(CANCERRESEARCH
54. 2808-2811,May15, 19941
Ultrastructural Localization of BHRF1: An Epstein-Barr Virus Gene Product
Which Has Homology with bcl-2'
Tamas Hickish,2 David Robertson, Paul Clarke, Mark Hill, Francesca di Stefano, Catherine Clarke, and
David Cunningham
Lymphoma Unit [T. H., M. H., D. C.J, CRC Section ofMedicune fT. H., P. C., M. H., F. di S., D. C.J, and Department
C. C.], Royal Marsden Hospital and Institute of Cancer Research, Downs Roa4 Sutton, Surrey, SM2 5PT United Kingdom
ABSTRACT
BHRF1 is an Epstein-Barr
virus encoded protein which has a 38%
sequence similarity with bcl-2 over the carboxyl portion. Like bcl-2,
BHRF1 has been shown to suppress programmed
cell death from apop
tosis. Previously BHRF1 has been detected in mitochondrial,
microsomal,
and nuclear compartments
by cell fractionation analysis. In this study we
have used the technique of immunoelectron microscopy to define the
ultrastructural
distribution of the BHRF1 product In the EBV converted
cell lines B95.8 and P3HR-l. The BHRF1 product was localized at the
periphery of the mitochondria in a pattern similar to that of bcl-2 and by
analogy with bcl-2 this is likely to be the functional destination. Sequence
analysis of the BHRF1 protein disclosed similarity with the recently
described bcl-2 homologues bcl-x (32%) and bax (34%) over the carboxyl
portion,
with several domains of complete identity. BHRF1 appears
to be
a member of a gene family Involved in the regulation of programmed cell
death. The Identity between BHRF1 and bcl-2, an apparent shared ability
to abrogate apoptosis, and the common ultrastructural localization is
compelling and suggests that bcl-2 and BHRF1 are both functionally
mechanistically
similar.
and
of Cell Biology and Experimental
Pathology [D. R.,
EBV-negative Burkitt's lymphoma cell line, raising the possibility
that it may also have a role in promoting cell survival (16).
By means of immunoelectron microscopy combined with the tissue
preparation technique of PLT, which maintains cell integrity and
enables scrutiny of the intracellular localization of a protein, our group
and others have demonstrated that bcl-2 is distributed primarily to the
periphery of the mitochondria but also to the nuclear envelope (17—
19). Recent data indicate that bcl-2 is anchored to the outer mitochon
drial membrane by its COOH terminus with the NH2 portion facing
the cytosol (18, 20). Cell fractionation studies also have identified
BHRF1
in the mitochondrial,
microsomal,
and nuclear
fractions
(6).
However, the utility of this technique may be restricted by the inabil
ity to produce pure fractions; ultracentrifugation at best produces
enriched samples. In order to pursue the apparent similarities between
bcl-2 and BHRF1 we have sought to define the ultrastructural distri
bution of the BHRF1 product by PLT immunoelectron microscopy
and have found that it is localized to the periphery of the mitochon
dna, in a pattern similar to that demonstrated for bcl-2.
MATERIALS AND METHODS
INTRODUCTION
The Epstein-Barr virus is a human B-cell lymphotropic virus which
typically establishes a persistent asymptomatic infection. It is the
causative agent of infectious mononucleosis and is also associated
with endemic Burkitt's lymphoma, undifferentiated nasopharyngeal
carcinoma, acquired immunodeficiency syndrome-related lymphoma,
subgroups of Hodgkin's disease, and recently gastric adenocarcinoma
(1—3).
BHRF1 (BamHI-H rightwardreadingframe1) is anEBV3gene
product of undecided function yet it is a constituent of all EBV
genomes, suggesting that it has an important role in the viral life cycle
(4). Spliced BHRF1 transcripts have been identified in latently in
fected B-cells, although the 17 kDa protein has yet to be detected in
these cells (5, 6). However, in the early phase of the viral replicative
cycle both BHRF1 protein and transcripts are expressed in abundance
(5, 6). However, activation of BHRF1 expression does not indicate
that the productive cycle will follow, and its expression is not required
for viral replication or B-cell transformation since recombinant virus
in which BHRF1 has been deleted is both transforming and able to
enter the replicative cycle (6—8).
