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J Virol, June 1998, p. 5182-5188, Vol. 72, No. 6
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Limited Transmission of Kaposi's
Sarcoma-Associated Herpesvirus in Cultured Cells
Rolf
Renne,1
David
Blackbourn,2
Denise
Whitby,3
Jay
Levy,2 and
Don
Ganem1,2,*
Howard Hughes Medical Institute and
Department of Microbiology and Immunology1
and
Department of Medicine,2
University of California, San Francisco, San Francisco, California
94143-0414, and
Chester Beatty Laboratories, Institute of
Cancer Research, London SW3 6JB, United Kingdom3
Received 29 December 1997/Accepted 10 March 1998
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ABSTRACT |
Kaposi's sarcoma-associated herpesvirus (KSHV) (also called human
herpesvirus 8) is a novel gammaherpesvirus strongly implicated in the
pathogenesis of Kaposi's sarcoma. Although virions can be produced in
high yield from latently infected B-cell lines treated with phorbol
esters, little is known about the infectivity of such virus, and
efficient serial propagation of KSHV has been problematic. Here we
report on the infectivity of KSHV produced from phorbol-induced BCBL-1
cells, employing an assay based on the detection of a spliced late mRNA
by a sensitive reverse transcriptase PCR (RT-PCR) method. The results
of this study confirm previous observations that 293 cells are
susceptible to viral infection; however, infection with BCBL-1-derived
virus is inefficient and the pattern of viral gene expression in
infected cells may not fully reproduce that of authentic lytic
infection. In keeping with this finding, serial propagation of
BCBL-1-derived virus could not be demonstrated on 293 cells. Eleven of
38 other cell lines tested also supported KSHV infection, as judged by
this RT-PCR assay, including cells of B-cell, endothelial, epithelial, and fibroblastic origin; however, in all cases, infection proceeded at
or below the levels observed in 293 cells.
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INTRODUCTION |
Kaposi's sarcoma-associated
herpesvirus (KSHV), also called human herpesvirus 8, is a novel
human gammaherpesvirus that is tightly linked to several human
diseases. As implied by its name, evidence of viral infection is found
in virtually all cases of Kaposi's sarcoma (KS), (reference
14 and references therein), and several lines of
evidence point to a key etiologic role in this disease. KSHV infection
precedes clinical KS, is highly associated with increased KS risk in
all populations thus far studied (15, 16, 30), and targets
the endothelial (spindle) cell thought to be the prime determinant of
KS tumorigenesis (7, 27). In addition, viral genomes are
regularly found in primary effusion lymphoma (PEL) (a rare,
AIDS-related B-cell neoplasm) (9) and in a second
lymphoproliferative disorder, multicentric Castleman's disease
(26); more indirect links to multiple myeloma have recently been suggested (22, 24).
Attempts to cultivate KSHV in vitro have met with mixed success. B-cell
lines derived from PEL are latently infected with KSHV (10,
21). One such line, BCBL-1, has been extensively characterized in
our laboratory (20, 21). Unlike many PEL-derived lines, it
harbors KSHV in the absence of concomitant Epstein-Barr virus (EBV)
infection; viral genomes are maintained in the nucleus as circular
episomes whose expression is highly attenuated (20, 21).
Treatment of these cells with phorbol esters results in dramatic
induction of lytic replication, with 20 to 30% of the cells displaying
evidence of late gene expression and with the accumulation of large
quantities of morphologically correct virions in the culture medium
(21). Several other PEL cell lines with similar properties
have since been described (2, 13), though other widely used
lines (e.g., BC-1) (10) appear to harbor rearranged genomes
that do not support virion production or do so very poorly (18, 19). These B-cell lines have been enormously useful
in KSHV research, allowing experimental study of latent and lytic infection as well as serving as sources of viral antigens for serologic
testing. However, they do not allow analysis of viral infectivity.
Since all cells in the culture are already infected and since most
cells die following induction (either from viral cytopathic effect or
from phorbol ester toxicity), there is little opportunity to observe
horizontal spread of infection in such cultures. Attempts to transmit
infection to other cell lines by inoculation with B-cell-derived
materials have been made but have not yielded a clear picture.
