119
463
90.1
46.0
Fluorescence Intensity in arbitrary units.
Amount bound divided by amount bound to control sample.


Figure Paqi
3-6. Effect of preincubation of BAT cells with 20mM ammonium chloride or
RPMI on relief of self-quenching of Relabeled MCUV5 virus bound to cells.. 62
3-7. Effect of preincubation of T-depleted leukocytes with 20mM ammonium
chloride or RPMI on relief of self-quenching of Relabeled MCUV5 virus
bound to cells 63
3-8. Effect of prelncubatlon of Rajl cells with chloroqulne or RPMI on relief of
self-quenching of Relabeled MCUV5 virus bound to cells 64
3-9. Effect of preincubation of BAT cells with chloroquine or RPMI on relief of
self-quenching of Relabeled MCUV5 virus bound to cells 65
3-10. Effect of preincubation of T-depleted leukocytes with chloroqulne or RPMI
on relief of self-quenching of Relabeled MCUV5 virus bound to cells 66
3-11. Effect of preincubation of Raji cells, BAT cells, and T-depleted leukocytes
with 5mM methylamine or RPMI on relief of self-quenching of Relabeled
MCUV5 virus bound to cells 67
3-12. Relative Intensity of fluorescein Isothlocyanate-dextran (FITC-dextran)
fluorescence as a function of pH 70
3-13. Excitation spectra at pH 7.4 of BAT cells containing FITC-dextran before
and after addition of monensin 71
3-14. Excitation spectra at pH 7.4 and pH 7.0 of NH4CI treated BAT cells
containing FITC-dextran before and after addition of monensin 72
3-15. Excitation spectra at pH 7.4 of chloroqulne treated and untreated BAT
cells containing FITC-dextran before and after addition of monensin 74
3-16. Excitation spectra at pH 7.0 of methylamine treated and untreated BAT
cells containing FITC-dextran before and after addition of monensin 75
3-17. Fluorescence properties of virus labeled with AF at pH 6.0 to pH 7.4. . 77
3-18. Relief of self-quenching of AF-labeled MCUV5 virus bound to Raji and
Molt 4 cells 79
3-19. Relief of self-quenching of AF-labeled MCUV5 virus bound to Raji cells
at pH 7.2 or pH 5.5 80
3-20. Relief of self-quenching of AF-labeled or Relabeled MCUV5 virus bound
to BAT cells at pH 7.2
81


61
O 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 3-5. Effect of preincubation of Raji cells with ammonium chloride (NH4CI) or
RPMI on relief of self-quenching of R1B-labeled MCUV5 virus bound to cells. Panel A,
20mM NH4CI; panel B, 10mM NHCI. Increase in fluorescence is expressed as a
percent of the maximum release obtained after addition of Triton-X-100 (infinite
dilution).


59
O 4 8 12 16 20 24 28 32 36
TIME (MINUTES)
Figure 3-4. Relief of self-quenching of Relabeled MCUV5 virus bound to T-depleted
leukocytes at pH 7.2 or pH 5.5. Virus was bound to cells at pH 7.2, cells were washed
to remove unbound virus and cells were resuspended in pH 7.2 or pH 5.5 Dulbecco's
saline. Increase in fluorescence Is expressed as a percent of the maximum release
obtained after addition of Triton X-100 (Infinite dilution).


26
0KB7 (Rao et al., 1985), which blocks virus binding, and HB5, which does not block
the virus binding site of CR2 (Nemerow et al., 1985a).
Virus Binding Assay
The ability of intrinsically radiolabeled virus to bind specifically to CR2 was
determined by use of receptor positive and negative cells that had been briefly fixed
with ice-cold 0.1% paraformaldehyde. Virus was incubated with 2 x 106 fixed cells for
60 minutes at 4C, cells were washed five times to remove unbound virus and the
acid-precipitable radioactivity that remained attached to cells was counted. The ability
of antibody to interfere with virus binding was determined by preincubation of virus
and antibody for 1 hour at room temperature.
Isolation of B-cell Enriched Leukocytes
Heparinized human peripheral blood was separated by flotation on
Lymphocyte Separation Medium (LSM; Litton Bionetics, Charleston, South Carolina).
T cells were depleted from the leukocyte fraction by a double cycle of resetting with 2-
aminoethyl isothiouronium bromide-treated sheep erythrocytes (Pellegrino et al., 1975)
and centrifugation over 60% isotonic Percoll (Pharmacia Fine Chemicals, Piscataway,
New Jersey). The nonrosetting cells remain at the RPMI-Percoll interface and are
collected and washed free from remaining Percoll.
Human tonsil tissue was teased apart with forceps and rinsed with RPMI to
collect single cells. Cells were washed and resuspended in RPMI and separated on
LSM. T cells were depleted by resetting as stated previously.
The T-depleted leukocytes were also depleted of monocytes in some
experiments by one of two methods. Monocytes were depleted by adherence to
plastic petri dishes in 10% RPMI for 1 hour at 37C and the nonadherent cells were


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111
antibodies. No cells stained delectably with the antibody OKB7 which is known to be
able to inhibit virus binding to B cells (Nemerow et al., 1985a) although the epitope
recognized by this antibody has been mapped very close to the EBV binding site and
the binding site of the natural ligand, C3d (Lowell et al., 1989). Of the other anti-CR2
antibodies that were used, only two, HB5 and B2 consistently gave positive staining.
These data indicate that there are parabasal and basal cells that express a molecule
that reacts with anti-CR2 antibodies and that the amount of this reactivity increases
from the basal to parabasal phenotype as judged by size of the cells.
Virus Binding and Fusion with Epithelial Cells bv Microscopic Analysis
Virus binding and fusion was measured by incubating cells with Relabeled
EBV and looking for rhodamine stained cells under the fluorescence microscope.
Cells were also fixed with 0.1% paraformaldehyde and assayed in parallel; fixing with
paraformaldehyde has been shown to prevent virus fusion as measured by relief of
self-quenching of Relabeled EBV bound to B cells. The data in Table 5-5 show that
20% of the parabasal cells and 26% of basal cells were positive for R!8 staining and
this was reduced to 1% and 3% respectively by fixation of the cells, confirming that
unfused virus was labeled with quenched amounts of R18. Cells were also stained in
parallel with the monoclonal antibody HB5, with which 27% of the parabasal cells and
8% of the basal cells were positive. A larger percentage of basal cells stained with
labeled virus than were identified as having a CR2-like molecule as determined by
reactivity with the HB5 antibody. Fixation of cells had little effect on parallel staining
done with HB5, 24% of parabasal and 7% of basal cells were positive, these results
assured that lower values obtained for HB5 staining of basal cells could not be
attributed to internalization of receptor by antibody.


106
Results
Isolation of Single Cells From Foreskin Tissue
The sequential digestion of the foreskin tissue with dlspase and trypsin allowed
disaggregation of the tissue and release of Individual cells. This treatment did not
result in complete digestion of the pieces of epidermis into single cells, but the cells
that were released from the tissue were healthy in terms of viability as determined by
exclusion of the dye trypan blue (Table 5-1). The mean value for the number of cells
isolated from tissue was 1.3 X 107 with a standard deviation of 4.7 X 106 cells. The
difference in the number of cells released from tissues in Table 5-1 primarily reflected
differences in the starting surface area of the tissue. The cells were separated by size
on discontinuous Percoll gradients in order to obtain populations of cells that would
reflect the normal differentiation process of the cells in the epidermis. The epidermis
consists of multiple layers of epithelial cells, called keratinocytes. Mitosis is restricted
to the basal layer and keratinocytes that leave this layer undergo terminal
differentiation as they move upwards towards the tissue surface. Typically, as the
cells differentiate, they increase in size and the shape of the cells changes from small
and rounded to enlarged, irregularly shaped cells. The cells obtained from the
interfaces of the Percoll gradient were evaluated separately on a morphological basis
into three categories, basal, parabasal and squamous cells and the results of this
analysis are presented in Table 5-2. The basal cells are the smallest cells and they
are the proliferative cells of the epidermal layer. These cells are found primarily at the
45/100% interface of the gradient, although a small percentage of these cells are
found throughout the gradient, most likely due to trapping by the larger cells.
Parabasal cells have a larger cytoplasm than the basal cells, but are distinguishable


57
TIME (MINUTES)
Figure 3-2. Relief of seif-quenching of Relabeled MCUV5 virus bound to Raji cells
at pH 7.2 or pH 5.5. Virus was bound to cells at pH 7.2, cells were washed to remove
unbound virus and cells were resuspended in pH 7.2 or pH 5.5 Dulbecco's saline.
Increase in fluorescence is expressed as a percent of the maximum release obtained
after addition of Triton X-100 (Infinite dilution).