BHRF1
has 38% similarity
in its carboxy
portion
with bcl-2 (bcl-2;
amino acids 97—239),originally identified as a result of its involve
ment in the 14;18 translocation characteristic of follicular B-cell
lymphoma, and which is the lead member of an emergent gene family
involved in the regulation of apoptosis (9—15).Recently a BHRF1
transgene has been shown to confer resistance to apoptosis in an
Cell Lines. B95.8 is an EBV-converted marmoset cell line (21). P3HR-1 is
an EBV-positive Burkitt's lymphoma cell line (22). SU-DHL4 (provided by
Dr. Alan Epstein) was derived from a diffuse histiocytic lymphoma and
expresses bcl-2 (9).
Cell Culture.
Cells in log phase growth were cultured in either standard
(10%fetalcalf serum)or stressed(fetal calf serumabsent)conditionsin RPMI
1640 in a humidified atmosphere with 5% CO2 for 3 days. Stressing serves to
induce the replicative cycle. Prior to harvesting, cells were counted and
assayed for viability by means of trypan blue exclusion.
Immunocytochemistry.
Cells were fixed in 4% formaldehyde in PBS for
20 min at room temperature, washed in PBS, permeabiized with 0.2% Triton
X-100 for 3 mm, and then incubated with either 5B11 [mwine monoclonal
antibody specific for BHRF1 (5—7');
provided by Dr. Gary Pearson] diluted to
1:32,000 or No. 100 (murine monoclonal
antibody specific for bcl-2 (23);
provided by Dr. David Mason) diluted to 1:4, followed by sheep anti-mouse
fluoresceun isothiocyanate conjugate. The pellet was then mixed 50:50 with
antiquenching
agent, mounted on a glass slide with a coverslip, and viewed on
a MRC-600 confocal imaging system with parallel phase.
Immunoelectron Microscopy. Cells were washed and pelleted, fixed, and
embedded in Lowicryl HM2O as described in detail elsewhere (24). Briefly,
cells were fixed for 1 h in 2.05 fix (2% formaldehyde + 0.05% glutaraldehyde
in PBS), dehydrated by progressive lowering of temperature, infiltrated with
Lowicryl HM2O, and polymerized
at -50°C with UV light. For immunola
belung, 100-nm sections were collected onto nickel grids, incubated overnight
in a 5B11 diluted to 1:16,000 followed by 5 am goat anti-mouse gold conjugate
(Amersham
International),
1:50, for 90 miii, and silver enhanced for 5 mm
(IntenSe; Amersham International). Grids were then double stained with uranyl
acetate and lead citrate using an LKB Ultrastainer and viewed in a Philips
CM1Oat 80 kV accelerating voltage. Immunolabelingwith No. 100 was
Received 12/13/93; accepted 3/21/94.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance with
performed similarly except it was used at a 1:8 dilution (data not shown).
18 U.S.C. Section 1734 solely to indicate this fact.
RESULTS
I This
2 To
3 The
work
whom
was
supported
requests
abbreviations
in part
for
reprints
used
are:
by
should
EBV,
the
be
Cancer
Epstein-Barr
temperature; PBS, phosphate-buffered saline.
Research
Campaign.
addressed.
virus;
PLT,
progressive
lowering
of
Immunocytochemistry.