Infection has generally been assayed by PCR for viral DNA. Using this
assay, several groups have described limited or transient transfer to
several recipient lines (12, 29), but interpretation has
been complicated by the fact that this assay does not reliably
distinguish authentic infection from persistence of viral DNA sequences
derived from the inoculum. For example, material derived from BC-1
cells, a line whose rearranged genome is likely to be too large to
permit efficient encapsidation, has been reported to be infectious in
Raji cells (EBV-positive Burkitt's lymphoma line) by this criterion
(19). More recently, Foreman et al. (12) have
examined the spread of KSHV isolates from primary explants of KS tissue
to cultured cell monolayers, again using DNA PCR to assay the transfer.
They showed that infection could be transferred to 293 cells, albeit
inefficiently, and reported that serial transmission from 293 cells was
observable. Based in part on their findings, we have explored the
infectivity of KSHV derived from the PEL cell line BCBL-1, using a
reverse transcriptase PCR (RT-PCR) assay that reliably distinguishes
authentic infection from persistence of the inoculum. Here we show that
BCBL-1-derived virus will reproducibly infect 293 cells and a variety
of other cell lines but that infection is both inefficient and
abortive, in that the products of infection cannot sustain further
transmission of infection.
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MATERIALS AND METHODS |
ORF29 RT-PCR assay.
Two primers at nucleotide position 53915 (29A [GCA CGT AGC CAA CTC CGT G]) and nucleotide position 50345 (29B
GCA GGA AAC TCG TGG AGC G) spanning both sides of open reading frame
29A (ORF29A) and ORF29B were synthesized to characterize the splice
donor and acceptor sites of this gene. Total RNA from BCBL-1 cells and
from KS biopsy material was extracted by using RNazol (Tel-Test, Inc., Friendswood, Tex.) according to the supplier's protocol. Total RNA
(500 ng) was reverse transcribed by using 200 U of Moloney murine
leukemia virus RT (Gibco BRL) in a total volume of 20 µl containing
125 µM dATP, dGTP, and dTTP, 20 U of RNasin (Promega), and 120 pmol
of random hexanucleotide primers (Boehringer Mannheim). After
incubation at 42°C for 35 min, the reaction was stopped by heating to
95°C for 5 min. To each RT reaction mixture, 80 µl of a PCR mix
containing 10× PCR buffer, 100 pmol of each primer, and 5 U of
Taq polymerase (Perkin Elmer) was added. Each reaction mixture was overlaid with 50 µl of mineral oil prior to amplification for 30 cycles of PCR (1 cycle consists of 30 s at 94°C, 1 min at
58°C, and 1 min 30 s at 72°C) (6). Amplification
products were directly ligated into pCRII (Invitrogen, San Diego,
Calif.), and derived clones were sequenced by using Sequenase
(Amersham) according to the supplier's protocols. For all infectivity
testings, 42 cycles of PCR (1 cycle consists of 30 s at 94°C, 1 min at 58°C, and 1 min 30 s at 72°C) were performed.
Amplification products were electrophoresed in 1.5% agarose gels, and
DNA was transferred to nylon membranes (Hybond-N). Filters were then
hybridized to probe generated by PCR using a nested pair of
ORF29-specific oligonucleotides at nucleotide position 50366 (29Bi
[CTG ACG AGT TCA CGG ATG]) and 53815 (29Ai [TAC ACG CGA CCC GGA
GGA]) at 65°C in Church buffer (11). Probes were
32P labeled by using a Random DNA Prime kit from Amersham.
Preparation of inoculum and infections.
BCBL-1 cells were
induced by treatment with tetradecanoyl phorbol acetate (TPA) as
previously described (21). KSHV virions were pelleted and
concentrated in three steps. First, induced-cell cultures were
centrifuged for 5 min at 4,000 rpm, and supernatants were transferred
to fresh tubes and centrifuged for 30 min at 4,000 rpm. KSHV virions
were then pelleted from these cell-free supernatants by
ultracentrifugation for 2 h at 15,000 rpm. Viral pellets were
resuspended in 1/30 of the original volume using RPMI 1640 without
fetal calf serum (FCS). To prepare virions from uninduced BCBL-1 cells,
the TPA induction step was omitted. For most infectivity assays, 1 ml
of inoculum (which equals 30 ml of supernatant) was added to monolayers
(30 to 50% confluency) or to suspension cultures at a density of
2 × 105 to 5 × 105 cells/ml. After
8 h, cells were washed with phosphate-buffered saline and overlaid
with fresh medium. In experiments in which TPA was used,
virus-inoculated cells were treated 8 to 12 h prior to harvest
with TPA at a concentration of 10 ng/ml. Total RNA was extracted 48 to
96 h after inoculation. To prepare inocula from cell extracts,
TPA-induced or uninduced BCBL-1 cells were lysed by three cycles of
freezing and thawing. These lysates were centrifuged at 10,000 × g for 10 min and finally filtered through a
0.45-µm-pore-size membrane.
