Download Restricted expression of Epstein–Barr virus (EBV)

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of General Virology (1998), 79, 1445–1452. Printed in Great Britain
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Restricted expression of Epstein–Barr virus (EBV)-encoded,
growth transformation-associated antigens in an EBV- and
human herpesvirus type 8-carrying body cavity lymphoma line
Laszlo Szekely,1 Fu Chen,1 Norihiro Teramoto,1 Barbro Ehlin-Henriksson,1 Katja Pokrovskaja,1
Anna Szeles,1 Agneta Manneborg-Sandlund,1 Mia Lo$ wbeer,1 Evelyne T. Lennette2 and George Klein1
1
2
Microbiology and Tumour Biology Centre (MTC), Karolinska Institute, S-17177 Stockholm, Sweden
Virolab Inc., Berkely, CA 94710-1509, USA
A body cavity lymphoma-derived cell line (BC1),
known to carry both Epstein–Barr virus (EBV) and
human herpes virus type 8 (HHV-8 ; or Kaposi’s
sarcoma-associated herpesvirus, KSHV), was analysed for the expression of EBV-encoded, growth
transformation-associated antigens and cellular
phenotype by immunofluorescence staining, Western blotting, RT–PCR and flow cytometry. A similar
phenotypic analysis was also performed on another
body cavity lymphoma line, BCBL1, that is singly
infected with HHV-8. Phenotypically, the two lines
were closely similar. Although both lines are known
to carry rearranged immunoglobulin genes, they
were mostly negative for B-cell surface markers.
Both expressed the HHV-8-encoded nuclear anti-
Introduction
Human herpes virus type 8 (HHV-8 ; or Kaposi’s sarcomaassociated herpesvirus, KSHV) is a recently discovered human
gammaherpesvirus that is the probable aetiological agent of
both AIDS-associated and sporadic Kaposi’s sarcomas (Chang
& Moore, 1996 ; Kedes et al., 1996 ; Miles, 1996). The virus has
also been found in primary effusion lymphomas (PEL), also
called body cavity lymphomas (Cesarman et al., 1995 a ; Nador
et al., 1996), in the multicentric Castleman’s disease (Gessain et
al., 1996) and in the dendritic cells of multiple myeloma
patients (Rettig et al., 1997). HHV-8 is not ubiquitous like
Epstein–Barr virus (EBV), but is present in a minority of people
in all human populations (Gao et al., 1996 b). Body cavity
lymphomas that are often, but not always, associated with
AIDS, frequently carry both EBV and HHV-8 (Carbone et al.,
1996 ; Said et al., 1996). Cell lines have been established from
both single HHV-8-positive and double HHV-8}EBV-positive
Author for correspondence : Laszlo Szekely.
Fax ­46 8 330498. e-mail lassze!ki.se
gen (LNA1). Similarly to Epstein–Barr nuclear antigen type 1 (EBNA1), LNA1 was associated with the
chromatin in interphase nuclei and the mitotic
chromosomes in metaphase. It accumulated in a few
well-circumscribed nuclear bodies that did not colocalize with EBNA1. BC1 cells expressed EBNA1,
LMP2A and EBV-encoded small RNAs but not
EBNA2–6, LMP1 and LMP2B. They were thus similar
to type I Burkitt’s lymphoma cells and latently
infected peripheral B-cells. Analysis of the splicing
pattern of the EBNA1-encoding message by RT–PCR
showed that BC1 cells used the QUK but not the YUK
splice, indicating that the mRNA was initiated from
Qp and not from Cp or Wp.
body cavity lymphomas (Arvanitakis et al., 1996). The latter
provide a unique opportunity to study the relations between
two latent and closely related herpesviruses.
EBV can establish at least three different types of virus
latency. The full expression of all nine growth transformationassociated viral proteins, EBNA1–6, LMP1, -2A and -2B is
only found in lymphoblastoid cell lines (LCLs), immunoblastic
lymphomas, and in Burkitt’s lymphoma (BL) lines that have
drifted to an immunoblastic phenotype after in vitro culturing.
