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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Herpesvirus 4, Human -- pathogenicity   ( mesh )
B-Lymphocytes -- virology   ( mesh )
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Epithelium -- virology   ( mesh )
Membrane Fusion   ( mesh )
Viral Fusion Proteins   ( mesh )
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Thesis:
Thesis (Ph.D.)--University of Florida, 1991.
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Bibliography: leaves 150-168.
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by Nancimae Miller.
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Vita.

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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




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