Download Induction of Lytic Epstein-Barr Virus (EBV) Infection in EBV

[CANCER RESEARCH 59, 1485–1491, April 1, 1999]
Induction of Lytic Epstein-Barr Virus (EBV) Infection in EBV-associated
Malignancies Using Adenovirus Vectors in Vitro and in Vivo1
Eva-Maria Westphal, Amy Mauser, Jennifer Swenson, Michelle G. Davis, Christine L. Talarico, and
Shannon C. Kenney2
UNC Lineberger Comprehensive Cancer Center [E-M. W., A. M., J. S., S. C. K.], and Department of Medicine [S. C. K.], University of North Carolina at Chapel Hill, Chapel
Hill, North Carolina 27599, and Department of Virology, Glaxo Wellcome Inc., Research Triangle Park, North Carolina 27709 [M. G. D., C. L. T.]
ABSTRACT
The consistent presence of EBV genomes in certain tumor types (in particular, AIDS-related central nervous system lymphomas and nasopharyngeal carcinomas) may allow novel, EBV-based targeting strategies. Tumors
contain the latent (transforming) form of EBV infection. However, expression
of either of the EBV immediate-early proteins, BZLF1 and BRLF1, is sufficient to induce lytic EBV infection, resulting in death of the host cell. We have
constructed replication-deficient adenovirus vectors expressing the BZLF1 or
BRLF1 immediate-early genes and examined their utility for killing latently
infected lymphoma cells in vitro and in vivo. We show that both the BZLF1
and BRLF1 vectors efficiently induce lytic EBV infection in Jijoye cells (an
EBV-positive Burkitt lymphoma cell line). Furthermore, lytic EBV infection
converts the antiviral drug, ganciclovir (GCV), into a toxic (phosphorylated)
form, which inhibits cellular as well as viral DNA polymerase. When Jijoye
cells are infected with the BZLF1 or BRLF1 adenovirus vectors in the
presence of GCV, viral reactivation is induced, but virus replication is inhibited (thus preventing the release of infectious EBV particles); yet cells are still
efficiently killed. Finally, we demonstrate that the BZLF1 and BRLF1 adenovirus vectors induce lytic EBV infection when they are directly inoculated
into Jijoye cell tumors grown in severe combined immunodeficiency mice.
These results suggest that induction of lytic EBV infection in tumors, in
combination with GCV, may be an effective strategy for treating EBVassociated malignancies.
INTRODUCTION
EBV is a human herpesvirus that infects .90% of humans (1). It
causes infectious mononucleosis and persists throughout life after the
primary infection, being contained by the immune system of the host (1).
The EBV genome has been found in a number of different tumor types,
including Hodgkin’s disease, B-cell lymphomas, nasopharyngeal carcinoma, gastric carcinomas, and leiomyosarcomas (1–9). EBV-associated
B-cell lymphomas are a particular problem in immunocompromised
hosts. In AIDS patients, essentially all primary CNS3 lymphomas contain
the EBV genome (6), as do many of the peripheral lymphomas (8, 9).
Although CNS lymphomas rarely metastasize, they have a very poor
prognosis and often respond minimally to conventional therapy.
Given the frequent presence of the EBV genome in certain tumor
types, we are developing therapeutic strategies that specifically target
EBV-containing cells for destruction. One such strategy is to convert
EBV infection in tumor cells from a latent into a cytolytic form. In
infected tumor cells, EBV normally exists in one of three latency
types (1). EBV-transforming proteins are expressed during latency
types II and III, whereas induction of lytic infection results in host cell
killing (1, 10, 11). The switch from latent to lytic EBV infection can
be induced by expression of either of the EBV IE proteins, BZLF1 and
BRLF1. BZLF1 and BRLF1 are transcriptional activators that activate
expression of EBV early genes (12–18). BZLF1 expression is sufficient to trigger induction of lytic infection in both B cells and
epithelial cells (12–14, 19 –22), and BRLF1 expression can induce
lytic EBV infection efficiently in epithelial cells (22). Thus, delivery
of either the BZLF1 or BRLF1 proteins into latently EBV-infected
tumors using gene delivery methods would be expected to result in
specific killing of tumor cells.
A potential problem with this approach is that lytic infection of
tumor cells may result in the release of infectious viral particles.
However, it is possible that the combination of lytic EBV infection
with the antiviral drug, GCV, could abort viral replication and prevent
release of infectious EBV, while still allowing EBV-specific killing.