The six EBNAs are spliced from a single giant message,
initiated from one of several alternative promoters in the C or
W region (W}C programme) (Bodescot & Perricaudet, 1986 ;
Bodescot et al., 1987). In type I latency, found in BL cells in vivo
and in derived cell lines that have maintained a representative
cellular phenotype (Rowe et al., 1986), as well as in latently
infected normal B-cells in vivo (Chen et al., 1995), only EBNA1
and LMP2 are expressed. The former is initiated from a
promoter in the Q region (Q programme) (Chen et al., 1995 ;
Schaefer et al., 1995). Latency type II, found in all non-B-cells
that carry the virus, including Hodgkin’s and T-cell
lymphomas, nasopharyngeal and other carcinomas, is identical
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L. Szekely and others
to type I latency as far as the EBNAs are concerned, but differs
by the constitutive expression of LMP1 (Fahraeus et al., 1988 ;
Hitt et al., 1989 ; Pallesen et al., 1991). The EBV-encoded small
RNAs (EBERs) are expressed in all three types of latency.
Latent EBV thus modifies its expression depending on the
phenotype of the host cell. Full expression of all six EBNAs and
the three LMPs is only known to occur in immunoblasts. The
resting B-cell and its closest counterpart, the BL cell that is
driven by the immunoglobulin}myc translocation, as well as
T-cell lymphomas and carcinoma cells, apparently lack some
immunoblast-specific component that is needed to activate the
full programme. Non-B-cells can express LMP1 constitutively.
This does not occur in lymphocytes of the B-lineage, due to a
cellular repressor that can be overridden by EBNA2 (Fahraeus
et al., 1993). In BL cells and normal resting B-cells, LMP1
remains suppressed.
The body cavity lymphoma lines BC1 and BCBL1, which
were the subjects of the present study, represent a different cell
type within the B-lineage, probably a pro- or pre-B-cell. It
appeared to be of interest to examine the EBV expression
pattern in the double-infected BC1 cell. We found that it
corresponds to type I latency, as in the BL cells but no other
lymphomas so far studied.
Methods
+ Cell culture and karyotype analysis. All cell lines were cultured
at 37 °C, in Iscove’s medium containing 10 % FCS and 50 mg}ml
gentamycin. Absence of mycoplasma contamination was monitored by
periodic staining with Hoechst 33258. The cell lines used in this study
have been described in the references (Cesarman et al., 1996 ; EhlinHenriksson et al., 1987 ; Renne et al., 1996 ; Pokrovskaja et al., 1996).
Karyotype analysis of G-banded chromosomes was done following
the protocol of Seabright (1971).
+ Immunofluorescence staining, image analysis and flow
cytometry. Indirect immunofluorescence staining and anti-complement
immunofluorescence reaction (ACIF) with monospecific absorbed human
sera or with unabsorbed serum with high anti-EBNA titre were performed
as previously described (Jiang et al., 1991 ; Szekely et al., 1991). Briefly,
cells were regularly fixed with methanol–acetone (1 : 1) for at least 10 min
and rehydrated in PBS. The first antibodies were diluted in blocking
buffer (2 % BSA, 0±2 % Tween-20, 10 % glycerol, 0±05 % NaN in PBS) in
$
the proportion of 1 : 1 and 1 : 10–50, respectively, depending on the
individual antibodies. The secondary antibody, FITC-conjugated rabbit
anti-mouse or anti-human IgG (Dakopatts), was diluted in blocking buffer
(1 : 20). The slides were mounted with 70 % glycerol, 2±5 % Dabco (Sigma)
pH 8±5 in PBS. For double immunofluorescence staining, serum of a
Kaposi’s sarcoma patient with high anti-LNA1 activity was used together
with mouse monoclonal anti-EBNA1 OT1x (1 : 200 ; provided by J. M.
Middlorp, Organon Teknika, The Netherlands) and followed by FITCconjugated anti-human IgG (1 : 100 ; Dakopatts) and biotinylated goat
anti-mouse IgG1 (1 : 100 ; Pharmingen). The latter was detected by
rhodamine-conjugated streptavidin (1 : 200 ; Dakopatts). The anti-EBNA
activity of the polyclonal human anti-LNA1 serum was eliminated by
absorption with acetone-fixed IB4 and IARC171 cells. No nuclear
staining was detected when these LCLs were stained with the absorbed
serum.
BEEG
Images were generated using a Leitz DM RB microscope, equipped
with Leica PL Fluotar ¬100, ¬40 and PL APO Ph ¬63 oil immersion
objectives. Leica L4, Tx and A composite filter cubes were used for the
FITC, Texas Red and Hoechst 33258 fluorescence, respectively. The
pictures were captured with a Hamamatsu dual-mode cooled CCD
camera (C4880), recorded and analysed on a Pentium PC (133 MHz, 32
Mb RAM) computer equipped with an AFG VISIONplus-AT frame
grabber board using the Hipic3.2.0 (Hamamatsu), Image-Pro Plus (Media
Cybernetics) and Adobe Photoshop image capturing and processing
software.