83
to an extent comparable to that found with Relabeled virus, whereas in the untreated
cells the virus fusion was marginally detectable due to the pH-dependent quenching of
the fluorophore. These data not only show that most of the virus fused from within an
acidic compartment, but that virus was not dependent upon acidic pH in order to fuse
and thus enter the cell to continue the infectious cycle.
Discussion
It is well established that enveloped viruses enter their host cells by membrane
fusion, either at the plasma membrane or from within an endocytic vesicle. In many
Instances, virus entry from within an endocytic vesicle Is catalyzed by the acidic
environment of the endosme (Blumenthal et al., 1987; White, 1990) and this acidic
environment is a requirement for successful virus entry into the cytoplasm of the cell.
In order to assess which conditions are necessary for entry of Epstein-Barr virus Into
lymphocytes, the effects of lysosomotropic agents and low pH treatment were
examined on fusion between virus and cellular membranes.
Altering the extracellular medium to pH 5.5 did not result in any increase in the
rate or extent of fusion of virus with the lymphoblastoid cells line Rajl, the recently
EBV-transformed B cell line BAT, or with fresh peripheral T-depleted leukocytes.
Viruses that are catalyzed by acidification fuse rapidly and efficiently once in the
endosme and this environment can be imitated at the cell surface by lowering the pH
of the extracellular medium. If viruses in this category, such as influenza, Semlikl
Forest, and West Nile virus are acidified before binding to their target membranes,
their fusion activity is irreversibly inactivated, presumably due to premature triggering
of the acid-activated conformational change in the viral fusion protein necessary for
fusion to occur.


117
TIME (MINUTES)
Figure 5-3. Relief of self-quenching of Relabeled virus bound to unsorted basal
epithelial cells (A) or basal epithelial cells from which HB5(+) cells were removed by
cell sorting (B and C). Fluorescence expressed in arbitrary units (a.u.). Arrow
Indicates point at which Triton X-100 was added to measure maximum relief of self
quenching of bound virus (infinite dilution).


91
0 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 4-3. Effect of preincubation of T-depleted leukocytes with sodium azide
(NaN3), chlorpromazine, or RPMI on relief of self-quenching of Rla-labeled MCUV5
virus. Increase in fluorescence is expressed as a percent of the maximum release
obtained after addition of Triton X-100 (infinite dilution).


FLUORESCENCE (a.u.)
77
WAVELENGTH (nm)
B
*
pH 6.0
pH 6.5
pH 7.0
pH 7.4
Figure 3-17. Fluorescence properties of virus labeled with AF at pH 6.0 to pH 7.4.
Measurements were taken at an excitation wavelength of 496nm. Fluorescence
intensity is expressed in arbitrary units (a.u.).


17
membrane fusion (Marsh et al., 1983a). Lysosomotropic agents, which elevate
endosomal pH, inhibit SFV penetration (Helenius et al., 1982). Semliki Forest virus
can fuse directly with the plasma membrane in vitro at low pH (White et al., 1980).
The SFV spike glycoproteins have been shown to be fusogenic in the absence of
other virus components (Marsh et al., 1983b). As far as the role of the glycopeptides
are concerned, it has been suggested that the peptide E1 may be directly involved in
the fusion activity (Kielian and Helenius, 1985). Both SFV and Sindbis, another
togavirus, have E1 proteins containing a hydrophobic peptide segment located close
to the N-terminus, and this segment has an external position in the virus membrane
(Garoff et al., 1980; White et al., 1983). Since E1 and E2 occur as a complex, E2 may
also participate in the fusion reaction. The role of E3 is not clear, it is a small
peripheral glycopeptide and there is no homologue in Sindbis virus (Welch and
Sefton, 1979).
Vesicular stomatitis virus (VSV), a rhabdovirus, has only one type of envelope
glycoprotein, designated the G-protein. The G-protein has a hydrophobic region near
the C-terminus forming the intramembranous domain. A small hydrophilic sequence
at the C-terminus is in contact with the cytoplasm. The larger N-terminal domain,
containing the oligosaccharide chains, is exposed to the exterior of the cell (Rose et
al., 1980; Rose and Gallione, 1981). The G-protein has been cloned and sequenced
(Rose and Gallione, 1981). Eukaryotic cells expressing the cloned G-protein gene
fuse, at low but not at neutral pH, indicating that this protein is both necessary and
sufficient for fusion activity (Reidel et al., 1984). In addition, at low pH, the G-protein
spikes reversibly aggregate at the ends of virus particles (Brown et al., 1988); this
observation may be potentially relevant to determining the mechanism of fusion for


8
et al., 1991), these findings suggest that the troplsm of EBV for B lymphocytes may
rely on factors other than receptor specificity.
Permissiveness of Virus Replication in Vitro
Cultures of EBV-infected lymphocytes vary in their permissiveness for viral
replication, most cultures being nonpermissive, but replication does occur in a small
fraction of cells In some cultures. The nonpermissiveness of EBV Infection has made
it difficult to study virus replication and also limits the amounts of purified virus
available for studying the components of mature virus particles. Clones of infected
lymphocytes that are more permissive of virus replication have been selected (Miller
and Llpman, 1973) and have facilitated studies of the virus replication cycle and
biochemical analyses. Two isolates of virus have been extensively studied, B95-8 and
P3HR1. The B95-8 strain Is produced by a cell line derived from a clone of marmoset
lymphocytes that were Infected with virus obtained from a culture of lymphocytes from
a patient with Infectious mononucleosis. The B95-8 viral DNA has been completely
sequenced (Baer et al., 1984) and has been the prototype used for gene mapping.
The P3HR1 cell line Is a clone of the Jijoye Burkitt-tumor derived cell line (Hlnuma et
al., 1967). The P3HR1 cells are more permissive than the parent clone for virus
replication and the virus produced by P3HR1 cells lacks the ability to growth-transform
nonlnfected B lymphocytes (Miller et al., 1974).
Genome Structure
The linear double stranded EBV genome contains nonrandom single stranded
breaks (Pritchett et al., 1975). When the genome is carried in the latent state it
circularizes via joining of the terminal repeated DNA sequences at either end of the
molecule (Dambaugh et al., 1980). The genome consists of five large regions of


BIOGRAPHICAL SKETCH
The candidate, Nancimae Miller, graduated in 1980 from Mother McAuley
Liberal Arts High School in Chicago, Illinois. She earned a Bachelor of Science
degree majoring In medical technology from Rush University at Rush-Presbyterian-St.
Lukes Medical Center In Chicago In 1985. Her graduate work at the University of
Florida began In 1985 as a student in the Department of Immunology and Medical
Microbiology from which she received a Master of Science degree in 1988. She
became a Ph.D. candidate In 1988 in the same department. Her research was
conducted under the excellent guidance of Dr. Lindsey Hutt-Fletcher in the
Department of Comparative and Experimental Pathology. She plans to pursue
additional research training working on herpes simplex retinitis as a postdoctoral
fellow In the laboratory of Dr. Sally Atherton at the University of Miami.
169


O 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 3-11. Effect of preincubation of Raji cells (panel A), BAT cells (panel B), and
T-depleted leukocytes (panel C) with 5mM methylamine or RPMI on relief of self
quenching of Relabeled MCUV5 virus bound to cells. Increase in fluorescence is
expressed as a percent of the maximum release obtained after addition of Triton-X-
100 (infinite dilution).


