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Journal of Virology, June 1999, p. 5149-5155, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Characterization of Monoclonal Antibodies Raised
against the Latent Nuclear Antigen of Human Herpesvirus 8
Paul
Kellam,1,*
Dimitra
Bourboulia,1
Nicolas
Dupin,1
Chris
Shotton,1
Cyril
Fisher,2
Simon
Talbot,1,
Chris
Boshoff,1,3 and
Robin A.
Weiss1
Institute of Cancer Research, Chester Beatty Laboratories,
London SW3 6JB,1 Royal Marsden Hospital,
London SW3 6JJ,2 and Department of
Oncology, Royal Free and University College Medical School, University
College London, London W1P 6BT,3 United
Kingdom
Received 8 October 1998/Accepted 16 February 1999
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ABSTRACT |
Human herpesvirus 8 (HHV-8; also designated Kaposi's
sarcoma-associated herpesvirus) is the likely etiological agent of
Kaposi's sarcoma (KS). HHV-8 encodes a latent nuclear antigen (LNA)
which is the product of the viral gene orf 73. LNA is recognized by most infected patient sera and is the basis of current
immunofluorescence assays used in epidemiological studies of HHV-8
infection. Here we describe the characterization of four monoclonal
antibodies raised to the C-terminal third of LNA-glutathione
S-transferase fusion proteins. These monoclonal antibodies
recognized discrete linear epitopes within the C terminus and
repetitive region of LNA, detected antigen in primary effusion lymphoma
(PEL) cells, and precipitated a 220- to 230-kDa protein doublet
corresponding to LNA from HHV-8-infected PEL cell lines. In situ
immunocytochemistry of KS lesions with these antibodies show that LNA
is extensively expressed in KS spindle cells.
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TEXT |
Human herpesvirus 8 (HHV-8; also
designated Kaposi's sarcoma-associated herpesvirus) is the first known
human member of the genus Rhadinovirus within the
Herpesviridae (9). HHV-8 is related to the New
World primate rhadinovirus herpesvirus saimiri (1), a rhesus
macaque rhadinovirus (10), and macaque retroperitoneal fibromatosis virus isolates Mn and Mm (25). More recently,
equine herpesvirus 2, bovine herpesvirus 4, alcelaphine herpesvirus 1, and murine herpesvirus 68 have been tentatively classified as rhadinoviruses. HHV-8 is associated with all epidemiological forms of
Kaposi's sarcoma (KS) (4, 6, 28). HHV-8 is also associated with primary effusion lymphoma (PEL) (7) and a subset of
multicentric Castleman's disease (12, 30). Epidemiological
studies using PCR detection of specific HHV-8 genomic sequences, and
immunological assays for antibodies to the major latent nuclear antigen
(LNA) and other antigens, have shown that HHV-8 largely fulfills
epidemiological criteria for causation in KS (5, 13, 14, 16, 19,
20, 22, 29, 32).
Sera from HHV-8-infected individuals react with a specific nuclear
antigen in latently infected PEL cell lines which is characterized by a
punctate nuclear immunofluorescence pattern (19). Screening of cDNA libraries with an HHV-8-positive patient serum identified this
nuclear antigen as the product of the viral gene open reading frame 73 (orf73), and the encoded protein was designated LNA or LANA (17,
18, 24). LNA is expressed from a latently controlled 5.32-kb
transcript that also encodes the viral cyclin (v-Cyc) and v-FLIP
(11, 18). The 5.32-kb latent transcript is spliced to form a
1.7-kb transcript that encodes only v-Cyc and v-FLIP (11, 18,
27). The predicted LNA protein is 1,162 amino acids long and has
a theoretical molecular mass of 135 kDa. In contrast to LNA's
predicted molecular size, the protein has an apparent molecular size of
220 to 230 kDa when analyzed by Western blotting (13, 18,
24). LNA can be divided into three domains: an N-terminal
337-amino-acid domain; an extremely hydrophilic central domain of 585 amino acids consisting of multiple repeat elements predominantly
containing the charge polar amino acids glutamine, glutamic acid, and
asparagine; and a C-terminal 240-amino-acid domain. In contrast to the
central domain, the N- and C-terminal domains are rich in basic amino
acids. The differently charged domains of the protein may in part
explain the aberrant running of LNA on sodium dodecyl sulfate
(SDS)-polyacrylamide gels.