Flow cytometry measurements were carried out as previously
described (Pokrovskaja et al., 1996).
+ Western blotting, RT–PCR and in situ hybridization. Detection of membrane-blotted proteins was carried out as previously
described (Pokrovskaja et al., 1996 ; Szekely et al., 1993) with the
modification that chemiluminescence (ECL kit, Bio-Rad) was used to
detect the peroxidase-conjugated goat anti-human IgG. RT–PCR for the
analysis of EBV gene expression as well as of promoter usage was carried
out as described (Chen et al., 1995).
For EBER in situ hybridization the cells were fixed in 4 % paraformaldehyde and were subsequently treated for 10 periods of 10 min
each in the following solutions : distilled water, 0±2 M HCl, twice in PBS,
PBS containing 5 µg}ml proteinase K, PBS, distilled water, 70 % ethanol
and finally 100 % ethanol. After drying, the cells were hybridized with
EBER probes (Dakopatts) following the manufacturer’s instructions. After
washing in 50 % fluoroescent antibody–2¬ SSC and 1¬ SSC, the EBER1
signal was detected by the combination of mouse monoclonal anti-FITC
(1 : 100 ; Dakopatts) and rabbit anti-mouse FITC-conjugated antibodies.
EBER2 was detected by the combination of rhodamine-conjugated sheep
anti-digoxigenin (1 : 10 ; BMH) and rhodamine-conjugated rabbit antisheep immunoglobulin (1 : 50).
Results
Phenotypic characterization and karyotype analysis
The expression of B-cell markers was analysed by immunofluorescence staining and flow cytometry. The LCLs IARC171
Table 1. Expression of B-cell markers in the BC1 and
BCBL1 lines
BC1
CD10
CD77
CD39
CD23
CD21
CD19
IgM
IgG
IgA
Kappa
Lambda
BCBL1
Control
%
mFI*
%
mFI
Cell line
%
mFI
10
0
4
0
0
0
0
0
0
0
0
5
–
1
–
–
–
–
–
–
–
–
3
8
29
3
0
0
0
0
0
0
0
3
11
7
3
–
–
–
–
–
–
–
BL29
100 55
P3HR1
97 977
P3HR1
99 151
IARC171 79 120
IARC171 76 33
P3HR1
96 53
Daudi
97 186
Rael
90 30
–
BL41
99 133
Rael
65 27
* mFI, mean fluorescence intensity (measured by flow cytometry).
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EBV in body cavity lymphoma
Table 2. Karyotype analysis
BC1 (43–51, XY)*
t(1 ; 3)(q12 ; q29)
9q­
12q®
13p­
15p­
16q­
20q­
Observed/analysed
metaphases
8}8
4}8
5}8
8}8
5}8
1}8
5}8
BCBL1 (41–48, XY)
Observed/analysed
metaphases
dup(1)(q21 ; q44)
2p®
6p­
8p­
10p­
11q­
14q­†
16q­
21p­
6}6
2}6
4}6
2}6
2}6
4}6
6}6
5}6
2}6
* ­ and ® indicate added and missing pieces to the given chromosome region, respectively.
† 14q­ does not resemble the myc}IgH translocation because the added piece is bigger than in the 14q­ in
BLs with t(8 : 14).
and Nad20 and the BL cell lines BL29, BL41, P3HR1, Daudi
and Rael were used as positive and negative controls,
respectively. The results are summarized in Table 1. Both body
cavity lymphoma lines were negative for most B-cell markers,
including immunoglobulins. Only a small fraction of the cells
showed some expression of B-cell markers, such as CD10,
CD39 and CD77. The mean fluorescence intensities of the few
positive cells were much lower than in the positive controls.
Karyotype analysis of Giemsa-stained metaphases revealed
multiple chromosomal aberrations in both lines. There was no
consistent, common change (Table 2).
Analysis of latency-associated EBV gene expression by
immunofluorescence, Western blot and in situ
hybridization
Immunofluorescence staining of BC1 cells with monoclonal
antibodies against EBNA1, EBNA2 and EBNA5 as well as with
monospecific absorbed human sera against EBNA1, EBNA3,
EBNA4 and EBNA6 detected only EBNA1. The staining
intensity showed some cell to cell variation but essentially
all BC1 cells were EBNA1-positive. Only a very small fraction
(! 0±1 %) of cells showed entry into the EBV lytic cycle.
EBV-negative BCBL1 cells and the EBV-carrying LCLs
IARC171 and CBMI-RAL-STO served as negative- and
positive-staining controls.