Phosphorylation of GCV into its triphosphate form is initiated by the
early (or lytic cycle) proteins of several herpesviruses, including the
HSV-TK protein and the CMV UL97 protein (23, 24). Phosphorylated
GCV is cytotoxic, and it inhibits not only virally encoded DNA
polymerases but also the cellular DNA polymerase, resulting in death
of the host cell (25–27). Delivery of the HSV-TK gene into tumor
cells, in combination with GCV, is, thus, being investigated as a
potential method for treating cancer (25–27). Although the EBV
genome contains homologues to both the HSV-TK and CMV UL97
genes, it is uncertain if either (or both) of these lytic EBV proteins can
phosphorylate GCV (28, 29). However, GCV is effectively phosphorylated in lytically EBV-infected cells (30) and lytic EBV replication
is inhibited by GCV (31). Thus, we hypothesized that the combination
of lytic EBV infection and GCV would result in cellular death, while
preventing the release of infectious EBV.
In this study, we have used adenovirus vectors expressing the BZLF1
or BRLF1 proteins to induce lytic EBV infection in a Burkitt lymphoma
line in vitro as well as in vivo. We demonstrate that GCV is phosphorylated into its active form in lytically EBV-infected Burkitt lymphoma
cells. The BZLF1 and BRLF1 adenovirus vectors can, thus, efficiently
kill EBV-positive Burkitt cells, without concomitant release of infectious
virus, when combined with GCV. The induction of lytic EBV infection,
combined with GCV administration, may be a promising therapeutic
approach for specifically killing EBV-positive tumors.
MATERIALS AND METHODS
Human Cell Lines. Jijoye is an EBV-positive Burkitt’s lymphoma cell line.
The CB95 cell line is an EBV-transformed lymphoblastoid B-cell line. CB95-TK
cells were stably transduced with a retroviral vector expressing the HSV-1 TK
gene, as described previously (32). The EBV-positive epithelial cell line, NPC-KT,
is derived from the fusion of a human adenoidal epithelial cell line and a primary
Received 6/29/98; accepted 2/3/99.
EBV-positive nasopharyngeal carcinoma (33). Primary human fibroblasts were
The costs of publication of this article were defrayed in part by the payment of page
established from neonatal human foreskin. Saos-2 is a human osteosarcoma line.
charges. This article must therefore be hereby marked advertisement in accordance with
B-cell lines were maintained in RPMI 1640 with 10% FBS and penicillin/
18 U.S.C. Section 1734 solely to indicate this fact.
1
streptomycin, and NPC-KT cells were maintained in DMEM with 10% FBS and
This work was supported by NIH Grants R01 CA 66519 and P01-CA19014.
2
To whom requests for reprints should be addressed. Phone: (919) 929-8356; Fax:
penicillin/streptomycin. Saos-2 cells were grown in McCoy’s 5A medium with
(919) 966-3015; E-mail: [email protected].
15% FBS and penicillin/streptomycin. Primary human fibroblasts were grown in
3
The abbreviations used are: CNS, central nervous system; IE, immediate-early; GCV,
MEM with 10% FBS and nonessential amino acids.
ganciclovir; HSV, herpes simplex virus; TK, thymidine kinase; CMV, cytomegalovirus;
Construction of Adenovirus Vectors. The EBV IE genes BZLF1 and
FBS, fetal bovine serum; FACS, fluorescence-activated cell sorter; MOI, multiplicity of
BRLF1 and the control lacZ gene were initially cloned under the control of the
infection; SCID, severe combined immunodeficient; AZT, 39-azido-39-deoxythymidine.
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INDUCTION OF LYTIC EBV IN TUMORS
Fig. 1. Construction of BZLF1 and BRLF1 adenovirus vectors. The BZLF1 and BRLF1 sequences (both under the control of the CMV IE promoter) were inserted into an E1- and
E3-deleted (replication-deficient) type 5 adenovirus vector via cre-loxP-mediated homologous recombination (34). The lacZ adenovirus vector (used as a control) was constructed
identically and contained the bacterial lacZ gene under the control of the CMV IE promoter. The positions of the adenovirus vector inverted terminal repeat (ITR) and packaging signal
(c) are indicated.