LNA is thus far the only latent nuclear antigen described for HHV-8 and
has no obvious sequence identity to known proteins. However, the
immunogenic nature, nuclear localization, and latent expression of LNA
suggest that this protein is analogous to Epstein-Barr virus (EBV)
nuclear antigens (EBNAs). EBNAs have a pivotal role in maintenance and
replication of the virus episome and transforming cells. Insights into
the function of LNA could be essential in the understanding of HHV-8
latency and tumor formation. Here we describe the characterization of
four monoclonal antibodies (MAbs) to the C terminus of orf73. These
antibodies recognize distinct linear epitopes of LNA, confirm that
orf73 encodes the major latent nuclear antigen of HHV-8, and detect LNA
expression in PEL cell lines and in KS nodules.
Generation of MAbs against LNA.
Glutathione
S-transferase (GST) proteins of the C terminus of orf73
(GST-C14 and GST-C17) were described previously (18). Briefly, GST-C14 contains amino acids 803 to 1113 of orf73 encompassing 126 amino acids of the central repeat domain as well as 184 amino acids
of the C-terminal basic domain (Fig. 1).
GST-C17 contains amino acids 803 to 942 comprised almost entirely of
repetitive coding sequence. GST-C14 served as the backbone for making
further defined deletions of the C terminus of orf73 by using three
unique restriction enzyme sites. GST-C7 was constructed by digesting GST-C14 with NruI (in orf73) and NotI (in
pGEX-4T3 [Pharmacia]), followed by end repair and ligation. GST-C11
was constructed by digesting GST-C14 with NruI and
NotI and cloning the restriction fragment into the
SmaI and NotI sites of the vector pGEX-4T1
(Pharmacia). GST-C11 served as the backbone for two further deletion
clones. GST-C9 and GST-C10 were constructed by restriction enzyme
digestion with StuI plus NotI and AgeI
plus NotI, respectively, followed by end repair and
ligation. Recombinant protein-expressing clones were confirmed by DNA
sequence analysis. In addition, two polyhistidine fusion proteins were
constructed. The entire BamHI-to-NotI C-terminal restriction fragment from GST-C14 was cloned into the polyhistidine fusion protein vector pTrcHis2C (Invitrogen) via BamHI and
HindIII following conversion of the NotI and
HindIII sites to blunt ends to produce clone TH-C14. The
NruI-to-NotI fragment corresponding to GST-C11
was PCR amplified and cloned to produce TH-NN. Large-scale production
and single-step affinity purification of fusion proteins (GST
[Pharmacia] and polyhistidine [Invitrogen]) were performed according to the manufacturer's instructions. All fusion proteins were
recognized by antibodies to GST (B14; Santa Cruz) or to the Myc epitope
present in the polyhistidine fusion proteins (data not shown). Pools of
affinity-purified GST fusion proteins were used for intraperitoneal
immunization of rats at 3-week intervals. Three days following the
final boost, rats were sacrificed to obtain splenocytes for hybridoma
production by standard procedures. Hybridomas were screened by
enzyme-linked immunosorbent assay (ELISA) and Western blotted against
the polyhistidine fusion protein TH-C14, and positive hybridomas were
subcloned. Following a second ELISA screening, reactive MAbs were bulk
cultured and antibodies were purified from culture supernatants by
antibody affinity chromatography. This procedure produced MAbs ranging
in concentration from 1 to 2 mg/ml. The isotype of each MAb was
determined by standard methods. Four MAbs, LN20, LN53, LN69, and LN72,
with isotypes immunoglobulin M (IgM), IgG2c, IgG1 and IgG2b,
respectively, were raised to these GST fusion proteins.
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FIG. 1.
Fusion protein mapping of LNA MAbs. The C-terminal
locations of six GST-LNA fusion proteins (GST-C14, -C7, -C17, -C11,
-C9, and -C10) and two histidine-tagged fusion proteins (TH-C14 and
TH-NN) are shown relative to that of full-length LNA. Numbers represent
amino acids relative to the putative start methionine at codon 127296 of the published HHV-8 genome (26). The hatched box
represents the extent of the repetitive coding region of LNA.