Immunoblotting of total cell lysates from IARC171 LCL
with a human serum with high anti-EBNA titres detected
EBNA1, EBNA2 and the high molecular mass EBNAs (-3, -4
and -6) (Fig. 1 A). Only a single specific band was identified in
the BC1 lysate, absent from the EBV-negative BCBL1. It was
approximately 75 kDa in size, well within the range of the
known EBNA1 size variation among the different EBV strains.
Testing the same cell lysates with affinity-purified human
antibodies that reacted only with the Gly–Ala repeats of
Fig. 1. Western blot detection of EBV- and HHV-8-encoded proteins in the
total cell lysates of BCBL1 and the BC1 lines, IARC171 (LCL) and BL28
line (EBV-negative control), using human sera with high anti-EBNA (A)
and anti-LNA1 (B) titres as well as affinity-purified human
immunoglobulins reacting with the Gly–Ala repeat of EBNA1 (C). Only
EBNA1 is detected in BC1 and IARC171 cells (positive control).
EBNA1 identified the single band in the BC1 lysate as EBNA1
(Fig. 1 C).
Fluorescent in situ hybridization with EBER1- and EBER2specific probes showed that both small RNA species were
present in the nuclei of BC1 but not in BCBL1 cells (Fig. 2 A).
RT–PCR assessment of viral gene expression and
splicing patterns
As shown in Figs 3 and 4, RT–PCR detected EBNA1 and
LMP2A mRNA expression in BC1 cells but not in EBNA2,
LMP1 or LMP2B. Promoter usage for EBNA1 transcription
was studied by primers detecting the YUK and QUK splicing
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L. Szekely and others
Fig. 2. For legend see facing page.
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EBV in body cavity lymphoma
C
Fig. 2. (A) Detection of EBER1 (green) and EBER2 (red) in BC1 but not in BCBL1 cells by fluorescence in situ hybridization.
DNA is stained with Hoechst 33258 (blue). (B) Double immunofluorescent staining of LNA1 (green) with LCL-absorbed
human HHV-8-positive polyclonal serum and of EBNA1 (red) with mouse monoclonal antibody. BC1 cells show no significant
overlap (white) between the two proteins. DNA is stained with Hoechst 33258 (blue). (C) Association of LNA1 (green) with
chromosomes in prometaphase and metaphase in BCBL1 cells. DNA is stained with Hoechst 33258 (blue).
Fig. 3. RT–PCR detects only LMP2A but not EBNA2, LMP1 or LMP2B
mRNA in BC1 cells. B95-8-, IB4-, CBMI-RAL-STO- and LMP2A-transfected
A431 cells served as positive and BCBL1 cells as negative controls.
Arrowheads point to the specific PCR products. Low molecular mass
bands on the LMP1 and LMP2B pictures are PCR primers.
patterns, characteristic for the Cp}Wp and Qp promoter
usage, respectively. BC1 cells were only positive for the QUK,
not the YUK, splice pattern. The B95-8 controls were positive
for both as previously shown (Chen et al., 1995). The BC1
pattern is the same as in phenotypically representative BL cells,
including in vivo tumours and recently established lines with
the corresponding phenotype.
Expression and distribution of the HHV-8-encoded
nuclear antigen (LNA1)
Western blotting with sera from human immunodeficiency
virus-positive Kaposi’s sarcoma patents with high anti-HHV-8
Fig. 4. BC1 cells use the QUK but not the YUK splice pattern, suggesting
that the latency-associated Qp, and not the Cp or Wp, promoter is used to
express EBNA1 in these cells, in contrast to the LCL control IB4 cell line
that uses Wp. B95-8 uses both Qp and Cp promoters.
titres showed that the major specific band corresponded to the
high molecular mass (p226 and p234) LNA1 (Fig. 1 B) (Rainbow
et al., 1997). The same sera detected, by immunofluorescence,
the coarsely speckled, sharply outlined blocks of nuclear
antigen for LNA1 present in all interphase nuclei (Gao et al.,
1996 b ; Simpson et al., 1996). LNA1 remained associated with
the chromosomes during mitosis (Fig. 2 C). There was no
difference in the staining pattern of the EBV-carrying BC1 and
EBV-negative BCBL1 lines.