Stressed conditions produced a 5—10-fold
increase in the number of B95.8 and P3HR-1 cells expressing the
2808
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ULTRASTRUCrURAL LOCALIZATION OF BHRF1
productive cycle (Fig. 3B). The association of BHRF1 to the periph
cry of the mitochondria is apparent at higher magnification (Fig. 3A)
and this pattern was identical in both unstressed and stressed cells. In
order to quantify the distribution of the anti-BHRF1 label a morpho
metric analysis was performed (Table 1). The number of gold particles
at the nucleus, nuclear membrane, mitochondrial periphery, mitochon
drial matrix (inner mitochondrial membrane), cytoplasm, and cell
membrane was counted in ten B95.8 cells expressing BHRF1. In total,
916 particles were counted. Ten apparently BHRF1-negative B95.8
cells were also examined and only 42 gold particles were counted. The
similar sensitivity displayed by immunocytochemistry and immuno
electron microscopy indicates that PLT did not have an effect on the
BHRF1 epitopes leading to the underscoring of the levels of label at
the various ultrastructural sites. The morphometric derived data dem
onstrate that BHRF1 is predominantly localized around the mitochon
drial periphery (indeed the association with other membranes is mm
imal) and the distribution is similar to that found for bcl-2 (17).
DISCUSSION
Using immunoelectron microscopy we have demonstrated that the
EBV-encoded protein BHRF1 is primarily localized to the mitochon
drial periphery in the EBV genome-positive cell lines B95.8 and
P3HR-1. The resolution of this technology is not sufficient to enable
the precise localization of the BHRF1 product in terms of the outer
mitochondrial membrane. The options lie between the outer aspect, a
%.;@.;Yrt.@4@!S%@
@
Fig. 1. Expressionof BHRF1 in P3HR-1 and B95.8 cells as visualized by immuno
fluorescence staining and confocal microscopy. Phase contrast (left) and confocal fluo
rescence (right) of B95.8 (a) and P3HR-1 (b) labeled with anti-BHRF1. Comparison of
the phase and fluorescent images clearly shows that not all the cells in the field are labeled.
In the fluorescentimage one can see cells thataredensely stainedandalso cells showing
a speckled pattern. Original magnification, X 850. Bar, 10 pm.
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c.@
BHRF1
product
and when detected,
expression
was always
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abundant
(Fig. 1). The confocal microscopy image in Fig. 1 shows that expres
sion is cytoplasmic and typically speckled in appearance, without any
background staining, in both B95.8 and P3HR-1 cells. The proportion
of positive
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cells was consistent with the predicted fraction of cells in
which EBV would be in the productive cycle and hence expressing
BHRF1. BHRF1 expression was not detected in SU-DHL4 cells and
bcl-2 was not detected in B95.8 or P3HR-1 but was found in all
SU-DHL4
@
@
@
cells
(data
not shown).
Negative
controls
with
no-first
antibody were performed and were consistently negative. The pattern
of anti-BHRF1 fluorescence was similar to that found for bcl-2 in the
SU-DHL4 cells (data not shown; Ref. 17) and suggests a mitochon
drial location.
,
.-...@.‘.
.@
.
‘4@ .:__•.@@‘
Immunoelectron
Microscopy. The proportion of the B95.8 and
@: ‘@
P3HR-1 cells in which the BHRF1 product was identified equated to
the iminunocytochemistry assay, indicating that in this system the two
p..
techniques had a similar sensitivity and specificity. Fig. 2 is a low
power electron micrograph and it is representive of the appearance of
both BHRF1-positive and -negative cells. In the upper BHRF1-neg
:
@-@-@:
ative cell, only 3 gold particles can be seen, the cell has a normal
chromatin pattern, and virions are not present. In the obviously
BHRF1-positive lower cell, gold particles are clustered around the
mitochondria, the chromatin architecture is disrupted, and virions are
Fig. 2. Electron micrograph of B95.8 cells immunostained for BHRFI. The upper cell
has a normal chromatun pattern, no visible virions, and no colloidal gold staining. In
just discernible in the nucleus; virions are clearly seen in Fig. 3B.
contrast the lower cell has a disrupted chromatin pattern and the positive staining is
BHRF1 was detected only in cells (either unstressed or stressed) in localized around the group of mitochondria in the cell. Original magnification, X 14,400.
which the presence of EBV virions indicated that the virus was in the Bar, 1 pm.
2809
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ULTRASTRUCI1JRAL LOCALIZATION OF BHRFI
loci is at the nuclear membrane where only 0.01% of BHRF1 was
found in B95.8 cells compared to 11% of bcl-2 label in SU-DHL4
cells.