102
State is consistent with the concept that the virus persists in part via low grade
replication in the oropharynx.
It is well accepted that virus infects epithelial cells, but the question of how the
virus gains access to epithelial cells remained unresolved. The membrane fusion
assay provides an opportunity for addressing this Important aspect of EBV infection.
The purpose of the work presented In this chapter was to extend studies of virus entry
into lymphocytes to Include epithelial cells.
Materials and Methods
Isolation of Epidermal Cells from Foreskin Tissue
Foreskin tissue from newborns was collected after surgery and placed in
DMEM (Sigma Chemical Co., St. Louis, Missouri) with 10% fetal calf serum and
antibiotics. The tissue was rinsed with PBS and subcutaneous fat was removed using
a small Iris scissors. The tissue was cut Into 3-5 mm2 pieces and incubated for 9-12
hours at 4C In 25ml F 12 media containing dlspase (Sigma) at 0.75 units/ml (Stenn
et al., 1989). After enzymatic treatment, the epidermis was easily peeled away from
the dermis using a scalpel and forceps. The pieces of epidermis were incubated with
15-20ml of 0.1% trypsin with EDTA (Sigma) for 35-40 minutes at 37C with stirring.
Epidermal cells were collected In the supernatant after Inactivation of trypsin with
serum. Cells were washed by centrifugation at 1500 r.p.m., resuspended In 4ml of
DMEM (Sigma) and layered onto a discontinuous 30 (3ml), 40 (3ml), 45 (3ml), and
100% (1ml) isotonic Percoll gradient. The gradient was centrifuged for 30 minutes at
1500 r.p.m. at 4C. Cells banded at the media/30%, 30/40%, 40/45% and the


37
subsequent event in virus replication. Since the labeled virus was being used for
studying events post binding it was necessary to examine the infectivlty of labeled
virus. A comparison was made of the ability of labeled and mock-labeled MCUV5
virus to Induce Immunoglobulin synthesis In cultures of T cell-depleted peripheral
leukocytes (Table 2-3). There was no Indication that incorporation of probe into virus
had any detrimental effect on its biologic activity.
Changes in Fluorescence after Interaction of R18-Labeled Virus with Lvmphoblastoid
cells
Figure 2-4 demonstrates the changes In fluorescence emission observed as
virus bound to Raji cells at 4C was warmed in the cuvette of the spectrofluorometer.
The fluorescence Increased gradually over approximately 28-32 minutes, after which
time a plateau was reached. At this time, Triton X-100 (1% v/v) was added to relieve
any residual self-quenching of the fluorophore and thus providing a rough
approximation of the percentage of bound virus that fused. In Figure 2-4, 56% of the
maximal fluorescence was reached, this value proved reproducible for this particular
batch of labeled virus. The maximal value obtained with any batch of virus was 75%.
Parallel analysis of the receptor negative Daudl cell line confirmed that Relabeled
virus failed to bind to these cells (Figure 2-4). This result also showed that there was
no significant diffusion of residual free or incorporated probe from the virus
preparation Into cell membranes during the 1 hour incubation at 4C.
Further data Indicating that relief of self-quenching was measuring a membrane
fusion event and not simple diffusion of probe from closely approximated membranes
were obtained using fixed Raji cells and the Molt 4 cell line (Figure 2-5). The increase
In fluorescence emission measured when virus was bound to Raji cells at 4C and
then warmed to 37C was almost completely eliminated if the cells were fixed with


12
the plasma membrane at physiologic pH. The alternative route, adsorptive
endocytosis followed by vesicle membrane fusion, Is utilized by Semllkl Forest virus
(SFV) (Helenlus et al., 1980a; Marsh and Helenius, 1980), Influenza A (Matlin et al.,
1981; White et al., 1981; White et al., 1983), Slndbls (Boggs et al., 1989) and vesicular
stomatitis virus (VSV) (Matlin et al., 1982; White et al., 1983). In most cases studied,
fusion Is Induced by a specific viral membrane 'fusion protein.
Adsorptive Endocytosis
Adsorptive endocytosis, also known as receptor-mediated endocytosis, Is a
process by which macromolecules are taken into cells. This process Is initiated by
binding of a ligand to a cell surface receptor followed by Invagination of the
membrane forming a vesicle (Goldstein et al., 1979; Sllversteln et al., 1977).
Specialized regions of the plasma membrane have been morphologically Identified as
sites for adsorptive endocytosis of some viruses (Goldstein et al., 1979). These
regions, the coated pits, are thought to concentrate receptors and receptor-ligand
complexes at sites of internalization. The protein dathrin Is a major component of the
coated pits (Pearse, 1975) and Is thought to participate In the early stages of
endocytosis (Doxsey et al., 1987). The process of adsorptive endocytosis as a
mechanism for virus entry has been documented for several viruses (White et al.,
1981; White et al., 1983; White, 1990). Semliki Forest virus (SFV), a togavlrus, has
been widely studied since Its isolation In 1944 (Smithbum and Haddow, 1944). It Is
one of the best characterized enveloped viruses due to its simple structure. The
nucleocapsid envelope Is a host derived lipid bilayer In which virus encoded
glycoproteins are Inserted. This virus gains entry Into cells by accumulation in coated
pits that are endocytosed. The endosomes become acidified, providing conditions


148
into the lymphoblastoid cell line Raji occurred at the plasma membrane and virus was
endocytosed into fresh B ceils and recently transformed cells. Virus that was
endocytosed was exposed to low pH as indicated by the results of fusion assays with
virus labeled with a pH sensitive fluorescent probe, but low pH was not a requirement
of the fusion event. The effect of chloroquine on virus fusion was attributed to
Inhibition of endocytosls. Virus entry into epithelial cells occurred at the surface of the
cells as Indicated by the lack of Inhibition of virus fusion under conditions when
endocytosls was inhibited and also by measuring virus fusion with a pH sensitive
probe.
The EBV receptor on B lymphocytes has been shown to be the CR2 protein.
A monoclonal antibody to this protein, HB5, was used to determine the presence of a
CR2-related receptor on epithelial cells. Basal epithelial cells expressed a CR2-related
molecule that was present in approximately 20% of the population as judged by
reactivity with the antibody HB5. Although almost all epithelial cells that bound virus
were found in the HB5 positive population, there was not good correlation between
expression of the epitope and the amount of virus binding. The epitope recognized
on gp350/220 by antibody 72A1 was involved In binding of some, but not all virus
binding to epithelial cells, and was possibly involved In virus fusion with this cell type.
These results were In contrast with those with B lymphocytes for which the antibody
72A1 was capable of completely inhibiting virus binding. A monclonal antibody to an
additional virus envelope glycoprotein, gp85, was shown to Inhibit virus fusion with
lymphocytes, but had no effect on virus fusion with epithelial cells. This finding
suggests that gp85 has an active role in fusion of EBV with B lymphocytes, but this Is
not necessarily true for fusion with epithelial cells.


116
Figure 5-2. Fluorescence profile of HB5 antibody binding to basal epithelial cells.
Antibody was visualized with FITC-conjugated anti-mouse antibody. Broken line is
control profile of cells incubated with FiTC-mouse anti-immunoglobulin alone. The
horizontal line indicates the portion of the gate used to sort cells into HB5(+) and
HB5() populations. The proportion of cells included in this gate was 18.99% of the
total.


105
Monoclonal Antibodies
Several antibodies that react with CR2 were used: OKB7, which blocks virus
binding to B cells and HB5, B2, 2G7, 6F7, 1F8, 1C8, and L02 which do not. The
antibodies OKB7, 2G7, 6F7, 1F8, 1C8, and L02 were gifts from Barry Myones
(Childrens Memorial Hospital, Chicago, Illinois) and the antibody B2 was a gift from
Stephen Pflugfelder (Bascom Palmer Eye Institute, University of Miami, Miami,
Florida). The HB5 hybridoma cell line was obtained from the American Tissue Culture
Collection (Rockville, Maryland) and the antibody was purified from culture medium by
immunoaffinity chromatography on protein A sepharose (Genzyme).
Fluorescence-Activated Cell Sorter Analysis fFACSl
Epithelial cells for FACS analysis were isolated from tissue and separated on
Percoll as previously described and the cells at the 45/100% interface were incubated
with HB5 antibody for 35 minutes at 37C followed by three washes. Cells were then
incubated with FITC-conjugated-sheep anti-mouse IgG for 35 minutes at 37C followed
by three washes. Control cells received the second antibody only. Samples of 1 x
106 control cells and 2 X 106 test cells were analyzed to determine the level of
fluorescence In order to position the gating for sorting and collection of the positive
and negative cells separately. The cells to be sorted were at a concentration of 3 X
106 per ml. Analysis was performed using the FACStar plus (Becton-Dickinson).
Preparation and Use of Lysosomotropic Agents and Sodium Azide
Cells were Incubated for 35 minutes at 37C with the lysosomotropic agents
ammonium chloride (NH4CI), chloroquine and methylamine, followed by incubation
with virus. Sodium azide, which inhibits oxidative phosphorylation was incubated with
cells for 35-40 minutes at 37C followed by incubation with virus.