CMV IE promoter into a shuttle vector containing a loxP site, the left
adenovirus terminal repeat, and a packaging signal (Fig. 1 and Ref. 34). The
BZLF1 shuttle vector contained the BZLF1 cDNA (a gift from Paul Farrell,
Ludwig Institute for Cancer Research, St. Mary’s Hospital, London, United
Kingdom), and the BRLF1 shuttle vector contains the genomic EBV BglIIHindIII fragment from position 105,413 to 103,080 (35). The shuttle vectors
were then inserted through cre-loxP-mediated recombination into an adenovirus type 5 derivative, which is missing the E1 and E3 genes and packaging
sequences. This parent adenovirus is a derivative of Ad d1309 (Stephen Hardy,
Cell Genesis; Ref. 34) and can only be packaged after recombination with the
shuttle vector. Virus stock was titered on the 293 cell line and purified by
double cesium chloride gradient, followed by dialysis.
Adenovirus Infection. Monolayer cells were plated 1 day prior to adenovirus infection and then overlaid with the appropriate medium containing 2%
FBS and adenovirus virions. B cells were concentrated at 1 3 106 per 100 ml
in RPMI 1640 with 2% FBS before adding adenovirus virions. After 3 h of
incubation at 37°C, fresh medium containing 10% FBS was added to the cells.
b-Galactosidase Staining. For in vitro staining, cells were fixed in 0.5%
glutaraldehyde and then stained for b-galactosidase expression, as suggested
by the manufacturer (Promega). Tumor tissue pieces were fixed in 2%
paraformaldehyde-0.2% glutaraldehyde prior to b-galactosidase staining.
Immunoblot Assays. Twenty to 50 mg of total cellular protein were loaded
onto a 10% denaturing polyacrylamide gel, and immunoblot analyses were
then performed as described previously (36). A 1:40 dilution of the monoclonal
antibody 9240 (Capricorn, Scarborough, ME) was used to detect induction of
the EBV lytic early protein, BMRF1. Proteins were visualized using a chemiluminescence kit from Amersham.
EBV Termini Assays. DNA was isolated from Jijoye cells 3 days after
adenovirus infection, cut with BamHI, run on a 0.8% agarose gel, and blotted
onto a Hybond nylon membrane. The filter was hybridized with a 32P-labeled
riboprobe spanning the EBV termini (1.9-kb XhoI fragment; a gift from Nancy
Raab-Traub; Ref. 37).
FACS Analysis. Cells were fixed in 60% ice-cold acetone and washed in
PBS with 1% BSA. The following dilutions of antibodies in PBS with 1% BSA
were used: monoclonal antibody 9240 (Capricorn) to detect BMRF1, 1:25;
monoclonal antibody BZ.1 (Dako, Carpinteria, CA) to detect BZLF1, 1:20;
monoclonal antibody 8C12 (Argene, Inc., North Massapequa, NY) to detect
BRLF1, 1:25; and monoclonal antibody B0172 (Virotech International, Inc.
Rockville, MD) to detect viral capsid antigen, 1:20. FITC-conjugated antimouse IgG (GAM-FITC, 1:100; Sigma Chemical Co., St. Louis, MO) was
used as a secondary antibody. FACS was performed on a Becton-Dickinson
apparatus. Annexin V binding was detected using the ApoDETECT Annexin
V-FITC kit (Zymed, South San Francisco, CA) following the manufacturer’s
instructions.
Intracellular Nucleotide (GCV) Anabolism Assays. GCV was synthesized at Wellcome Research Laboratories (Research Triangle Park, NC). A
total of 2 3 105 Jijoye or NPC-KT cells were infected with a MOI of 50 of the
BZLF1 or lacZ adenovirus vectors or were mock-infected. The cells were
pulse-labeled with 27 mM [8-3H]GCV (Moravek Biochemicals, Brea, CA) at
various time points postinfection in the appropriate medium. After incubation
at 37°C for either 6 or 18 h, the pulse media were removed, and the cells were
rinsed with ice-cold PBS. Following extraction with ice-cold 0.5 M perchloric
acid, the cellular extracts were clarified by centrifugation (770 3 g, 10 min).
GCV anabolites were quantitated by passage through a cation exchange
column, as described previously (38). Column effluent was counted using a
Packard 2500TR liquid scintillation counter.
Ganciclovir Viability Assays. Cells were infected with adenovirus particles as described above. After 3 h of virus incubation, 10 mM ganciclovir was
added to the appropriate medium containing 10% FBS. Cells were maintained
under optimal growth conditions (logarithmic phase) with and without the
drug. The number of live cells was determined by trypan blue exclusion.