Analysis of infected 293 cell expression pattern.
Total RNA
was extracted with RNazol (Tel-Test, Inc.) according to the supplier's
protocol, and poly(A)+ RNA was selected by using the
Oligotex mRNA Kit (Qiagen). To generate radiolabeled cDNA probes, 100 ng of poly(A)+ RNA from either TPA-induced BCBL-1 cells or
KSHV-infected 293 cells was reverse transcribed by using 200 U of
Moloney murine leukemia virus RT in a total volume of 50 µl
containing 0.5 mM dATP, dGTP, and dTTP, and 0.1 mM dCTP, 50 µCi (1 Ci = 37 Gbq) of [
-32P]dCTP, 50 U of RNasin
(Promega) and about 50 pmol of random hexanucleotide primers
(Boehringer Mannheim). The reaction mixture was incubated at 37°C for
1 h, and the reaction was stopped by heating to 95°C for 5 min.
The labeled cDNA probes were hybridized to filters containing cloned
KSHV DNA at 65°C overnight in Church hybridization buffer
(11). To produce filters, nine lambda clones containing ca.
120 kbp of KSHV DNA were digested with SalI or
SacI and 2 to 4 µg of each clone was loaded on a 0.8%
agarose gel, separated by electrophoresis, and transferred to nylon
membranes (Hybond-N).
Cell cultures.
All Burkitt's lymphoma cell lines (BL30,
BL41, BL41B95-8, Namalwa, Ramos, Raji, Daudi, Loukes, IB4, and BJAB)
were a kind gift of Eliott Kieff and were grown in RPMI 1640 supplemented with 10% FCS, 0.05 mM 2-mercaptoethanol, 1 mM sodium
pyruvate, and 2 mM L-glutamine. BPH-1, ND-1, and Ln-Cap
were grown in RPMI 1640 supplemented as described above but with only
5% FCS. 293 cells, Vero, BHK-21, OMK637, and COS-7 cells were obtained
from the American Type Culture Collection (ATCC) and were grown in Dulbecco modified Eagle medium (DMEM) supplemented with 10% FCS and 2 mM L-glutamine. A549 and CHELI (kindly provided by E. Lenette) cells were cultured in DMEM supplemented with 10% FCS, 2 mM
L-glutamine and antibiotics. CEMx174, U937, HCT-8, HT29,
343MG, and HUf23 cells were cultured in RPMI 1640 medium supplemented
with 10% FCS, 2 mM glutamine, and antibiotics. The endothelial
cells HUVEC, CNVEC, AEC, BB19, and 181GB1-4 were transformed with the
E6 and E7 genes of human papillomavirus (HPV) type 16. These
transformed endothelial cells (kindly provided by Ashlee Moses and Jay
Nelson) were cultured in Endothelial Cell Growth Medium (Clonetics
Corporation), which was supplemented with G418 (200 µg/ml). Primary
neonatal and adult capillary endothelial cell cultures (kindly provided by Scott Heron) were also grown in Endothelial Cell Growth Medium (Clonetics Corporation). FaDu, RPMI2650, and SCC15 were obtained from
ATCC and cultured according to ATCC instructions.
Patient KSHV isolates and coculture with 293 cells.
KS
biopsies were previously quick-frozen in liquid nitrogen. Biopsy tissue
was cocultivated with subconfluent 293 cells in six-well plates.
Supernatant was harvested at 3 and 7 days, and cells were passaged
every 7 days. Virus-containing supernatants were filtered through a
0.45-µm-pore-size filter before storage at 
70°C or reinfection of
293 cells. For passage of cell-free virus supernatant, filtered stocks
were added to freshly trypsinized 293 cells. The medium was changed
after the cells were allowed to adhere overnight.