Restriction enzyme sites NruI, AgeI, and
StuI used to construct the fusion protein clones are
indicated. The reactivity of each MAb to each fusion protein is shown
as A410 above background of greater than 0.5 absorbance units (+++), between 0.1 and 0.5 absorbance units (++), or
between 0.02 and 0.1 absorbance units (+). The epitope-mapped position
of each MAb is indicated on TH-C14. The epitopes are EQEQE for LN53,
EVDYPV for LN72, and THPKKPHPRYQQ for LN69.
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Mapping of the epitopes recognized by the MAbs.
The antigenic
epitopes recognized by these MAbs were mapped by using the panel of GST
and polyhistidine fusion proteins (Fig. 1) in an ELISA format. Briefly,
fusion proteins were immobilized onto 96-well plates (Immulon 2; Dynex
Technologies) in 10 mM sodium phosphate buffer (pH 7.0) by overnight
incubation at 4°C. A standard ELISA protocol was followed, using
appropriate dilutions of anti-LNA MAbs and 1:2,000 dilution of goat
anti-rat alkaline phosphatase-conjugated secondary antibody (Sera-Lab)
as described elsewhere (15). Reactions were visualized by
using p-nitrophenyl phosphate (Sigma Fast) according to the
manufacturer's instructions. Antibody LN20 recognized all recombinant
proteins to various degrees, as shown by reactivity to clones of the
repetitive and nonrepetitive C-terminal regions (Fig. 1). The
recombinant proteins represent a nonoverlapping set and contain no
apparent common epitopes, which suggests that LN20 is a mixed antibody
population, requiring further rounds of monocloning. Antibody LN53
recognized recombinant proteins GST-C14, GST-C17, GST-C7, and TH-C14
(Fig. 1), demonstrating that the minimum epitope recognized by this
antibody is the repetitive region of orf73 encompassing GCT-C17 or the
13 amino acids C terminal to the repetitive region. Antibody LN69
recognized only GST-C9, GST-C10, TH-C14, and TH-NN. This mapped the
LN69 minimal epitope to the 37 amino acids (amino acids 981 to 1018 of
LNA) present in GST-C10 (Fig. 1). It is unclear why LN69 did not
recognize GST-C14 or GST-C11, as both contained the minimal epitope.
Antibody LN72 recognized GST-C14 and -C7 but not GST-C17 and thus
recognizes a 39-amino-acid region (from amino acids 942 to 981) of LNA.
To confirm and further refine the minimal epitopes for each MAb, multiple overlapping peptides corresponding to the regions of LNA
identified by ELISA were synthesized by using the SPOTS system (Genosys). Peptide mapping revealed that LN69 recognized the epitope THPKKPHPRYQQ, LN72 recognized the epitope EVDYPV, and LN53 recognized the epitope EQEQE. These epitopes are contained within the minimal LNA
recombinant proteins identified by ELISA, thus confirming the binding
sites of the three MAbs. The LN53 epitope is contained within the
repetitive region of LNA, resulting in 23 copies of the LN53 epitope in
full-length LNA. Database searching revealed that this epitope is
present in human proteins, including the alpha-type calcitonin
gene-related peptide precursor (P06881), drebrin (Q16643), translation
initiation factor IF-2 (P46199), and aldehyde dehydrogenase (P30838),
but only in single copy. The LN69 and LN72 epitopes have only partial
homologies to known cellular proteins.
Immunoprecipitation and immunofluorescence assay (IFA) of HHV-8
LNA.
Previous studies have shown that the authentic LNA protein
encoded in PEL cell lines is a doublet with an apparent molecular mass
of 220 to 230 kDa (13, 18, 24), in contrast to the predicted
molecular mass of 135 kDa. The reasons for the size difference are
unclear. MAbs LN53, LN69, and LN72 produced in this study were used to
immunoprecipitate LNA from the PEL cell line BC-3 (2).