Animal Experiments. Four- to 5-week-old SCID mice (NIH) were injected s.c. with 5 3 107 Jijoye cells into both flanks. After 10 –14 days, tumors
developed and were injected with 2 3 109 adenovirus particles containing
BZLF1, BRLF1, or lacZ. Two days later, the mice were sacrificed, and the
Fig. 2. The BZLF1 and BRLF1 adenovirus vectors disrupt EBV latency. A, NPC-KT
cells (left) or Jijoye cells (right) were infected with the BZLF1-, BRLF1-, or lacZexpressing adenovirus vectors (MOI of 50). Two days after infection, cellular extracts
were examined for expression of the early lytic EBV protein, BMRF1, using immunoblot
analysis. B, Jijoye cells were infected with the BZLF1, BRLF1, or lacZ adenovirus vectors
(MOI of 50 and 150) and examined for expression of IE (BZLF1 or BRLF1), early
(BMRF1), and late (VCA) lytic EBV proteins using FACS analysis.
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INDUCTION OF LYTIC EBV IN TUMORS
Fig. 3. Lytic EBV infection increases phosphorylation of
GCV. Epithelial NPC-KT and Jijoye lymphoma cells were
either mock-infected or infected with BZLF1- or lacZ-expressing adenovirus vectors. Cells were incubated with
[8-3H]GCV, and protein extracts were prepared. GCV anabolites were quantitated as described previously (38). The
amount of phosphorylated GCV (pmol of total phosphorylates per 1 million cells) for each condition is shown at days
2– 4 after infection with the adenovirus vectors in NPC-KT
cells and at day 2 after adenovirus infection in Jijoye cells.
GCV anabolites were also quantitated in latently infected,
EBV-positive lymphoblastoid cells (CB95) transduced with
either a retroviral vector expressing the HSV-1-TK gene
(HSV-TK) or transduced with a control retroviral vector
(control).
tumors were removed. Tumor tissue was frozen in methanol/dry ice and used
for protein extraction and immunoblot analysis or b-galactosidase staining.
RESULTS
The BZLF1 and BRLF1 Adenovirus Vectors Disrupt Latency
in EBV-positive B Cells and Epithelial Cells. In transfection experiments, we have previously shown that BZLF1 disrupts latency in
EBV-positive epithelial cells and B cells, whereas BRLF1 disrupts
latency in an epithelial cell-specific manner (22). To determine the
effect of the BZLF1 and BRLF1 adenovirus vectors, we infected the
nasopharyngeal carcinoma cell line NPC-KT and the Burkitt lymphoma line Jijoye with the adeno-BZLF1, adeno-BRLF1, and adenolacZ vectors (using a MOI of 50) and examined the expression of the
early lytic cycle EBV protein BMRF1 2 days later. In the absence of
adenovirus infection, both NPC-KT cells and Jijoye cells are primarily
latently EBV infected and, thus, do not express BMRF1. As shown in
Fig. 2A, infection with the lacZ adenovirus vector did not activate
lytic EBV infection in either cell type. In contrast, infection with
either the BZLF1 or BRLF1 adenovirus vectors induced BMRF1
expression in both NPC-KT cells and Jijoye cells. Thus, in the context
of an adenovirus vector, BRLF1 expression (like BZLF1) disrupts
EBV latency in both B cells and epithelial cells.
Lytic EBV infection follows a set pattern of gene expression: IE
genes are expressed first, then early viral genes are expressed, and
finally, late genes are expressed. To determine whether infection with
the BZLF1 and BRLF1 adenoviruses results in fully lytic infection,
we performed FACS analysis to quantitate early and late lytic EBV
gene expression. As shown in Fig. 2B, when Jijoye cells were infected
with the BZLF1 and BRLF1 adenovirus vectors at an MOI of 50,
essentially all of the cells expressing the BZLF1 or BRLF1 IE proteins
also expressed the early BMRF1 protein. In addition, within 2 days
after infection, a late EBV protein, viral capsid antigen, was also
clearly induced by the BZLF1 adenovirus vector using an MOI of 50
and by both the BZLF1 and BRLF1 adenovirus vectors using an MOI
of 150. Thus, infection of Jijoye cells with either the BZLF1 or
BRLF1 adenovirus vectors results in fully lytic infection and, hence,
would be expected to result in release of infectious EBV.