123
Discussion
Epstein-Barr virus can be found in saliva in most patients with infectious
mononucleosis and at low levels in almost all healthy EBV seropositive persons
(Chang et al., 1973; Golden et al., 1973; Miller et al., 1973; Niederman et al., 1976;
Yao et al., 1985). The ease with which EBV is recovered from oral secretions of
persons with primary or reactivated EBV infections suggests that a cell type freely
permissive of EBV replication exists in the oropharynx (Morgan et al., 1979).
Increasing evidence has suggested that EBV can infect and replicate in epithelial cells
(Greenspan et al., 1985; Lemon et al., 1977; Sixbey et al., 1983, 1984, 1987). Even
though is it well accepted that EBV infects two lineages of cells, B lymphocytes and
epithelial cells, the question of how EBV gains access to epithelial cells is still not
resolved.
Initially, the anti-CR2 antibody HB5 was used to determine which cells from the
epidermal tissue were expressing a receptor for virus. This antibody has been used
by other investigators to locate CR2-positive cells in epithelium from various areas of
the body (Sixbey et al., 1987; Thomas and Crawford, 1989; Young et al., 1986, 1989).
By using this reagent it was determined that the highest amount of reactivity was
located in the parabasal cell population. Many other antibodies that detect the CR2
molecule on B cells were also tested for their ability to detect this molecule on
epithelial cells. Of the other antibodies tested, only one, B2 (Nadler et al., 1981; lida
et al., 1983) reacted with epithelial cells. However, the B cell CR2 molecule is
composed of 15 or 16 repetitive elements and is a member of a gene family which
has a high degree of polymorphism (Fujisaku et al., 1989; Toothtaker et al., 1989). It
is then perhaps not surprising that the epithelial CR2-related molecule might not be


113
Relative Kinetics of Virus Fusion bv Soectrofluorometric Analysis
The kinetics of relief of self-quenching of virus bound to parabasal and basal
cells was analyzed on a population basis in the spectrofluorometer. Figure 5-1 shows
the fluorescence dequenchlng curves of parabasal and basal cells which were
essentially identical for the two cell populations. Approximately 50-60% of the bound
Relabeled virus was dequenched within 35 minutes. These values are comparable to
those that we have previously obtained with lymphocytes. The amount of labeled
virus that was bound to cells could be evaluated by comparing the values for
maximum fluorescence upon addition of Triton X-100 to cells and virus. These values
were about twice as high for the basal cells as for parabasal cells despite the fact that
fewer of the basal cells stained with HB5 or B2 antibody. In comparison to a
population of Raj'i cells, in which greater than 90 per cent of the cells stain with HB5
antibody, the amount of virus binding to basal cells, on a per cell basis for cells
reacting with HB5, is two to three times higher for the basal cells than the Raji cells.
FACS Analysis of Basal Epithelial Cells
The cell sorting capability of a flow cytometer makes it possible to isolate
relatively pure populations of cells with defined characteristics. This technique of cell
sorting first requires that the stream containing the sample of cells is broken into
droplets that are a fixed distance from the observation point so that individual cells
can be analyzed. Between the time a cell transverses the observation point and the
time it reaches the droplet breakoff point, the cell is analyzed for fluorescence and
based on the signal level that has been determined as positive or negative from an
analysis of a sample of the population to be sorted. As each droplet breaks off from
the stream it Is electrostatically charged If it is to be collected. Charged droplets are


153
Davies, P.J.A., D.R. Davies, A. Levitzki, F.R. Maxfield, P. Milhaud, M.C. Willingham,
and I.H. Pastan. 1980. Transglutaminase is essential in receptor-mediated
endocytosis of alpha2-macroglobulin and polypeptide hormones. Nature (London)
283:162.
de Duve, Christian, T. de Barsy, B Poole, A. Trouet, P. Tulkens, and F. Van Hoof.
1974. Lysosomotropic agents. Biochem. Pharm. 23:2495-2531.
de-The, G. 1982. Epidemiology of Epstein-Barr virus and associated malignant
diseases in man, p. 25-103. Jn: B. Roizman (ed.), The herpesviruses, vol. 1. Plenum
Publishing Corp., New York.
de-The, G. and Y. Zeng. 1987. Population screening for EBV markers: toward
improvement of NPC control, p.237-249. In: M. Epstein and B. Achong (ed.), The
Epstein-Barr virus: recent advances. Wiley and Sons, New York.
Dickson, R.B., R. Schlegel, M.C. Willingham, and I.H. Pastan. 1982. Involvement of
Na+ and HC03 in receptor-mediated endocytosis of alpha2-macroglobulin, epidermal
growth factor, and vesicular stomatitis virus. J. Cell Physio. 113:353-358.
Dorns, R.W. and A. Helenius. 1988. Properties of a viral fusion protein, p. 385-398.
Jn: S. Ohki, D. Doyle, T.D. Flanagan, S.W. Hui, E. Mayhew (ed.), Molecular
mechanisms of membrane fusion. Plenum, New York.
Dorns, R.W., A. Helenius, and J. White. 1985. Membrane fusion activity of the
influenza virus hemagglutinin: the low pH-induced conformational change. J. Biol.
Chem. 260:2923-2931.
Dorns, R.W., J. White, F. Boulay, and A. Helenius. 1990. Influenza virus
hemagglutinin and membrane fusion. Jn: D. Hoekstra and J. Wilschut (ed.), Cellular
membrane fusion: fundamental mechanisms and applications of membrane fusion
techniques. Dekker, New York.
Doxsey, S.J., F.M. Bridsky, G.S. Blank, and A. Helenius. 1987. Inhibition of
endocytosis by anti-clathrin antibodies. Cell 50:453-463.
Edson, C.M. and D.A. Thorley-Lawson. 1981. EBV membrane antigens:
characterization, distribution and strain differences. J. Virol. 39:172-182.
Epstein, M.A. and Y. Barr. 1964. Cultivation in vitro of human lymphoblasts from
Burkitts malignant lymphoma. Lancet 1:252-253.
Epstein, M.A., G. Henle, B.G. Achong, and Y.M. Barr. 1965. Morphological and
biological studies on a virus in cultured lymphoblasts from Burkitt's lymphoma. J.
Exp. Med. 121:761-770.
Evans, A.S. 1984. New discoveries in infectious mononucleosis. Mod. Med. 1:18-24.


CHAPTER 7
SUMMARY AND CONCLUSIONS
Recapitulation
It Is clearly evident that in order to understand the biologic activity of EBV, one
must understand how this virus infects cells, and what components, such as surface
membrane proteins are important to the process. The studies described here have
focused on establishing an assay which measures membrane fusion of EBV with host
cells in order to analyze the early events of Epstein-Barr virus infection. The viral
envelope of herpesviruses are more complex than viruses such as influenza, vesicular
stomatitis, Sendai, and Semliki Forest virus and it is likely that several herpesvirus
glycoproteins are involved in virus entry. Epstein-Barr virus is unique in that it exhibits
specific tropism for two target cells types, lymphocytes and epithelial cells, which
provide discrete and distinct opportunities to study the complexities of virus entry.
Over recent years, the octadecylrhodamine (R18) fluorescence dequenching
assay has become widely used and accepted as a means of monitoring membrane
fusion. The quantitation of membrane fusion by lipid mixing between labeled and
non-labeled membranes resulting in dilution of R,a is an Indirect approach, but it is a
current technique of choice in membrane research.
The work in this dissertation has shown that the entry of EBV into lymphocytes
and epithelial cells occurs Independent of triggering by exposure to a low pH
environment. In confirmation of previous studies with electronmicroscopy, virus entry
147


60
Effect of Lysosomotropic Agents on Virus Fusion
Although fusion of EBV with lymphoblastoid cell lines occurs at the plasma
membrane and therefore presumably does not require exposure to low pH, virus has
been reported to fuse with normal B cells after endocytosis and certain
lysosomotropic agents capable of altering the pH of intracellular compartments have
been shown to Inhibit virus infectlvity (Nemerow and Cooper, 1984a). These agents
have been used In many virus systems to determine the mechanism by which virus
enters cells (Andersen and Nexo, 1983; Blumenthal et al., 1987; Cassel et al., 1984;
Gilbert et al., 1990; Gollins and Porterfield, 1986; Stein et al., 1987).
The effects of ammonium chloride (NH4CI), methylamine and chloroqulne on
three cell types, Raji, BAT, and fresh T-depleted human leukocytes were studied in the
fluorescence dequenching assay. Figures 3-5, 3-6 and 3-7 demonstrate that 20mM
NH4CI did not have any effect on fusion of virus with any of the three cell types. Three
concentrations of chloroqulne were tested with Raji cells and did not effect fusion
(Figure 3-8). In contrast, chloroqulne BAT cells and fresh T-depleted leukocytes
exhibited dose-dependent inhibition of fluorescence dequenching shown in Figures 3-
9 and 3-10. Chloroqulne inhibited fusion of virus with BAT cells by 34% at 1mM and
by 30% at 0.5mM. For peripheral B cells, the Inhibition was 60% at 1mM, 50% at
0.5mM, and 24% at 0.2mM. The third agent used, methylamine, which in addition to
elevating the endosomal pH also is an inhibitor of transglutaminase which has been
suggested to be Involved in receptor-mediated endocytosis (Davies et al, 1980), did
not Inhibit relief of self-quenching with any of the three cell types (Figure 3-11), thus
paralleling the data for the NH4CI-treated cells. In confirmation that these