Briefly, cells were starved for 1 h in methionine- and
cysteine-free RPMI (Sigma) supplemented with 10% dialyzed fetal calf
serum (FCS; Gibco), washed, and then labeled for 4 h in fresh
medium supplemented with a mixture of [35S]methionine and
[35S]cysteine (70 µCi/ml in medium; Amersham Pro-mix)
plus 10% dialyzed FCS. After being washed, the cell pellets were lysed
for 30 min on ice in 1 ml of lysis buffer (150 mM NaCl, 1.0% Nonidet
P-40, 50 mM Tris-HCl [pH 8]) supplemented with the protease inhibitor phenylmethylsulfonyl fluoride (100 µg/ml), pepstatin A (0.7 µg/ml), and leupeptin (5 µg/ml), and lysates were cleared by centrifugation at 14,000 × g for 10 min at 4°C. Extracts from the
equivalent of 107 cells were precleared for 1 h with
protein G-Sepharose beads (Sigma) equilibrated in PBST
(phosphate-buffered saline [PBS] plus 1% Triton X-100) and then
immunoprecipitated overnight at 4°C with protein G-Sepharose beads
precoated with saturating amounts of the indicated MAbs.
Immunoprecipitates were washed and loaded onto an SDS-8%
polyacrylamide gel. Only MAb LN53 was able to precipitate a doublet
protein of 220 to 230 kDa. LN69 and LN72 precipitated proteins present
in both BC-3 and control Ramos cells but did not precipitate LNA (Fig.
2a). The identity of the major 105- to
110-kDa protein precipitated by LN69 from both cell types is unclear,
and a database search using the LN69 epitope failed to identify any
candidate proteins. Antibodies LN20, LN53, and LN72 reacted with a
protein doublet of 220 to 230 kDa by Western blotting (Fig. 2b). In
addition, a protein of approximately 180 kDa was recognized by the
antibodies by Western blotting (Fig. 2b). Reactivity to proteins of
this size had previously been seen in Western blots with HHV-8-positive
patient sera but had not been assigned as LNA specific (13, 18,
24).
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FIG. 2.
Immunoprecipitation and Western blot analyses using LNA
MAbs. (a) Immunoprecipitates from 107 BC-3 cells or Ramos
cells per lane were resolved on SDS-8% polyacrylamide gels. LN53 was
able to precipitate a 220- to 230-kDa doublet corresponding to LNA.
Pooled antibody isotype controls to HIV gp120 for each LNA MAb as well
as Protein G-Sepharose-only immunoprecipitation controls (Control and
Protein G lanes, respectively) were used in identical
immunoprecipitations. (b) Western blot of 2 × 105
cell equivalents of total protein extract from BC-3 (2) and
Ramos cells detected with LNA MAb LN20. A doublet of 220 to 230 kDa,
and a smaller reactive band of approximately 180 kDa (marked on blot)
are seen. Identical lanes were detected with an anti-rat alkaline
phosphatase-conjugated secondary antibody as a control. The same
results were obtained for LN53 and LN72 (data not shown).
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IFAs are used extensively in the seroepidemiology of HHV-8
(
6). Under standard IFA conditions, MAbs LN20, LN53, and
LN72
were able to produce the punctate nuclear fluorescence pattern
associated with HHV-8 LNA in PEL cell lines (Fig.
3a to c). The
antibodies were specific
for HHV-8 LNA, with no apparent cross-reaction
to other proteins
present in control B-cell lines Daudi and Ramos
(Fig.
3d and e). The
best antibody for IFA was LN53, which could
be diluted to 1:10,000
without significantly affecting the characteristic
LNA IFA pattern in
BCP-1 cells. All antibodies were also able
to recognize LNA expressed
in BCP-1 cells by fluorescence-activated
cell sorting analysis (FACS)
analysis (Fig.
4a). No cross-reactivity
was observed for each antibody
with the control B-cell lines Ramos
(EBV negative) and Daudi (EBV
positive) (representative controls
[Fig.
4b]). FACS analysis was also performed
with LN53 on one
other PEL, BC-3, and on the HHV-8/EBV-coinfected
B-cell lymphoma
line HBL-6 (Fig.
4b). The BC-3 cell line exhibited the
characteristic
FACS pattern showing that the majority of cells express
LNA. Analysis
of HBL-6 showed that most cells expressed LNA, but a
subpopulation
appeared to be nonexpressing. Whether this is a
consequence of
HHV-8/EBV coinfection is currently being studied.
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FIG. 3.