Lytic EBV Replication Induces GCV Phosphorylation. We next
investigated whether induction of lytic EBV replication in Jijoye and
NPC-KT cells using the BZLF1 and BRLF1 adenovirus vectors results in
phosphorylation of GCV. NPC-KT and Jijoye cells were either mockinfected or infected with the BZLF1 or lacZ adenovirus vectors, and 2
days later, they were incubated with 3H-labeled GCV. The amount of
mono-, di-, and triphosphorylated GCV was determined as described in
“Materials and Methods” and compared with levels obtained in a lymphoblastoid cell line (CB95-TK) stably transduced with the HSV-1 TK
gene. As shown in Fig. 3, when ;50% of NPC-KT and Jijoye cells were
infected by the BZLF1 adenovirus vector, there was a 4–5-fold increase
in GCV phosphorylation in comparison to lacZ-infected cells (correlating
to an 8–10-fold increase if 100% of cells were lytically infected). Furthermore, the amount of GCV phosphorylation induced by lytic EBV
infection was comparable to that observed in lymphoblastoid cells stably
transduced with the HSV-1 TK gene, which we have previously shown to
Fig. 4. GCV inhibits lytic EBV replication. Jijoye cells were infected with either the
BZLF1 and lacZ adenovirus vectors (MOI of 50; A) or the BRLF1 and lacZ adenovirus
vectors (MOI of 150; B) in the presence or absence of 10 mM GCV or 100 mM acyclovir
(ACV). Three days later, DNA was isolated, cut with BamHI, and hybridized to a probe
spanning the EBV termini (37). The episomal (fused termini) form of the EBV genome
is seen in latent infection, whereas the linear form of the genome (replication intermediates) is produced by lytic EBV replication.
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INDUCTION OF LYTIC EBV IN TUMORS
Fig. 5. The BZLF1 and BRLF1 adenovirus vectors induce killing of Jijoye cells in the
presence or absence of GCV. Jijoye cells were infected with the BZLF1-, BRLF1-, or
lacZ-expressing adenovirus vectors (MOI of 50) with or without concomitant administration of 10 mM ganciclovir. Cell viability was determined by trypan blue exclusion 9
days after infection. Results are normalized such that growth of Jijoye cells infected with
the lacZ vector (in the absence of GCV) is set at 100%. Similar results were obtained in
several independent experiments.
be extremely susceptible to GCV-induced cell killing (32). Therefore, the
level of GCV phosphorylation induced by the BZLF1 and BRLF1 adenovirus vectors in EBV-positive tumor cells should be sufficient to
promote GCV-mediated cell death.
GCV Inhibits Lytic EBV Replication. We next determined
whether GCV inhibits lytic EBV replication in cells infected with the
BZLF1 and BRLF1 adenovirus vectors. Lytic replication was quantitated
by Southern blot analysis using the termini assay (37). As shown in Fig.
4, Jijoye cells infected with the lacZ adenovirus contain only the latent
(episomal) form of the EBV genome. Infection with the BZLF1 adenovirus vector efficiently induces the lytic (linear) form of the EBV genome
in Jijoye cells (Fig. 4A). The BZLF1 adenovirus vector induces early lytic
EBV protein expression in Jijoye cells in the presence or absence of
acyclovir or GCV (data not shown). However, treatment with either
acyclovir or GCV completely abolishes the ability of BZLF1 to form
linear EBV genomes, which result from lytic viral replication and are
required for productive EBV infection. Similar results were obtained
using the BRLF1 adenovirus in Jijoye cells (Fig. 4B), although BRLF1
was less efficient than BZLF1 in inducing fully lytic replication. EBV
cannot be packaged into infectious mature virions unless it is lytically
replicated (1). Therefore, concomitant GCV administration should prevent the release of infectious EBV particles when tumor cells are treated
with the BZLF1 or BRLF1 adenovirus vectors.
The BZLF1 and BRLF1 Adenovirus Vectors Kill Jijoye Cells in
the Presence or Absence of GCV. The above results predict that the
BZLF1 and BRLF1 adenovirus vectors will induce cell killing in Jijoye
cells in the presence or absence of GCV, although the mechanism of cell
death is presumably different. To confirm this, we infected Jijoye cells
with the BZLF1, BRLF1, or lacZ adenovirus vector and examined cell
viability in the presence and absence of GCV. As shown in Fig. 5, in the
absence of lytic EBV infection, GCV has little effect on the viability of
Jijoye cells because latent EBV infection does not phosphorylate GCV.