Peripheral blood mononuclear cells (PBMCs) from KS patients were
separated from 30-ml samples of EDTA-treated whole blood on Ficoll
gradients. T cells were then depleted using CD4- and CD8-specific
magnetic beads (MACS) (Miltenyi Biotech Inc). The remaining cells were
cocultivated with freshly trypsinized 293 cells as described above,
with and without 5 mM N-butyrate. For PCR analysis, 500 µl
of filtered supernatant was pelleted at 100,000 rpm for 30 min in a
Beckman Optima TLX benchtop ultracentrifuge. DNA was extracted from the
pellet, and nested PCR was performed as described by Boshoff et al.
(8).
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RESULTS |
Infectivity assay.
To develop a more reliable assay for
KSHV infectivity, we decided to assay for the production of viral mRNA,
which can occur only following viral particle uptake and nuclear
delivery of the viral genome. For this purpose, we chose to
employ RT-PCR, which can sensitively detect even small quantities of
viral transcripts. To allow rigorous discrimination from input DNA and
to provide information about the nature of the infection in the
recipient cell, we selected as the RT-PCR target a gene (KSHV ORF29)
which in other herpesviruses is (i) spliced and (ii) expressed only late in the lytic infection cycle, following viral DNA replication. The
product of this gene is involved in DNA packaging, a step late in the
capsid assembly pathway (3, 4). The recently published
sequence of the KSHV genome suggested that ORF29 has two exons which
are separated by a 4-kbp-long intron harboring two viral genes on the
opposite strand (a similar arrangement exists in other herpesviruses,
e.g., herpes simplex virus, herpesvirus saimiri, and EBV) (Fig.
1A) (23). The presence of such
a large intron in ORF29 makes this gene an ideal candidate for the
development of an RT-PCR assay, since the spliced RT-PCR product is
smaller than any amplification product from contaminating genomic DNA. To validate this assumption, we prepared primers from regions flanking
the proposed splice donor and acceptor regions (see Materials and
Methods) and used them to amplify cDNA prepared from BCBL-1 cells as
well as from a solid KS tumor. As shown in Fig. 1B (lanes 5 and 7), a
PCR product approximately 300 bp long was produced only from RNA which
had been reverse transcribed (lanes 5 and 7) but not from RNA in the
absence of RT (lanes 6 and 8). To confirm its spliced nature, we cloned
the amplified fragment and determined its sequence. The cloned
fragment contained sequences from both exons (ORF29A and ORF29B) and
mapped the splice donor to nucleotide position 53755. This splice
donor was spliced to a splice acceptor site at position 50504 in the
KSHV genome (Fig. 1A and C). The intron termini (5' GT and 3' AG) are
in agreement with the conventional splicing consensus sequence and are
similar to those observed in UL15, the ORF29 homolog of herpes simplex
virus (Fig. 1D) (4). Therefore, ORF29 of KSHV is expressed
from a spliced mRNA which is readily detectable in BCBL-1 cells and
also in RNA extracted from KS biopsy material.
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FIG. 1.
Mapping of the splice donor and acceptor sites of ORF29
in BCBL-1 cells and KS tumors by RT-PCR. (A) Genomic map of the region
containing both exons of ORF29 divided by a 3.2-kbp region encoding
four ORFs on the opposite strand. (B) Agarose gel showing the RT-PCR
products. Lanes 5 and 7 show products derived from KS tumor RNA and
BCBL-1 cells after 30 cycles. Lanes 1 and 2 show products derived from
mock-infected or KSHV-infected 293 cells after 42 PCR cycles. As an
additional control for DNA contamination, all samples were amplified in
the presence (+) and absence () of RT. (C) After cloning of the
RT-PCR product into the vector pCRII (Invitrogen), clones derived from
BCBL-1 and 293 cells were sequenced and showed identical results. The
sequence of the exon border between ORF29A and ORF29B is outlined. (D)
Comparison of the exon and intron borders of KSHV ORF29 and herpes
simplex virus (HSV) UL15; both show the conserved GT/AG major class
intron consensus sequence.
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Next we assessed if this ORF29-directed RT-PCR assay can be used to
screen cell lines for their susceptibility to KSHV derived
from
TPA-induced BCBL-1 cells. 293 cells (a transformed human
kidney cell
line) were first chosen as a target cell line, since
Foreman et al.