125
indicate that virus fusion was not dependent on low pH in order to occur and this
parallels what has been found with the lymphoblastoid cell line Raji. Virus entered Raji
cells by fusion at the plasma membrane, in contrast with freshly isolated T-depleted
leukocytes, in which virus was endocytosed and virus fused from within an endocytic
vesicle. T-depleted leukocytes were not inhibited by methylamine or NH4CI, but virus
fusion with chloroquine treated cells was inhibited possibly due to reduced
endocytosis of these cells after chloroquine exposure. Sodium azide, which also
inhibits endocytosis because of its effects on oxidative phosphorylation of the cell,
was able to inhibit virus fusion with T-depleted leukocytes, but did not inhibit fusion of
virus with basal epithelial cells. Further confirmation that virus fusion occurs at the
plasma membrane of basal epithelial cells was obtained from fusion assays in which
fluorescence dequenching of virus labeled with R18 was compared with that of virus
labeled with the pH sensitive probe AF. The dequenching curves were
superimposable, indicating that virus is not exposed to a low pH environment before it
fuses with the epithelial cell.
Further work will be necessary to understand why the level of virus binding to
basal cells does not parallel the amount of receptor detectable with known anti-CR2
reagents.


9
unique DNA domains, U1-U5, which are separated by four regions of internal repeats,
IR1-IR4, and flanked on both ends with tandem repeats (Cameron et al., 1987;
Dambaugh and Kieff, 1982; Given et al., 1979). Latently infected cells usually contain
more than one copy of the complete EBV genome, which can be integrated, but is
most often found to exist as a covalently closed circular episome (Lindahl et al.,
1976). Episomes are replicated once per cell cycle by DNA polymerase early in S
phase (Adams, 1987; Hampar et al., 1974). Replication of the eplsomal DNA is
proposed to occur from a circular form in a manner similar to that of SV40 DNA
(Gussander and Adams, 1984).
At least two EBV types have been identified In human populations (Rowe et al.,
1989; Sculley et al., 1988). The two strains have significantly divergent EBNA 2
sequences. These were designated EBV type A and B, but are more appropriately
designated EBV-1 and EBV-2, so as to parallel the HSV-1 and HSV-2 nomenclature.
EBV-1 and EBV-2 are considerably more closely related to each other than are HSV-1
and HSV-2. Analysis of hosts shedding both EBV types in the oropharynx revealed
only type 1 In peripheral blood lymphocytes (Slxbey et al., 1989). Oral hairy
leucoplakia lesions consistently contain EBV DNA of the type 2 (Raab-Traub and
Slxbey, personal communication). The type 2 strain transforms B lymphocytes less
efficiently than the B95-8 type 1 strain and B cell transformants of the type 2 are more
difficult to maintain In culture (Rlckinson et al., 1987).
Membrane Proteins
The membrane antigen complex was initially described by surface fluorescence
of EBV-producing cells using human immune sera. The complex was further resolved
by analysis of Infected cell membranes into three major envelope glycoproteins of


48
peripheral blood for each experiment. In future experiments virus entry Into BAT cells
will be studied In parallel and compared to entry Into fresh normal B cells.
Discussion
The fluorescent amphiphile octadecyl rhodamine B chloride (R18) has been
used by several groups to study interactions of virus with biological membranes and
liposomes (Blumenthal et al., 1987; Gilbert et al., 1990; Hoekstra et al., 1984, 1985;
Lapidot et al., 1987; Morris et al., 1989; Slnangil et al., 1988; Stegmann et al., 1986;
Wunderli-Allenspach and Ott, 1990). The results from these papers indicate that
fluorescence dequenching reflects the occurrence of virus membrane fusion and when
discussing the results of the experiments In this dissertation, fluorescence
dequenchlng and membrane fusion will be considered interchangeable terms. The
behavior of Relabeled EBV, as demonstrated by relief of self-quenching of virus
bound to Raji cells, and the absence of fluorescence of virus bound to fixed Raji cells
or Molt 4 cells, provides strong corroborative support for this conclusion. Fixed cells
are resistant to virus membrane fusion (Gilbert et al., 1990; Lapidot et al., 1987) and
Molt 4 cells are reported to bind but not internalize virus (Menezes et al., 1977).
The R,e labeling procedure did not affect the binding specificity or the amount
of EBV that bound to lymphoblastoid cells. This Is in agreement with the effect of
labeling on attachment of Sendai virus (Hoekstra et al., 1985). Labeling of VSV with
R,e has been reported to enhance virus binding by twofold, possibly because the
probe Is positively charged and Increases the net charge of the virus (Blumenthal et
al., 1987). Labeled virus retained Its infectlvity as indicated by Its ability to Induce
Immunoglobulin synthesis by cultured T-depleted peripheral leukocytes.


30
membranes by modification of the method of Hoekstra et al. (1984). Three microliters
of stock probe were dried under nitrogen and solubilized in 39ul absolute ethanol and
15ul of this solution, containing 15nmole R18, were added to 250ul of concentrated
virus under vortexing. For mock-labeled virus the same volume of absolute ethanol
was added to the virus as used in the labeling procedure. Probe and virus were
Incubated at room temperature in the dark for 1 hour. Virus and nonincorporated R18
were separated by chromatography on Sephadex G-75 (Sigma Chemical Co., St.
Louis, Missouri). Labeled virus was aliquoted and stored at -70C.
Fluorescence Deauenchina Assay
An Aminco-Bowman spectrofluorometer (SLM Amino Bowman Instrument Co.,
Urbana, Illinois), equipped with a chart recorder was used for continuous monitoring
of fluorescence. The cuvette chamber was equipped with a magnetic stirrer and held
In a temperature controlled circulating water bath. For fluorescence measurements,
the Instrument was calibrated such that any residual fluorescence of membranes at
time zero was set at the zero level. At the end of the assay, Triton X-100 (Sigma)
(1.0% v/v, final concentration) was added to allow measurement of the maximum
obtainable fluorescence for the virus bound upon infinite dilution of the fluorophore.
Relabeled virus (volume not exceeding 100ul) were added to pellets of 2 x 106
cells and incubated for 1 hour at 4C on ice and in the dark. Cells were washed four
times with ice cold Dulbecco's saline and transferred to the microcuvette of the
spectrofluorometer. The principle of the assay relies upon the self-quenching
properties of R,8 when inserted into the virus membrane. When the two fusing
membranes come into molecular contact their lipid components must mix and this
mixing dilutes the R,8 allowing relief of the self-quenching. The relief of self-quenching


32
540 560 580 600 620
WAVELENGTH (nm)
Figure 2-2. Excitation ( -o ) and emission ( ) spectra of Ria-containing
virions relieved of self-quenching with Triton X-100 (infinite dilution). Relative
fluorescence expressed in arbitrary units (a.u.).


75
2000
1500
LU
O
o
T¡
LU
ce
o
3
1000
500
0
450 460 470 480 490 500 510 520
WAVELENGTH (nm)
CONTROL
CONTROL-M
METHYLAMINE
METHYLAMINE-M
Figure 3-16. Excitation spectra at pH 7.0 of methylamine treated and untreated
(control) BAT cells containing FITC-dextran before and after addition of monensin (M).
Measurements were taken at a fixed emission wavelength of 522nm and fluorescence
is expressed in arbitrary units (a.u.).


110
Table 5-3. Reactivity of epithelial cells with the monoclonal anti-CR2 antibody HB5.
gradient fraction
percentage positive cells
1 -media/30% Interface
53
2-30/40% interface
29 8
3-40/45% interface
21 6
4-45/100% interface
63
Table 5-4. Reactivity of epithelial cells with antl-CR2 monoclonal antibodies.
Monoclonal
antibody
% positive cells
parabasal
basal
HB5a
27
8
B2
66
45
OKB7
0
0
2G7
0
0
1F8
0
0
1C8
0
0
L02
0
0
aAntlbody binding was visualized with FITC-labeled anti-mouse antibody.