Infection of Jijoye cells with either the BZLF1 or BRLF1 adenovirus
vectors (MOI of 50) induced dramatic cell killing (97–99%) in the
presence or absence of GCV. Interestingly, this amount of cell killing was
significantly greater than the percentage of infected cells (;35% as
Fig. 6. Effect of BZLF1 and BRLF1 on EBV-negative cells. A, primary human fibroblasts
and Saos-2 osteosarcoma cells were infected with the BZLF1, BRLF1, or lacZ adenovirus
vectors (MOI of 50). Cell viability was determined 3 days later by trypan blue exclusion.
BZLF1- and BRLF1-induced growth inhibition was calculated by comparing the growth rate
of BZLF1 and BRLF1 adenovirus vector infected cells to that of lacZ adenovirus vectorinfected cells. Both the primary human fibroblasts and Saos-2 cells were efficiently infected
by the adenovirus vectors (80% infectivity or more) in these experiments. B, normal human
fibroblasts were infected with the BZLF1 or lacZ adenovirus vectors (MOI of 50) and
examined for annexin V binding using FITC-labeled annexin V in FACS analysis. The level
of annexin V binding is shown on the X axis, and propidium iodide (PI) staining is shown on
the Y axis. The position of cells that are apoptotic (bottom right) and necrotic (top right) is
indicated (41). BZLF1 expression did not significantly increase annexin V binding in normal
human fibroblasts, although essentially all cells were infected with the adenovirus vector (data
not shown). In contrast, the BRLF1 adenovirus induced annexin V binding and/or other
markers for apoptosis in a variety of cell types (data not shown).4
4
J. Swenson and S. C. Kenney, submitted for publication.
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INDUCTION OF LYTIC EBV IN TUMORS
DISCUSSION
Fig. 7. Induction of lytic EBV infection in Jijoye tumors in vivo. Jijoye cell tumors
were grown s.c. in SCID mice and directly inoculated with the lacZ-, BZLF1-, or the
BRLF1-expressing adenovirus vectors. Two days later, the mice were sacrificed and
tumor extracts were examined for expression of the lytic EBV protein, BMRF1, using
immunoblot analysis.
determined by FACS analysis using BZLF1- and BRLF1-specific antibodies). Thus, the induction of lytic EBV infection in tumor cells results
in cell death, in the presence or absence of GCV.
Effect of the BZLF1 and BRLF1 Adenovirus Vectors in EBVnegative Cells. Although it has been reported previously that BZLF1
expression is nontoxic in EBV-negative cells (39, 40), BZLF1, in fact,
induces a cell cycle block in these cells (39). The effect of BRLF1
expression in EBV-negative cells is unknown. Unfortunately, we were
unable to efficiently infect a variety of EBV-negative B-cell lines with
adenovirus vectors (data not shown). Therefore, to examine the effect
of BZLF1 and BRLF1 adenovirus vectors in EBV-negative cells, we
infected two different cell types, normal human fibroblasts, and the
osteosarcoma cell line, Saos-2, with the lacZ, BZLF1, and BRLF1
adenovirus vectors and quantitated cell numbers 3 days later. These
two cell lines were chosen because they represent different extremes
in terms of tumor suppressor dysregulation; Saos-2 cells are deleted in
both p53 and Rb, whereas normal human fibroblasts are not. As
shown in Fig. 6A, infection of Saos-2 cells with either the BZLF1 or
BRLF1 adenovirus vectors had relatively little effect upon cell growth
in comparison to the lacZ control vector. However, infection of
primary human fibroblasts with either the BZLF1 or BRLF1 adenovirus vectors resulted in significant growth inhibition.
Because a BZLF1-induced cell cycle block could cause growth
inhibition in EBV-negative cells without killing them, we examined
the level of annexin V binding in primary fibroblasts infected with the
lacZ or BZLF1 adenoviruses. Annexin V binds to cells expressing
phosphatidylserine on the outer membrane, which is a sensitive
marker for cells undergoing early apoptosis or necrosis (41). As
shown in Fig. 6B, BZLF1 adenovirus infection of normal human
fibroblasts does not significantly increase the binding of FITC-labeled
annexin V. Therefore, BZLF1 expression inhibits growth of primary
human fibroblasts (due to a cell cycle block) but does not kill the cells.