(
12) had earlier reported this cell line as susceptible
to
KSHV derived from KS biopsies. As inoculum, cell-free
supernatants
of TPA-induced BCBL-1 cells containing approximately
10
7 viral genome equivalents/ml were prepared as
previously described
(
21). 293 cells were seeded into
six-well plates 24 h prior
to inoculation with KSHV virions. After
adsorption for 8 to 12
h, monolayers were washed twice before with
fresh growth medium
(Materials and Methods) was laid over the
monolayers. Total cellular
RNA was extracted 48 to 72 h after
exposure, and 0.5 µg of RNA
was used for RT-PCR analysis as described
in Materials and Methods.
As shown in Fig.
1B, cells which were mock
infected did not produce
a PCR band (lanes 1 and 2), whereas 293 cells
exposed to KSHV
showed a band which comigrated with the PCR products
obtained
by amplifying cDNA from either BCBL-1 cells or a KS tumor
(lanes
5 and 7). To confirm these results, these bands have also been
cloned and sequenced as described earlier and showed the same
sequence
and splice junctions as found in BCBL-1-derived ORF29
transcripts (data
not shown). These data indicate that BCBL-1-derived
virus is capable of
entering 293 cells, delivering its genome
to the nucleus, and allowing
a series of transcriptional events,
including expression of genes
belonging to the late phase of the
lytic cycle.
If infectious virus particles were generated in these 293 cells, then
it should be possible to serially passage KSHV derived
from these
cells. When we inoculated fresh 293 cells with supernatants
of 293 cells which had scored positive in the ORF29 RT-PCR assay,
we were not
able to demonstrate serial passage, suggesting that
infection of 293 cells was abortive (data not shown). We also
did not observe any
cytopathic effect in 293 cells infected with
KSHV, in contrast to
previously reported observations (
12).
We therefore decided
to analyze the transcriptional program of
KSHV in 293 cells in more
detail.
Characterization of 293 cell infection.
In infected 293 cells
which were positive in the ORF29 RT-PCR, we were not able to detect
individual viral transcripts by Northern blot analysis with
virus-specific probes, including the 1.1-kb nut-1 transcript
which is the most abundant RNA produced in the lytic infection cycle of
KSHV (28, 31, 32). To examine further the pattern of viral
transcription in 293 cells, we employed a cDNA assay we had earlier
developed to study viral gene expression in BCBL cells and KS tumors.
Briefly, poly(A)+ RNA was prepared from the infected cells
and used as the template for randomly primed cDNA synthesis in
the presence of 32P-labeled deoxynucleoside triphosphates.
This labeled cDNA probe, which represents all viral and cellular
RNA in the population was then used to probe a filter bearing
restriction fragments spanning most of the genome of KSHV. As
previously reported (21), when such an experiment is
performed with RNA from TPA-induced BCBL-1 cells, strong hybridization
to many bands is observed (Fig. 2A).
KSHV-infected 293 cells, however, showed a different expression pattern
of viral genes. Using RNA from three independent infections which
scored positive in the ORF29 RT-PCR assay, only very faint signals in
this cDNA hybridization assay were detected in two of these three
experiments. The third assay generated stronger autoradiographic
signals; results of this assay are shown in Fig. 2B. Two points are
noteworthy. (i) The exposure time employed here was 20 times that in
the BCBL-1 cell experiment of Fig. 2A. (ii) The pattern of positive
bands is not identical to that generated in BCBL-1 cells. The most
striking difference is seen in lanes 1 and 9. These lanes contain
fragments corresponding to the nut-1 (T1.1) gene of KSHV,
whose product accumulates to ca. 105 copies per cell in
lytic infection (28, 32). As expected, a prominent band is
observed in this lane with cDNA from BCBL-1 cells (Fig. 2A, lanes 1 and
9); however, no comparable signal is generated by cDNA from infected
293 cells (Fig. 2B, lanes 1 and 9). Taken together, these results show
that (i) expression of KSHV genes by 293 cells is extremely inefficient
compared to that generated by induction of BCBL-1 cells, with variable
RNA levels hovering around the detection threshold of the cDNA
hybridization assay, and that (ii) the transcriptional program in 293 cells is not identical to that observed in BCBL-1 cells or in KS
lesions (32). Several possible explanations for the
differences in the transcriptional program (and the failure to serially
passage the virus) can be envisioned. (i) 293 cells may be only
semipermissive and may support only abortive infection. (ii) KSHV
virions from BCBL-1 cells may harbor mutations that affect
infectivity. (iii) Cell-free virus does not efficiently infect using
these cell culture conditions. (iv) The transcriptional program
following de novo infection may differ from that following induction
from latency.