Taken together the data indicate that BHRF1 is predominantly
associated with the mitochondria, and by analogy with bcl-2 this is
likely to be the functional destination.
However, localization to the mitochondria (and nuclear envelope) is
not crucial for bcl-2 function per se since bcl-2 mutants lacking the
COOH terminus are cytosolic but retain some ability to protect against
apoptosis (25). Furthermore, while the COOH terminus sequence
appears to be important for bcl-2 membrane localization it can be
substituted with a heterologous transmembrane domain and still lo
calize to the mitochondria with maintained function (26).
$@@st
A precise biochemical activity has yet to be ascribed to bcl-2. bcl-2
has been proposed to modulate an antioxidant pathway since expres
sion is associated with a reduction of lipid peroxidation (25). Alter
b5@%
;.
natively bcl-2 may abrogate apoptosis by regulating intracellular
@4-11@
,,
calcium homeostasis, since apoptosis-inducing cytokine withdrawal is
associated with partitioning of calcium to the mitochondria which can
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be blocked by bcl-2 expression (27). The intracellular distribution of
bcl-2 (and by implication BHRF1) is consistent with a role in either of
these processes.
Recently three additional human bcl-2 homologues have been iden
..
::
@:
, :..:@
. :@
tified; bcl-x, bax and md-i (13—15).bcl-x and bax both regulate
apoptosis while md-i is expressed in programmed myeloid cell
differentiation but its impact on apoptosis is unknown. Fig. 4 displays
a sequence comparison among BHRF1, bcl-x, bax, bcl-2, and md-i.
,.
@,.
-‘c'@i@!1
There are regions of major sequence similarity and identity among all
proteins in the carboxyl portions suggesting that these are the func
tional domains. This analysis indicates that BHRF1 is a member of a
burgeoning gene family involved in the regulation of apoptosis.
Fig.3. Electronmicrographsof B95.8cellsimmunostained
for BHRF1.a, clustering
of the labelarounda groupof mitochondriain the cell whereasan areaof roughand BHRF1 has greatest similarity to bcl-2 and is therefore most likely to
be a viral homologue of bcl-2 in origin rather than of bcl-x or bax.
endoplasmic reticulum on the left of the micrograph is free from label. In b positively
a
@
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f
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stained mitochondria can be seen in the lower part of the micrograph and in the upper part
viral particles within the nucleus are clearly visible. Original magnification: a, X 38,750;
1
b, X 56,700. Bar: a, 0.25 pm; b, 0.2 pm.
hbhrf
hbcl2a
hbclxl
hbaxa
celLsTo
Table 1 Morphometric analysis ofBHRFJ localization in B95.8
determine the proportions of colloidal gold label bound to various cell compart
wereanalyzed
ments, electron micrographs of B95.8 cells showing positive labeling with BHRF1
andassigned
as described previously (17). Every gold particle was counted, marked,
themitochondrial
to a cell compartment.
hbhrf
Ithcl2a
hbclxl
hbaxa
periphery, it had to be within one gold particle diameter of the mitochon
(normalchromatin
drial
periphery. As an internal control an equivalent area of negative cells
yielded42 pattern, no virions visible) was scored in the same manner. The count
BHRF1in
grains as compared to 916 grains on BHRF1-positive cells. The distribution of
goldparticles
of total
(541)Mitochondrial
periphery
(66)Cytoplasmmatrix
(293)Cell
membrane
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51
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semetpsain
llqgfiqdra
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48%; cytoplasm, 36%; nuclear membrane, 11%; nucleus, 0%; inner mitochon
drial membrane, 5%; cell membrane, 0% (17)].%
(8)Mitochondrial
membrane
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hincil
[mitochondrialperiphery,
B95.8 cells was similar to that found for bcl-2 in the SU-DHL4 cell line
(8)Nuclear
cir
hmcl 1
For a particle to be scored as associated with
countedCell
no.)Nucleus
compartment
50
Ma .
ye tREILLa1 .