In contrast, BRLF1 induced apoptosis in a variety of cell types.4 Thus,
BZLF1-induced cell killing appears to be more EBV-dependent than
BRLF1-induced killing, suggesting that BZLF1 may be preferable for
treatment of tumors in vivo.
Disruption of EBV Latency in Vivo. To determine whether the
BZLF1 and BRLF1 adenovirus vectors can disrupt viral latency in
vivo, SCID mice were injected s.c. with Jijoye cells, resulting in the
formation of palpable tumors 2 weeks later. The Jijoye tumors were
directly inoculated with 2 3 109 plaque-forming units of the lacZ,
BZLF1, or BRLF1 adenoviruses. Tumor extracts were prepared 2
days after injection and analyzed for expression by immunoblot
analysis of the early lytic EBV BMRF1 protein. As shown in Fig. 7,
injection of tumors with either the BZLF1 or BRLF1 adenoviruses
induced BMRF1 expression from the EBV genome, whereas tumors
injected with the lacZ adenovirus express little (if any) BMRF1. Thus,
both the BZLF1 and BRLF1 adenoviruses can disrupt EBV latency in
lymphoma cells in vivo. To our knowledge, this is the first demonstration that EBV latency can be purposefully disrupted in vivo.
The consistent presence of EBV in certain tumor types may open
novel, EBV-based targeting strategies. Here, we have investigated the
feasibility of converting EBV latency into lytic EBV infection in
Burkitt lymphoma cells. Using adenovirus vectors, we demonstrated
that lytic infection can be induced successfully in vitro and in vivo
with either of the two EBV IE proteins. We have also demonstrated
that lytic EBV infection phosphorylates GCV, converting it into a
cytotoxic agent that kills the host cell while simultaneously preventing
completion of the viral replicative cycle and release of infectious EBV
particles. Therefore, disrupting EBV latency by gene delivery methods, with concomitant GCV administration, may be an effective
method for treating EBV-associated tumors.
Because we were previously unable to show that transfected
BRLF1 protein disrupts EBV latency in B cells (22), we were surprised to discover that the BRLF1 adenovirus vector efficiently induces lytic infection in Jijoye cells. The BRLF1 adenovirus vector
likewise disrupts viral latency in another Burkitt cell line, Akata.5
Because another group has recently reported that BRLF1 expression
(delivered by transfection) in B cells disrupts EBV latency (42), the
ability of the BRLF1 adenovirus vector to induce lytic infection in
Burkitt lines is probably due to a greatly increased level of BRLF1
expression from the adenovirus vector versus our plasmid vectors
rather than a requirement for an adenovirus-encoded “helper” effect.
In any event, our finding that the BRLF1 adenovirus vector disrupts
EBV latency in both B cells and epithelial cells indicates that either
BZLF1 or BRLF1 gene delivery vectors can be considered for the
induction of lytic infection of EBV-associated B-cell lymphomas
in vivo.
Although both BZLF1 and BRLF1 disrupt viral latency, they are
significantly different in other respects. For example, BZLF1 blocks
the cell cycle (39), whereas BRLF1 activates cell cycle progression.4
There may also be differences in the in vivo toxicity of BZLF1 versus
BRLF1 in EBV-negative cells. Therefore, it will be important to
compare the efficacy and toxicity of BZLF1 versus BRLF1 adenoviruses in vivo to determine which is the better vector for disrupting
tumor latency. Should the expression of the BZLF1 or BRLF1 proteins prove to be excessively toxic to EBV-negative cells in vivo, these
proteins could be placed under the control of EBV-specific promoter
elements (such as the EBNA-1-dependent oriP enhancer or the
EBNA-2-responsive BamHI C promoter; Refs. 43 and 44) to make
this system even more EBV dependent.
Previous investigators have delivered the HSV-TK gene to promote
GCV-induced killing of tumor cells. Here, we have devised a novel
strategy in which we do not deliver a virally encoded TK gene but
instead induce the EBV genome within tumor cells to express proteins
that phosphorylate GCV. This strategy has the advantage of being
completely EBV specific, because the expression of BZLF1 or
BRLF1 in EBV-negative cells obviously can not induce virally encoded proteins. Furthermore, because phosphorylated GCV has been
shown to produce bystander killing in certain cell types (25–27), the
BZLF1/GCV (or BRLF1/GCV) combination could potentially kill
more cells than delivery of the IE proteins alone. Although we have
been unable to observe bystander killing with the HSV-TK/GCV
combination in EBV-positive B-cell lines in vitro, bystander killing
occurred with the HSV-TK/GCV combination in EBV-positive lymphomas in vivo (32). Thus, the use of GCV with EBV IE gene delivery
vectors will not only prevent release of infectious EBV but may
actually improve the efficiency of tumor cell killing in vivo.