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FIG. 2.
Gene expression in BCBL-1 cells compared to that in
KSHV-infected 293 cells. Phage DNA from the indicated lambda clones,
which together span ca. 120 kb of the KSHV genome, was cleaved with the
indicated restriction enzyme, electrophoresed through 0.8% agarose
gels, stained with ethidium bromide, and transferred to duplicate nylon
filters. To these filters was annealed radiolabeled cDNA probes
corresponding to poly(A)+ transcripts from TPA-treated
BCBL-1 cells or KSHV-infected 293 cells prepared as described in
Materials and Methods. As a control for integrity of the probe as well
as an index of the amount of poly(A) RNA used in the assay, a plasmid
encoding glyceraldehyde phosphate dehydrogenase (GAPDH) was also
electrophoresed in parallel (lane 10). Blot A was exposed for 8 h;
in contrast, blot B was exposed for 1 week. Exp. time, Exposure time.
(A) Hybridization of the filter with cDNA prepared from mRNA from
TPA-treated BCBL-1 cells. (B) Hybridization of an identical filter with
cDNA probe prepared from mRNA from KSHV-infected 293 cells. The
positions of molecular size standards (in kilobases) are shown at the
sides of the gels.
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Cell-free virus preparations versus cellular extracts and
cocultivation.
To address the possibility that cell-associated
rather than cell-free virus might be the more infectious inoculum, we
cocultivated 293 and BCBL-1 cells and also prepared inocula from
extracts from BCBL-1 cells, by using techniques similar to those
described by Foreman et al. (12). We inoculated 293 cells
either with virion particles purified from the media of TPA-induced
BCBL-1 cells or from extracts prepared by freeze-thaw cycles from
induced and uninduced BCBL-1 cells. In addition, we tested supernatants
of uninduced BCBL-1 cells and also performed coculture experiments with these cells. Total RNA and supernatants were harvested 72 h
after exposure, and the RT-PCR assay for ORF29 mRNA was performed. To
increase sensitivity, we blotted the PCR products and hybridized them
to a probe spanning the splice sites of ORF29 (see Materials and
Methods). The experimental results (Fig.
3) clearly demonstrate that cell-free
virus derived from TPA-induced or uninduced BCBL-1 cells is much more
infectious than extracts prepared from these cells (Fig. 3, compare
lanes 4 and 8 to lanes 6 and 10). Cocultivation of BCBL-1 and 293 cells
seems to be as efficient as cell-free virus preparations; however, in
this assay we cannot exclude the possibility that some of the signal is
due to contamination with input BCBL-1 cells, which can stick to the
293 monolayer. The lower panel of Fig. 3 shows a similar experiment
with BHK-21 (baby hamster kidney cells) which are also infectible with
KSHV, though at a lower level than 293 cells. In BHK-21 cells, we were
not able to detect any signal by using cell extracts as the inoculum (Fig. 3, lanes 19 and 23). However, cell-free virus of either TPA-induced or uninduced BCBL-1 cells lead to a detectable signal (lanes 17 and 21). This result clearly shows that cell-free virus preparations of KSHV are more infectious than cellular extracts prepared from BCBL-1. The observation that supernatants from uninduced BCBL-1 cells have infectious material is not surprising, since 1 to 3%
of the cells in these cultures spontaneously enter the lytic cycle. The
fact that cell-free virus of both TPA-induced and uninduced cells give
rise to comparable signals (Fig. 3, lanes 4, 8, 17, and 21) suggest
that the virus titer might not be the limiting factor in the
infectivity of KSHV for 293 and BHK-21 cells.
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FIG. 3.