It has been previously shown that EBNA1, alone among
the EBV-encoded growth transformation-associated proteins,
is chromatin-associated both in interphase and metaphase
nuclei. This was also true in the BC1 line, where the EBNA1
staining was mainly restricted to well-defined nuclear dots,
detected both by ACIF staining with monospecific human sera
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L. Szekely and others
and by indirect immunofluorescence with rabbit polyclonal or
mouse monoclonal antibodies. Double staining of BC1 nuclei
showed that LNA1 and EBNA1 occupy different nuclear
domains, without any colocalization (Fig. 2 B).
Discussion
EBV expression in latently infected cells is regulated in a
host cell phenotype-dependent fashion. Immunoblasts induced
by EBV transformation in vitro, established LCLs and the
immunoblastic lymphomas of immunocompromised individuals express the full set of EBNAs and all three LMPs. We can
refer to this as the full programme. BL cells in vivo and derived
cell lines that have maintained the BL-representative (type I)
phenotype, as well as normal B-cells that carry latent virus, use
a restricted programme and express only EBNA1 and LMP2A.
Non-B-cells that harbour the virus genome, such as certain Tcell lymphomas, Hodgkin’s lymphoma and nasopharyngeal
carcinoma, express a variant of the restricted programme,
characterized by the additional expression of LMP1.
Change from restricted to full expression occurs in B-cells
in connection with immunoblastic transformation. Examples
include the phenotypic drift of EBV-carrying BL lines from
type I to a more immunoblastic (type II–III) phenotype during
prolonged in vitro culturing (Rowe et al., 1987) or by the
outgrowth of latently EBV-infected normal B-cells or chronic
lymphocytic leukaemia cells into established lymphoblastoid
lines (Lewin et al., 1995).
Cell fusion experiments between LCLs and different B- and
non-B-cells showed that the full expression programme
manifested in the Cp promoter-initiated polycistronic
EBNA1–6 transcript was switched off in hybrids between
LCLs and non-B-cells that have acquired the phenotype of the
non-B-parent, while the restricted monocistronic EBNA1
transcript was turned on (Altiok et al., 1992 ; Contreras-Brodin
et al., 1991 ; Contreras-Salazar et al., 1989).
Our phenotypic characterization of the two BC lines,
together with earlier evidence (Gao et al., 1996 a), showed that
both lines have a closely similar phenotype. Their B-cell origin
is supported by the IgH rearrangement, previously detected
with a JH probe (Cesarman et al., 1995 b). Most B-cell markers
were absent, however, including the pan-B marker CD19,
CD21 and immunoglobulin heavy and light chains.
A minority of the cells expressed some B-cell markers, such
as CD10, CD39 and CD77, in a small fraction of the cells and
at a low level. This phenotype does not correspond to any
conventional B-cell subtype. It is possible that the cells of the
two BC lines correspond to an unusual pro- or pre-B-cell.
However, EBV transformed pro- and pre-B-cells have been
grown in culture previously, but their phenotype was similar
to immunoblastic LCLs and they expressed the full EBV
programme (Altiok et al., 1989).
Recently, in parallel experiments, Horenstein et al. (1997)
analysed five EBV}KSHV double-infected primary effusion
BEFA
lymphomas in order to determine the EBV gene expression
pattern and had the same results as those presented above.
Two conclusions can be drawn from these findings. First,
the use of the restricted, rather than the full, programme by the
virus in the BC1 cell is consistent with the widely accepted
notion that the full programme can only be expressed in
immunoblasts. This is the first confirmation of that conclusion
in a non-immunoblastic B-cell-derived lymphoma line that is
not of BL origin. Second, the finding further reinforces the
impression that B-cell clones that maintain a restricted
programme during proliferation can only be derived from
established malignancies in vivo. In vitro transformation selects
for proliferating immunoblasts that express the full programme.
Immunoblastic proliferation can occur in vivo, but only in
immunodeficient systems. This is consistent with the high
immunogenicity of EBNA2–6 for T-cells.
Cells infected in vivo that have a restricted programme can
only proliferate in vitro if they are driven by some EBVunrelated stimulus. BL cells are driven by c-myc that has been
constitutively activated by immunoglobulin}myc translocation. It may be surmised that the BC lines are driven by
HHV-8. This is consistent with the existence of the EBVnegative, HHV-8-positive BCBL1 line. Alternatively, their
immortality may stem from genetic changes, as suggested by
the numerous, but essentially non-overlapping, chromosomal
anomalies found in the two lines.
This work was supported by the Department of Health and Human
Services and by grants from the Swedish Cancer Society (Cancerfonden),
Swedish Medical Research Council and Concern Foundation}Cancer
Research Institute. We thank Mika Popovic for providing the cell lines.
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BEFC
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Received 5 November 1997 ; Accepted 27 January 1998
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