LNA MAbs react with nuclear bodies in PEL cells. MAbs
LN20 (a), LN53 (b), and LN72 (c) all react by IFA to nuclear antigenic
structures in BCP-1 cells (3, 14) identical to those
detected with HHV-8-positive KS patient serum (f). Antibody LN53 did
not react with nuclear antigens present in control Daudi (d) or Ramos
(e) cells. LN20 and LN72 were also negative on control cells (not
shown). The bar in panel a applies to all panels.
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FIG. 4.
FACS profile of LNA MAbs on HHV-8-infected cells. The
MAbs were tested by FACS on cell lines BCP-1, BC-3, HBL-6, Daudi, and
Ramos; 5 × 105 cells were permeabilized, and LNA
antibodies were used at the dilution of 1:100. The cells were incubated
for 1 h with individual antibodies in PBS-0.05% saponin-1%
FCS), washed three times in PBS-0.1% (vol/vol) Tween, and incubated
for 1 h with rabbit anti-rat fluorescein isothiocyanate-conjugated
(DAKO) secondary antibody. Analysis was performed on a FACS 440 (Becton
Dickinson). Fluorescent intensity is expressed on an arbitrary
logarithmic scale. Black histograms represent antibody-stained cells,
and white histograms represent secondary antibody conjugate controls.
(a) All antibodies detected the LNA-expressing cell population in BCP-1
cells. (b) Antibody LN53 does not detect LNA in the negative control
cells Ramos (EBV negative) and Daudi (EBV positive) but does detect the
LNA-positive population in the HHV-8-infected BC-3 and HBL-6 cell
lines. All other antibodies are negative for the control cell lines
(data not shown).
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HHV-8 LNA is abundantly expressed in KS lesions.
To date few
protein-specific antisera or MAbs have been raised to HHV-8 proteins.
Antibodies to virus-encoded interleukin-6 (vIL6) (21) and to
orf26 (23) and orf59 (8) protein products have
been used to study protein expression in a variety of HHV-8-infected cell types. However, no antibodies to latent proteins have been used in
such studies. LN53 and LN72 but not LN20 and LN69 reacted with antigen
in paraffin-embedded KS tissue. As MAb LN53 was assessed to be the best
antibody raised, it was used further in the examination of the
expression of HHV-8 LNA in KS lesions. LNA was extensively expressed in
late-stage nodular KS, predominantly in cells of spindle-shaped
morphology (Fig. 5b to d). LNA-positive
cells account for more than 90% of all cells present in nodular KS
lesions. The staining was exclusively nuclear, and in most cases a
punctate nuclear staining, reminiscent of the nuclear IFA staining seen in PEL cell lines (Fig. 3), was seen (Fig. 5d). No expression of LNA
was detected in surrounding normal dermis (Fig. 5b). No staining was
observed in the control tumors angiosarcoma and hemangioma (not shown).
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FIG. 5.
LN53 reactivity to LNA in nodular KS.
Immunocytochemistry was performed on a paraffin-embedded tumor from
classical KS. Permeabilization was performed by microwave. Negative
control biopsies were from angiosarcomas and hemangiomas (not shown).
After incubation with normal rabbit serum (DAKO), MAbs were applied for
1 h at 22°C followed by two washes in PBS-0.1% (vol/vol)
Tween. The secondary biotin-conjugated antibody (rabbit anti-rat; DAKO)
was applied for 30 min followed by washing. Antibody reactions were
visualized with streptavidine-alkaline phosphatase (Vector
Laboratories) and a substrate red chromogen (Vector). Adjacent sections
were stained by standard methods. (a) Hematoxylin and eosin staining
showing KS nodule (left) and surrounding dermis (right). (b) Adjacent
sections stained with LN53 showing clear demarcation between LNA
expression in the KS lesion and not in the surrounding dermis
(magnification, ×40). Almost all spindle-shaped cells are positive for
LNA (×60) (c), with a stippling pattern reminiscent of the staining of
PEL cells (×160) (d).
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Conclusions.