Figure
page
3-21. Effect of preincubation of BAT cells with ammonium chloride or RPMI
on relief of self-quenching of AF-labeled MCUV5 virus bound to BAT cells
at pH 7.2 82
4-1. Effect of preincubation of Raji cells with sodium azide (NaNJ or RPMI on
relief of self-quenching of Relabeled MCUV5 virus 89
4-2. Effect of preincubation of BAT cells with sodium azide (NaN3) or RPMI on
relief of self-quenching of Relabeled MCUV5 virus 90
4-3. Effect of preincubation of T-depleted leukocytes with sodium azide
(NaNJ, chlorpromazine, or RPMI on relief of self-quenching of Relabeled
MCUV5 virus 91
4-4. Effect of preincubation of BAT cells with chlorpromazine or RPMI on relief
of self-quenching of Relabeled MCUV5 virus 93
4-5. Effect of preincubation of Raji cells with chlorpromazine or RPMI on relief
of self-quenching of Relabeled MCUV5 virus 94
4-6. Effect of preincubation of Raji cells with leupeptin or RPMI on relief of
self-quenching of Relabeled MCUV5 virus 95
4-7. Effect of preincubation of BAT cells with leupeptin or RPMI on relief of
self-quenching of Relabeled MCUV5 virus 96
4-8. Effect of preincubation of T-depleted leukocytes with leupeptin or RPMI
on relief of self-quenching of Relabeled MCUV5 virus 97
5-1. Relief of self-quenching of Relabeled MCUV5 virus bound to parabasal
and basal epithelial cells 114
5-2. Fluorescence profile of HB5 antibody binding to basal epithelial cells. ... 116
5-3. Relief of self-quenching of Relabeled MCUV5 virus bound to unsorted
basal epithelial cells or basal epithelial cells from which HB5 (+) cells were
removed by cell sorting 117
5-4. Effect of preincubation of basal cells with chloroquine or RPMI on relief
of self-quenching of Relabeled MCUV5 virus bound to cells 119
5-5. Effect of preincubation of basal cells with methylamine, NH4CI, or RPMI
on relief of self-quenching of Relabeled MCUV5 virus bound to cells 120
5-6. Effect of preincubation of basal cells with sodium azide or RPMI on relief
of self-quenching of Relabeled MCUV5 virus bound to cells 121
IX


107
Table 5-1. Cell counts and viability of cells recovered from infant foreskin epidermis.
number of cells
viability8
4.95 X 106
89%
1.84 X 107
93%
2.17 X 107
94%
1.90 X 107
86%
1.14 X 107
96%
1.22 X 107
98%
1.29 X 107
95%
1.54 X 107
93%
9.10 X 106
96%
1.41 X 107
95%
1.26 X 107
87%
7.42 X 106
87%
1.02 X 107
99%
1.74 X 107
95%
8.6 X 106
95%
determined by trypan blue exclusion


13
that trigger fusion of the virus envelope with the vesicle membrane (Helenius et al.,
1980b; Kielian and Helenius, 1985; Marsh and Helenius, 1980; White and Helenius,
1980; White et al., 1980). The entry of vesicular stomatitis virus (VSV) has been
reported to resemble that of SFV (Clague et al., 1990; Matlln et al., 1982). Influenza
virus, an orthomyxovirus, is also taken Into cells by adsorptive endocytosis followed
by fusion of the viral membrane with the endosomal membrane (Matlin et al., 1981;
White et al., 1981; Yoshimura and Ohnlshi, 1984).
Fusion at the Plasma Membrane
Fusion directly at the plasma membrane Is utilized by paramyxoviruses
(Choppin and Compans, 1975a). The best studied member of this group is Sendai
virus, whose glycoproteins have been extensively characterized. The entry of Sendai
involves Initial attachment of virions to the cell surface and subsequent fusion between
the viral envelope and plasma membrane (Choppin and Scheid, 1980; White et al.,
1983). It is well established that binding is mediated by the HN protein and fusion Is
initiated by the F protein, both of which are spike-like projections on the surface of the
virus (Choppin and Scheid, 1980). Fusion activity has been shown to be critically
temperature dependent, optimally occurring at 37C, while fusion Is insignificant at
temperatures below 23C (Hoekstra et al., 1984).
The major entry mechanism for human immuno deficiency virus (HIV), a T-
lymphotropic retrovirus, is reported to be fusion with the plasma membrane at the cell
surface (Maddon et al., 1988; McClure et al., 1988; Stein et al., 1987). Previous data
from Maddon et al. (1986), proposed that HIV entry into T lymphoblastoid cells
occurred after endocytosis because the virus receptor, CD4, was internalized. The
key findings of Stein et al. showed that the entry of HIV was not low-pH-dependent,


40
0 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 2-5. Comparison of relief of self-quenching of R18-labeled P3HR1-CI13 virus
bound to Raji cells, fixed Raji cells, or Molt 4 cells. Increase in fluorescence is
expressed as a percent of the maximum release obtained with each cell line after
addition of Triton X-100 (Infinite dilution). The average maximum fluorescence for
each cell line was: Raji, 100a.u.; fixed Raji, 97 a.u.; Molt 4, 75 a.u.. Vertical lines
Indicate the standard deviation of the mean of experiments with the same batch of
labeled virus.


Henle, G. and W. Henle. 1979. The virus as a etiologic agent of infectious
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Henle, G., W. Henle, and V. Diehl. 1968. Relation of Burkitt tumor associated herpes-
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Hoffman, G.J., S.G. Lazarowitz, and S.D. Hayward. 1980. Monoclonal antibody
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Hsu, M., A. Scheid, and P. Choppin. 1982. Enhancement of membrane fusing activity
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Johnson, D.C. and M.W. Ligas. 1988. Herpes simplex viruses lacking glycoprotein D
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62
Figure 3-6. Effect of preincubation of BAT cells with 20mM ammonium chloride
(NH4CI) or RPMI on relief of self-quenching of Relabeled MCUV5 virus bound to
cells. Increase in fluorescence is expressed as a percent of the maximum release
obtained after addition of Triton-X-100 (infinite dilution).


143
O 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 6-5. Effect of preincubation with 100ug of normal mouse immunoglobulin or
antibody E1D1 on relief of self-quenching of Relabeled MCUV5 virus bound to basal
epithelial cells. Increase in fluorescence is expressed as a percent of the maximum
release after addition of Triton x-100 (infinite dilution).


20
(Nemerow et al., 1987; Tanner et al., 1987), and possibly also by attachment of gp220
(Wells et al., 1982).
Penetration of virus has been studied In normal B cells and lymphoblastoid cell
lines. Virus fuses with the membrane of the lymphoblastoid cell line Raji at the cell
surface and CR2 Is not internalized (Nemerow and Cooper, 1984a; Tedder et al.,
1986). In normal B cells, both receptor and virus are endocytosed into thin-walled
nonclathrin coated vesicles before fusion occurs (Ibid).
The virus envelope protein mediating the fusion event has not been
conclusively identified. The EBV envelope glycoprotein, gp85, which has been
recently mapped to the BXLF2 open reading frame of EBV DNA does, however have,
characteristics of a fusion protein (Oba and Hutt-Fletcher, 1988; Heineman et al.,
1988). Computer assisted analysis of the sequence Indicates that it is overall a
hydrophobic molecule with a potential N-terminal signal sequence and a C-terminal
anchor sequence. The sequence also includes a stretch of 16 extremely apolar amino
acids that could be a fusion sequence (Oba and Hutt-Fletcher, 1988). The gp85
glycoprotein has homology with the herpes simplex virus glycoprotein gH, and the
varicella-zoster virus gplll, which are involved in cell to cell fusion.
Measuring Fusion
The common procedures used to examine fusion of biological membranes,
such as microscopic or cytochemical techniques, are frequently difficult to quantitate
and have low sensitivity; extensive fusion activity may be required before it can be
detected. The use of radioisotopes to measure fusion does not permit continuous
monitoring of the fusion process and it is necessary to separate fused and nonfused


52
(HA) undergoes an irreversible conformational change upon exposure to mildly acidic
pH within acidic organelles after endocytosis. If virus is bound to the ceil surface and
the extracellular pH is briefly lowered to pH 5.0, fusion of the virus can occur at the
plasma membrane. If the virus alone is exposed to acidic pH the conformation
occurs prematurely and the virus is unable to fuse. Treatment of cells with
lysosomotropic agents inhibited influenza infectivity. The unprotonated form of these
lipophilic amines crosses cell membranes but the protonated form does this far less
efficiently. When the uncharged form enters acidic compartments it becomes
protonated, thereby raising the pH and inhibiting its own escape across the
membranes of the vacuoles.
Vesicular stomatitis virus (VSV) is another example of a virus that fuses from
within an acidic compartment after endocytosis (Dahlberg, 1974; Dales, 1973; Dickson
et al., 1982; Matlin et al., 1982). The fusion activity can be shown to take place on the
plasma membrane if cells with VSV attached to their surfaces are placed in pH 5.9
medium (Matlin et al., 1982; Blumenthal et al., 1987). Lysosomotropic agents were
also shown to inhibit fusion from with an endocytic vesicle, but had no effect on fusion
at the plasma membrane at pH 5.9 (Blumenthal et al., 1987).
The work described here sought to determine whether EBV fusion is a truly pH
dependent event and where fusion takes place in lymphoblastoid cell lines, freshly
isolated human B cells and recently transformed human B cells.
Materials and Methods
Membrane Fusion Assay
Virus that has been labeled with R18 at self-quenching concentration was added
to 2 X 106 cells and incubated for 1 hour on ice in the dark. Cells were washed four


100
personal communication) and it is reported that the endosomal membrane has a
different lipid composition from that of the plasmalemma. One possibility that will
require further examination is that the lipid composition of the normal B cell
plasmalemma (but not the endosme) is different from that of the Raji cell and is thus
not capable of fusing with the envelope of EBV.