EBV contains homologues to both the HSV-TK gene and the CMV
5
E-M. Westphal and S. C. Kenney, unpublished observations.
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INDUCTION OF LYTIC EBV IN TUMORS
UL97 gene. It is currently unknown whether one or both of these EBV
proteins phosphorylate GCV. A recent report did not find that bacterially expressed EBV-TK phosphorylates GCV (29). Nevertheless,
stable expression of the EBV-TK in melanoma cells significantly
sensitizes the cells to GCV toxicity (29). Interestingly, bacterially
expressed EBV-TK phosphorylates AZT much more efficiently than
HSV-TK and results in increased sensitivity to AZT toxicity when it
is stably expressed in melanoma cells (29). AZT has also been
reported to inhibit lytic EBV replication (45). Therefore, it is possible
that AZT, which can be given orally and is less toxic than GCV,
would be as effective as GCV in killing EBV-positive tumors inoculated with the BZLF1 or BRLF1 adenovirus vectors.
Although we presume that expression of the BZLF1 and BRLF1
proteins in EBV-positive lymphoma cells results in cell death due to
an EBV-mediated mechanism, we were, unfortunately, unable to use
adenovirus vectors to efficiently infect the most biologically relevant
control cell, an EBV-negative lymphoma. Interestingly, it has been
recently shown that EBV infection increases the ability of adenovirus
vectors to infect B cells, which otherwise do not express the adenovirus receptor (46). We believe that the discordance between the
number of EBV-positive Jijoye cells killed using the BZLF1 and
BRLF1 adenovirus vectors (.90%) versus the number of cells supposedly infected (,50%) likely reflects the relative insensitivity of
our imunofluoresence assays (such that the adenovirus infectivity may
be, in fact, higher). However, it remains possible that BZLF1 and/or
BRLF1 expression can also induce death in B cells through an
EBV-independent mechanism (for example, by induction of toxic
cytokines).
Although we successfully used an adenovirus vector to efficiently
deliver EBV IE genes to a Burkitt tumor in vivo, it is likely that the
infectivity of EBV-associated lymphomas by adenovirus vectors will
vary. Although some B-cell lines are susceptible to efficient adenovirus infection (47), we and others have found that EBV-transformed
lymphoblastoid B-cell lines (the closest in vitro equivalent to primary
CNS lymphomas) are rather resistant to adenovirus infection (46).
Therefore, retroviral vector expression of the BZLF1 and BRLF1
proteins may prove to be more efficient for in vivo delivery to CNS
lymphomas. Alternatively, bispecific antibodies could be used to
increase the infectivity of adenovirus vectors for B cells, as has
previously been accomplished with T cells (48).
A variety of EBV-based strategies have recently been proposed for
treating EBV-associated malignancies (32, 40, 44, 49 –52). For example, we have reported that EBV-based delivery vectors expressing
cellular toxins kill tumor cells in an EBV-specific fashion (51). The
lytic strategy presented here is even more EBV dependent. Ironically,
although EBV induces development of tumors in immunocompromised hosts, its very presence may now be used to mark such cells for
destruction.
ACKNOWLEDGMENTS
Much of this work was performed while S. C. K. was on sabbatical at
Glaxo-Wellcome (Research Triangle Park, NC), working with Drs. Brian
Huber and Steven Davis. We thank the Glaxo-Wellcome group for many
helpful suggestions and Drs. Joseph Pagano and Beverly Mitchell for reviewing the manuscript. We thank Kay Neal for secretarial support and Drs. R. Jude
Samulski and Douglas McCarty at the UNC Gene Therapy Core for preparing
adenovirus-lacZ, adenovirus-BZLF1, and adenovirus BRLF1.
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Induction of Lytic Epstein-Barr Virus (EBV) Infection in
EBV-associated Malignancies Using Adenovirus Vectors in
Vitro and in Vivo
Eva-Maria Westphal, Amy Mauser, Jennifer Swenson, et al.
Cancer Res 1999;59:1485-1491.
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