One of the predominant proteins encoded by HHV-8
during the latent phase of the virus life cycle is LNA, the product of
orf73 (17, 18, 24). Here we describe the characterization of
four MAbs to the C terminus of LNA. The properties of the MAbs are summarized in Table 1. These antibodies
recognize LNA in immunofluorescence, immunoprecipitation, in situ
immunocytochemistry, and FACS analyses. Three of the MAbs recognized
different discrete epitopes in the C terminus of LNA. The most reactive
MAb, LN53, recognized the minimal epitope EQEQE present as 23 copies in
LNA. This provides an explanation for this antibody's high reactivity
in all assays used, as the repeat units of LNA should provide multiple
epitopes for the antibody. The majority of the EQEQE epitopes are
located in the extensive leucine zipper motif of LNA. Structure
predictions of this region and knowledge of coiled-coil leucine zipper
structures suggest that the EQEQE motif is located in a regular pattern
on the leucine zipper alpha helices, allowing accessibility of the antibody under native protein folding conditions. This correlates with
the ability of LN53 to immunoprecipitate LNA from cell extracts.
Immunoprecipitation studies confirmed that LNA exists as a protein
doublet of 220 to 230 kDa in BC-3 cells (Fig.
2a). A protein
of similar
size was recognized by Western blotting (Fig.
2b).
As with
HHV-8-positive patient sera, the LNA antibodies produce
a punctate
immunofluorescence pattern in PEL cell lines (Fig.
3). Using LN53, we
demonstrated extensive LNA expression in nodular
KS. There is a clear
demarcation between the KS nodule and surrounding
dermis (Fig.
5b). The
majority of cells showed punctate nuclear
staining reminiscent of the
nuclear staining in HHV-8-positive
PEL cells (Fig.
5d), which suggests
that the nuclear structures
containing LNA present in PEL cells are
also present in HHV-8-infected
KS spindle cells. LNA expression has
recently been demonstrated
in all tissue and cell types associated with
HHV-8 infection and
disease (
12). At present it is unclear
what LNA associates with
in the nuclei of latently infected cells.
Studies have shown that
LNA remains associated with chromosomes during
mitosis (
31).
This feature is reminiscent of EBNA1, which is
chromatin associated
in both interphase and metaphase nuclei
(
31). However, in cells
coinfected with EBV and HHV-8, EBNA1
and LNA do not colocalize,
indicating that they occupy different
nuclear domains (
31).
A careful study of colocalization and
protein association with
LNA by using MAb LN53 should provide insights
into the function
of LNA in HHV-8
latency.
The expression pattern of all known HHV-8 genes in the PEL cell line
BC-1 has shown that relatively few transcripts are abundantly
produced
during HHV-8 latency (
27). Using antibody LN53, we
show
expression of one such latent protein, LNA, in both PEL cell
lines and
KS tumors. Antibodies to other, predominantly lytic
HHV-8 proteins,
namely, the products of orf59 (
8), a putative
DNA polymerase
accessory protein, and orf26, a 32-kDa inducible
protein
(
23), have been described. Antisera to vIL6 showed
expression
in approximately 50% of uninduced BCP-1 cells and primary
ascitic
PELs, suggesting constitutive vIL6 expression in latently
infected
PEL cells (
21). However, vIL6 protein was rarely
detected in
KS lesions, and when detected, it accounted for less than
2% of
HHV-8-infected cells, indicating that vIL6 could be
differentially
expressed in hematopoietic versus mesenchymal cells.
Whether LNA
is involved, through interaction with different
tissue-specific
cellular factors, in the differential expression of
HHV-8 genes
and whether this contributes to HHV-8 pathogenesis remain
to be
determined. LNA is the first latent protein described for HHV-8,
and use of our MAbs to understand its function is likely to be
crucial
to the understanding of HHV-8 cellular transformation
and virus
pathogenesis.
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ACKNOWLEDGMENTS |
We thank, Y. Chang, S. J. Gao, and P. S. Moore for
providing the BCP-1 cell line. BC-3 cells were kindly provided by E. Cesarman. The LNA MAbs were provided by Advanced Biotechnologies Inc.,
Columbia, Md.
This work was supported by the Cancer Research Campaign and GlaxoWellcome.
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FOOTNOTES |
*
Corresponding author. Present address: Windeyer
Institute of Medical Sciences, University College London, 46 Cleveland
St., London W1P 6DB, United Kingdom. Phone: 44-171-504-9343. Fax:
44-171-387-3310. E-mail: p.Kellam{at}ucl.ac.uk.
Present address: Department of Medical Microbiology, Edinburgh
University Medical School, Edinburgh EH8 9AG, United Kingdom.
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Journal of Virology, June 1999, p. 5149-5155, Vol. 73, No. 6
0022-538X/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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