80
Figure 3-19. Relief of self-quenching of AF-labeled MCUV5 virus bound to Raji cells
at pH 7.2 or pH 5.5. Virus was bound to cells at pH 7.2, cells were washed to remove
unbound virus and cells were resuspended In pH 7.2 or pH 5.5 Dulbecco's saline.
Increase in fluorescence is expressed as a percent of the maximum release obtained
after addition of Triton X-100 (infinite dilution).


45
Figure 2-7. Comparison of relief of self-quenching of Relabeled P3HR1-CI13 virus
bound to tonsil derived B cells pre and post monocyte depletion by adherance to
plastic. Increase in fluorescence is expressed as a percent of the maximum release
obtained after addition of Triton X-100 (infinite dilution).


71
WAVELENGTH (nm)
Figure 3-13. Excitation spectra at pH 7.4 of BAT cells containing FITC-dextran before
(-G-) and after (--) addition of monensin. Measurements were taken at a fixed
emission wavelength of 522nm and fluorescence is expressed in arbitrary units (a.u.).


96
O 4 8 12 16 20 24 28 32
TIME (MINUTES)
Figure 4-7. Effect of preincubation of BAT cells with leupeptin or RPMI on relief of
self-quenching of Relabeled MCUV5 virus. Increase in fluorescence is expressed as
a percent of the maximum release obtained after addition of Triton X-100 (infinite
dilution).


56
virus membranes by modification of the method used to incorporate R,a into virus
membranes. Briefly, 2ul of the stock AF was added to 250ul of the MCUV5 strain of
EBV that had been collected from culture supernatant and concentrated 250-fold.
Virus and AF were vortexed immediately after addition of the fluorescent probe and
incubated at room temperature in the dark for 1 hour. Virus and non-incorporated AF
were separated by chromatography on Sephadex G-75 (Sigma Chemical Co., St.
Louis, Missouri) recovering the AF-labeled virus in the void volume. Labeled virus was
aliquoted and stored at -70C.
Fluorescence measurements were made at an excitation wavelength of 496nm
and an emission wavelength of 522nm using a SLM SPF500C spectrofluorometer
equipped with a thermostatically-controlled cuvette chamber and magnetic stirrer
(SLM Aminco, Urbana, Illinois).
Results
Effect of Lowering Extracellular oH on Fusion
Previous studies with viruses that are known to be dependent on the low pH of
the endosme in order to fuse have shown that they are also able to fuse at the
plasma membrane if the pH of the extracellular medium if briefly lowered (Blumenthal
et al., 1987; Marsh et al., 1983a; White et al., 1980). Experiments were therefore done
to see if the rate or extent of fusion of EBV would be affected if the pH of the
extracellular media was decreased in order to drive low pH-dependent fusion to occur
at the plasma membrane. The results In Figures 3-2, 3-3, and 3-4 indicate that
altering the extracellular pH from 7.4 to 5.5 did not effect virus fusion with Raji, BAT, or
fresh T-depleted leukocytes.


99
with the agent were inhibited in their ability to permit virus membrane fusion. The
phenothiazine drug, chlorpromazine, has also been reported to inhibit endocytosis of
some ligands although by a different mechanism than NaN3 (Salisbury et al., 1980,
1981; Cheung et al., 1983). Chlorpromazine binds to calmodulin, which is a calcium
binding protein that has multifaceted involvement in regulation of cellular processes
(Cheung, 1980). Chlorpromazine exerts its activity by blocking the stimulation of
Ca2*-dependent phosphodiesterase by calmodulin. This drug inhibited fusion of virus
with BAT cells by 40% and fusion of virus with T-depleted leukocytes was decreased
from 48.8% to 15.6%. Raji cells were marginally effected by treatment with this drug.
Chlorpromazine was also shown to block virus infectivity of human B cells as
measured by outgrowth of transformed cell colonies or by stimulation of DNA
synthesis (Nemerow and Cooper, 1984b).
The second possibility was tested with the protease inhibitor leupeptin which
has potent activity for inhibiting cathepsin B along with possessing protease inhibiting
activities for many other enzymes. Leupeptin failed to inhibit virus fusion with any cell
types.
These experiments strongly suggest that inhibition of endocytosis in BAT cells
and normal B cells compromises their ability to fuse with EBV. It is not clear why virus
should be able to fuse efficiently at the plasmalemma of Raji cells and apparently not
at the plasmalemma of normal B cells but this perhaps reflects differences in the
composition of the membranes of these different cells. Fusion of viruses with any
membrane is dependent on many factors including the lipid composition of the
membrane (Bramhall and Wisnieski, 1981). Altering the lipid composition of the Raji
cell membrane has been shown to inhibit virus fusion (Patel, Hutt-Fletcher, and Crews;


63
70
60
ID
O
z
LLI
O 30
cc
§ 20
Li-
10
0
Figure 3-7. Effect of preincubation of T-depieted leukocytes with 20mM ammonium
chloride (NH4CI) or RPMI on relief of self-quenching of R18-labeled MCUV5 virus
bound to cells. Increase in fluorescence is expressed as a percent of the maximum
release obtained after addition of Triton-X-100 (Infinite dilution).
0 4 8 12 16 20 24 28
TIME (MINUTES)


46
O 4 8 12 16 20 24 28 32 36
TIME (MINUTES)
Figure 2-8. Relief of self-quenching of Relabeled MCUV5 virus bound to fresh T-
depleted peripheral leukocytes expressed as a percent of the maximum release
obtained after addition of Triton X-100 (infinite dilution).


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127
Materials and Methods
Cells
The lymphoblastoid cell lines used, Rajl and BAT, were grown at 37C and
diluted at least biweekly in RPMI 1640 (Sigma Chemical Co., St. Louis, Missouri)
supplemented with heat-inactivated fetal calf serum, 100 IU of penicillin and 100ug of
streptomycin per ml. Both cell lines are latently infected with EBV and express the
virus receptor CR2.
Fresh human peripheral leukocytes were obtained by flotation on LSM followed
by depletion of T lymphocytes by rosettlng with sheep erythrocytes. These cells were
used in assays directly after Isolation.
Human epithelial cells were isolated from foreskin tissue by a sequential
enzymatic digestion with dlspase (Stenn et al., 1989) to separate the epidermis from
the dermis, followed by digestion of the epidermal sheets with trypsin. The
suspension of epidermal cells were separated further by centrifugation on a 30, 40,
45, 100% discontinuous Percoll gradient. Cells from the 40/45% Interface and the
45/100% interlace were collected and used as two separate population of cells,
respectively, parabasal and basal cells.
Monoclonal Antibodies
Monoclonal antibodies were purified from hybridoma culture supernatants by
chromatography on protein A-Sepharose. Five anti-viral antibodies were studied, two
of the antibodies recognize gp85; F-2-1 (Strnad et al., 1982), of the lgG2a subclass,
and E1D1 (Balachandran et al., 1987), of the lgG1 subclass. E8D2 (Balachandran et
al., 1986) Is an lgG2a antibody that recognizes the EBV-Induced early membrane