Xahagrtgyd
nRIIVMkyih
(Absolute
0.01
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60
7
33
0
hbhrf
hbcl2a
hbclxl
hbaxa
hmcll
yhv11E@Iie
qa ..GDDfar
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ri . . ODILDa
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hbhrf
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Rnsetltltw
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aaVd . . tDSp
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rgrFatVVel LflDGV .Nib
yqsFeqVvnN LFP.DOV.NIIG
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MPeDOnfNWO
vkBlsrVMih
VVaDOVtNIG
RaLkWmaWcmhaC . RTLccN
RIVAPFeFOG vMCVeSV. .N
RIVAFFaFOG aLCVeSV. . D
RWALFyFAS kLvLXaLc . .
qetpyyVvdL
rJKspLVdnI
klXqvLVsrI
tkVpeLlrtl
svrgunleaaN gLdgWIhQQG
A1WMtNYLnrHLhtWIQDNG
AaWMatYLnDHLepWXQINO
ngWtlDFLrZ RL1gWIQDQG
RIVtlisFQ&
qiac. .IepL
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151
fVakHlktiN
200
201
hbhrf
transmembrane position, the inner aspect, or the points where the
inner and outer membranes are in contact. The distribution around the
periphery of the mitochondria was similar to that found with bcl-2
(i7). These data are consistent with a recent confocal microscopy
based study which demonstrated that bcl-2 and BHRF1 exhibit sub
stantial colocalization in cells in which they are both constitutively
expressed (16). The morphometric data indicate that the only differ
ence between bcl-2 and BHRF1 levels at the various ultrastructural
250
@(at1IRdhi pgarr. ..fB WtlfLag.LT
hbcl2a
OIbDAPVE1YO
hbclxl
hbaxa
hmcll
@iDtFVE1YO nnaaaesrkg qlrfnawfLT g.NtVAGVV1 Lgalf aRK
@iDO1LsyFO tp
T WQt
VT ..IfVAOVLt aaltlwXxmg
G1IDOFVIfFh vedi
IggIRnvLl afagVkoVga glayLiR
pamrplfdfS
W.lsLXtlLS
Fig. 4. Primary sequence alignmentofhuman
bcl-2a and related proteins was computer
generatedby the BESTFIT,LINEUP,andPRE'LTYprograms(GeneticsComputerGroup
software package) and by visual analysis. The single amino acid code is used. Bold,
ca@lized letters, identical or similar amino acids in the compared primary sequences.
Amino acid similarities are defined as follows: aromatic, F, Y, w; aliphatic, L, I, V. M,
and G, A; basic, K, R, H; acidic and derivatives, D, E, N, 0; alcoholic, S, T. Sequence
similarity over whole protein BHRF1 versus: bcl-2, 77 of 239 (32%); bcl-x, 60 of 234
(26%); bax, 54 of 192 (28%), md-i, 34 of 350 (10%, or 25% with carboxyl portion of
md-i). We compare only human sequences with BHRF1 but others exist, e.g., African
swine fever virus (31) and Al (murine) (32).
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ULTRASTRUCI1JRAL LOCALIZATION OF BHRF1
BHRF1
was expressed
only in cells in which the presence
11. Hockenbery, D., Nunez, D., Milliman, C., Schreiber, R. D., and Korsmeyer, S. J. bcl2
is an inner mitocbondrial protein that blocks programmed cell death. Nature (Lond.),
of virions
signified EBV had entered the replicative cycle. While BHRF1 ap
pears to be dispensable for viral replication under certain conditions
(7, 8) this observation lends support to the notion that BHRF1 has a
role in enabling maximum virus yield before cell death (16). EBV
gene expression has been demonstrated to have an impact on the host
cells' vulnerability to apoptosis (16, 28). Similarly, expression of the
EBV latent genes, LMPJ and EBNA-2, has been shown to up-regulate
bcl-2 and thereby to protect B-cells from apoptosis (29, 30). Hence
expression of BHRF1 may be an option in the context of a general
viral strategy to protect the host cell from apoptosis and ultimately
ensure survival of self.