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Relationship between the Epstein-Barr virus genome and nasopharyngeal carcinoma
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components on the surface of leukemia cells and of lymphocytes transformed by
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sequence and expression of the B95-8 Epstein-Barr virus genome. Nature (London)
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Balachandran, N., D.E. Oba, and L.M. Hutt-Fletcher. 1987. Antigenic cross-reactions
amongst HSV type 1 and 2, EBV, and CMV. J. Virol. §1:1125-1135.
Balachandran, N., J. Plttari, and L.M. Hutt-Fletcher. 1986. Detection by monoclonal
antibody of an early membrane protein Induced by EBV. J. Virol. 60:369-375.
Beisel, C., J. Tanner, T. Matsuo, D. Thorley-Lawson, F. Kexdeg, and E. Kleff. 1985.
Two major outer envelope glycoproteins of Epstein-Barr virus are encoded by the
same gene. J. Virol. 54:665-674.
Berg, T. and H. Tolleshaug. 1980. the effects of ammonium ions and chloroquine on
uptake and degradation of 'l-labeled aslalo-fetuin in isolated rat hepatocytes.
Blochem Pharmacol. 29:917-925.
Blumenthal, R., A. Ball-Puri, A. Walker, D. Covell, and O. Eldelman. 1987. pH-
dependent fusion of VSV with vero cells. J. Biol. Chem. 262:13614-13619.
Boggs, W.M., C.S. Hahn, E.G. Strauss, J.H. Strauss, and D.E. Griffin. 1989. Low pH-
dependent Sindbis virus-induced fusion of BHK cells: Differences between strains
correlate with amino acid changes in the E1 glycoprotein. Virology 169:485-488.
150


64
O 4 8 12 16 20 24 28 32
A TIME (MINUTES)
Figure 3-8. Effect of preincubation of Raji cells with chloroquine or RPMI on relief of
self-quenching of Relabeled MCUV5 virus bound to cells. Panel A, 1mM; panel B,
0.5mM; panel C, 0.2mM. Increase In fluorescence Is expressed as a percent of the
maximum release obtained after addition of Triton-X-100 (infinite dilution).


10
300-350 kda, 200-220 kDa, and 85 kDa (Edson and Thorley-Lawson; Thorley-Lawson
and Edson, 1979). Three additional membrane associated proteins, p105, gp78/55,
and the product of the BDLF3 open reading frame, have also been studied. The p105
protein is not glycosylated and differs from the other membrane proteins in that its
synthesis is not influenced by the viral DNA Inhibitor phosphonoacetic acid
(Balachandran et al., 1986). Glycoprotein gp78/55 is the product of the BILF2 open
reading frame (Mackett et al., 1990). Antibodies to a bacterially expressed BDLF3
protein reacted with virus and with the plasma membrane of virus infected cells.
Additional membrane proteins are likely to exist since there are many unassigned
open reading frames which have characteristics of those encoding membrane
proteins.
Glycoproteins gp300-350 and gp200-220 are present in large amounts in the
virus envelope and have been extensively characterized. Glycoprotein gp350 and
gp220 are encoded by the same open reading frame from which an Intron is
removed, without change In reading frame to produce gp220 (Belsel et al., 1985;
Hummel et al., 1984). Monoclonal antibodies that recognize gp350/220 are capable
of Inhibiting virus binding (Nemerow et al., 1987). Binding of EBV to CR2 is mediated
by attachment of gp350 (Nemerow et al., 1987; Tanner et al., 1987) and possibly also
by attachment of gp220 (Wells et al., 1982). A common epitope in gp350 and gp220
has been identified as a primary region responsible for virus binding to B lymphocytes
by attachment to CR2 (Nemerow et al., 1989).
Glycoprotein gp85 Is also present In the envelope but In less abundant
amounts than gp350/220. Glycoprotein gp85 has been recently mapped to the
BXLF2 open reading frame in two independent studies (Heineman et al., 1988; Oba


90
Figure 4-2. Effect of preincubation of BAT cells with sodium azide (NaN3) or RPMI on
relief of self-quenching of Relabeled MCUV5 virus. Increase in fluorescence Is
expressed as a percent of the maximum release obtained after addition of Triton X-
100 (infinite dilution).


3
Infectious Mononucleosis
Epstein-Barr virus (EBV) persists in those individuals it infects and induces
permanent seroconversion. The virus is transmitted horizontally and primary infection
usually takes place in childhood without apparent disease (Henle and Henle, 1979). if
the primary infection is delayed until adolescence or young adulthood, which happens
at a higher incidence in developed countries, infection leads to infectious
mononucleosis (I.M.) in about 50% of cases (Niederman et al., 1970). The first
suggestion that EBV was the cause of I.M. came when a laboratory technician (in the
laboratory of Drs. W. and G. Henle) who had previously lacked EBV antibodies,
seroconverted in the course of I.M.. Her circulating lymphocytes failed to grow in vitro
prior to the illness, but gave rise to permanent cultures when collected during the
acute phase or during early convalescence (Henle et al., 1968). Following this
discovery, a prospective study was conducted at Yale where it was found that all pre-
I.M. sera collected lacked antibodies to EBV, while the corresponding acute and
convalescence phase sera contained EBV antibodies (Niederman et al., 1968).
The initial step in pathogenesis of any primary infection with EBV, whether
symptomatic or not, is entry of the virus into the oropharynx and subsequent
replication at that site. The clinical manifestations of EBV-induced I.M. are thought to
be caused by a rapid polyclonal T and B cell proliferation. Primary replication of virus
occurs in pharyngeal epithelium (Sixbey et al., 1983, 1984) from which circulating B
cells are infected and transformed allowing their rapid proliferation (Rickinson et al.,
1987; Svedmyr et al., 1984). The symptoms that the I.M. patient experiences are
thought to result from the conflict in the immune system as an aggressive T cell
response is mounted in order to keep the B cell proliferation in control. Since the


27
collected. Alternatively, cells were incubated with iron filings in a 15 ml polypropylene
tube at 37C on a rotator for 1 hour and the Iron containing cells were removed with a
magnet. The remaining cells were layered on lymphocyte separation medium for
additional removal of iron containing cells. The extent of monocyte depletion was
determined by cell counts and nonspecific esterase staining of cells prior to and after
depletion.
Nonspecific Esterase Stain
Nonspecific esterase is contained in the granules of monocytes and was
stained with a solution of Sorensen's buffer, hexazotised pararosanillne and alpha-
naphthyl butyrate (Li et al., 1973). Sorensens buffer consists of 0.2M Na^HPO,, and
0.2M NaH2P04 at pH 6.3. Hexazotised pararosanlline was prepared by mixing equal
volumes of pararosaniline HCL and 4% sodium nitrite. One gram of alpha-naphthyl
butyrate was dissolved in 50ml of dimethyl formamlde and stored at -20C, protected
from the light. To prepare the primary stain, 0.25ml of hexazotised pararosanillne and
3.0ml of alpha-napthyl butyrate were added to 44.5ml of Sorensen's buffer and the
solution was filtered through a Whatman #1 filter and 5 X 106 fixed cells were stained
for 30 to 45 minutes at 37C. The slides were rinsed with deionized water and
counterstained in methyl green for 15 seconds. The slides were rinsed again in
deionized water and air dried. Monocytes were identified by the brown coloration of
their cytoplasm.
Initiation of an Immortalized B Cell Line
T-depleted leukocytes were isolated from peripheral blood as previously
described. The cells were plated In a 24-well tissue culture plate at a concentration of
2 X 106 per milliliter and 10Oul of virus were added to each well using a twofold


31
of the R,u was continuously monitored at excitation wavelength of 560nm and
emission wavelength of 585nm and documented with a chart recorder.
Immunoglobulin Assay
Immunoglobulin in culture supernatants was measured by a double sandwich
micro-enzyme-linked immunosorbent assay (Voller et al., 1976) using rabbit anti
human immunoglobulin as the immobilized antigen. Antibody in the culture
supernatants was allowed to bind to the immobilized antigen followed by horseradish
peroxidase-conjugated rabbit anti-human Ig (Cooper Biomedical Inc., Malvern,
Pennsylvania). The substrate, hydrogen peroxide with 5-amino salicylic acid was
degraded by the enzyme and the colorimetric change was measured at 492 nm.
Results
Fluorescence Properties of R,0 Labeled Virus
Figure 2-2 shows the excitation and emission spectra of octadecyl rhodamine
B chloride (R1S) incorporated into virus membranes when relieved of self-quenching
with Triton X-100 (1% v/v final concentration). The excitation spectrum exhibits a
maximum peak at 560 nm. The peak emission wavelength displayed a maximum at
585 nm. The emission wavelength has been shown to be dependent upon the
environment of the probe (Hoekstra et al., 1984) with variance between 569 and 590 in
different solvents. Figure 2-3 demonstrates the stability of the quenching of the
fluorophore within the virus membrane and subsequent relief of quenching upon
addition of Triton X-100 (1% v/v).