Deregulation of bcl-2 expression is considered to be a key onco
genic event in lymphoma. The role of BHRF1 in EBV-associated
tumors is unknown. The possibility that aberrant BHRF1 could pro
mote cell survival and contribute to the development of the malignant
phenotype remains to explored.
348: 334—336,1990.
12. Hockenbery, D. M., Zutter, M., Hickey, W., Nahm, M., and Korsmeyer, S. J. bcl-2
proteinis topographically
restrictedin tissuescharacterizedby apoptoticcell death.
Proc. Natl. Acad. Sci. USA, 88: 6961-6965, 1991.
13. Kozopas, K. M., Yang, T., Buchan, H. L, Thou, P., and Craig, R. W. mcli, a gene
expressed in programmed myeloid cell differentiation, has sequence similarity to
bcl2. Proc. NatI. Acad. Sci. USA, 90: 3516—3520, 1993.
14. Boise, L, Gonzalez-Garcia, M., Postema, C., Ding, L, Lindsten, T., Turka, L, Mao,
x., Nunez, 0., and Thompson,C. bcl-x, a bcl-2-relatedgene that functions as a
dominant regulator of apoptotic cell death. Cell, 74: 597—608,1993.
15. Oltvai, Z., Milliman, C., and Korsmeyer, S. bcl-2 heterodimerizes in vivo with a
conserved homolog, Bax, that accelerates programmed cell death. Cell, 74: 609—619,
1993.
16. Henderson, S., Huen, D., Rowe, M., Dawson, C., Johnson, 0., and Rickinson, A.
Epstein-Barr virus-coded BHRF1 protein, a viral homologue of bcl-2, protects human
B cells from programmed cell death. Proc. NatI. Acad. Sd. USA, 90:8479-8483,
1993.
17. Monaghan, P., Robertson, D., Amos, T. A., Dyer, M. J., Mason, D. Y., and Greaves,
M. F. Ultrastructural localisation of bcl-2 protein. J. Histochem. Cytochem., 40:
1819—1825, 1992.
18. Krajewski, S., Tanaka, S., Takayama, S., Schibler, M. 1., Fenton, W., and Reed, J. C.
Investigation of the subcellular distribution of the bcl-2 oncoprotein: residence in the
nuclear envelope, endoplasmic reticulum, and outer mitochondrial membranes. Can
ACKNOWLEDGMENTS
cer Res., 53: 4701—4714, 1993.
19. De Jong, D., Prins, F. A., Mason, D. Y., Reed, J. C., van Ommen, G. B., and KIuin,
P. M. Subcellular localisation of the bcl-2 protein in malignant and normal lymphoid
We wish to thank Hugh F. Paterson for excellent assistance with the
confocal microscopy, Gary Pearson for providing the 5B11 antibody, Dorothy
Crawford for providing the P3HR-1 cell line, Paul Farrell and Gordon Allen
cells. Cancer Res., 54: 256—260,1994.
20. Nguyen, M., Millar, D. G., Yong, V. W., Korsmeyer, S. J., and Shore, 0. C. Targeting
for providing the B95.8 cell line, and David T. Rowe and Martin Rowe for
helpful discussions.
of bcl-2 to the mitochondrial outer membrane by a COOH-termunal signal-anchor
sequence. I. BioI. Chem., 268: 25265-25268, 1993.
21. Miller, 0., and Lipman, M. Release of infectious Epstein-Barr virus by transformed
marmoset leukocytes. Proc. Natl. Acad. Sci. USA, 70: 190—194,1973.
22. Hinuma, Y., and Grace, J. T. aoning of immunoglobin-producing human leukemic
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Downloaded from cancerres.aacrjournals.org on June 17, 2017. © 1994 American Association for Cancer Research.
Ultrastructural Localization of BHRF1: An Epstein-Barr Virus
Gene Product Which Has Homology with bcl-2
Tamas Hickish, David Robertson, Paul Clarke, et al.
Cancer Res 1994;54:2808-2811.
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