Cell-free virus is more infectious than inocula prepared
from extracts. ORF29-specific RT-PCR products after 42 cycles of PCR
were electrophoresed on a 0.8% agarose gel and transferred to a nylon
membrane. Filters were hybridized to an ORF29-specific probe as
described in Materials and Methods. As additional control for
contamination, all samples were amplified in the presence (+) and
absence () of RT. 293 and BHK-21 cells were infected with (i)
cell-free virus from TPA-induced (lanes 4, 5, 17, and 18) or uninduced
(lanes 8, 9, 21, and 22) BCBL-1 cells or (ii) with extracts prepared
from TPA-induced (lanes 6, 7, 19, and 20) or uninduced (lanes 10, 11, 23, and 24) BCBL-1 cells. 293 and BHK-21 cells were seeded at 4 × 105 cells/well in six-well plates. Monolayers were then
incubated for 8 h with either cell-free virions concentrated from
10 ml of supernatant (TPA induced and uninduced) or extracts prepared
from 10 ml of BCBL-1 cells at a density of 5 × 105
cells/ml (TPA-induced and uninduced) as described in Materials and
Methods. Monolayers were washed three times and overlaid with fresh
medium, and total RNA was harvested 72 h later. In addition, 293 and BHK-21 cells were cocultivated with 106 BCBL-1 cells
for 24 h (lanes 12, 13, 25, and 26). As a positive control, a
diluted RT-PCR amplification product derived from BCBL-1 RNA was loaded
in lanes 1 and 14.
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BCBL-1-derived KSHV virions versus virus preparations obtained from
KS biopsies.
The fact that we could not observe serial
transmission of BCBL-1-derived KSHV virions in 293 cells raised the
possibility that such virus stocks might harbor mutations reducing
infectivity, despite the efficient production of morphologically
correct virions. Such infectivity defects have been reported for
virus from EBV-infected Burkitt's lymphoma cell lines (17).
Accordingly, we determined whether KSHV isolates from other sources
might be more readily serially passaged. We examined four primary KSHV
isolates that had been obtained from KS patients; two isolates were
obtained by cocultivating the PBMCs from KS patients with 293 cells
and two isolates were obtained by inoculating 293 cells with KS biopsy material as described in Materials and Methods. Supernatants of these
cocultures were passaged several times on 293 cells and tested positive
for the presence of viral genomes by DNA-based nested PCR. When
cell-free supernatants of these cultures were used to infect fresh 293 cells, no signals were generated in the ORF29 RT-PCR assay (Fig.
4). This result suggests that the block to serial transmission of KSHV in 293 cells is not unique to
BCBL-1-derived virus.
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FIG. 4.
KSHV isolates from four different patients behave like
BCBL-1-derived virus in the ORF29-directed infectivity assay. Fresh 293 cells were incubated with 293 cell supernatants from serially passaged
KSHV isolates. All supernatants which have been serially passaged up to
three times scored positive in a nested DNA PCR assay (Materials and
Methods). Inoculation and RT-PCR assay was done as described in the
legend to Fig. 3. None of these isolates scored positive even after
prolonged exposure times (lanes 1 to 10).
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Screening of additional cell lines for KSHV susceptibility.
To
search for cell lines that might be more permissive for KSHV infection,
we examined a total of 38 cultured cell lines and primary cells of
different origin for KSHV susceptibility, using the ORF29-specific
RT-PCR assay. Cell lines or primary cells tested for KSHV
susceptibility are listed in Table 1,
which also indicates their cell type of origin (where known). Based on
the known phylogeny of KSHV as a gammaherpesvirus, and given its
association with B-cell lymphomas, considerable emphasis was placed on
infection of B cells, including numerous B-cell lines (either positive
or negative for EBV infection) and also primary, CD19-positive B cells
isolated from human peripheral blood. A complete summary of the results
of this infectivity testing is presented in Table 1, and selected
examples of these assays are shown in Fig.
5. Since ORF29 is a lytic gene, most
tested cells were examined in the presence and absence of TPA; however,
this treatment did not increase signal strength in any of the cell
lines which tested positive for ORF29 transcripts (e.g., Fig. 5,
lanes 10, 12, 24, and 26). Of the B-cell lines tested, only primary
B-cells (CD19-positive PBMCs) supported KSHV infection, as
has previously been reported by Blackbourn et al. (5). We
also tested many endothelium-derived cell populations, given the known
presence of viral DNA in KS spindle cells, which are thought to be
of endothelial lineage (7, 27). Several established
endothelial lines, whether derived from large vessels (e.g., HUVEC) or
capillaries (e.g., HMEC) were nonpermissive. However, primary neonatal
microvascular endothelial cells did support viral entry and
transcription in this assay (Fig. 5, lanes 10 and 12); BB19 and
181GB1-4 cells, two human brain endothelial lines, were also
susceptible at very low levels. Other positive cell lines included
Ln-Cap, a line derived from cancerous human prostatic epithelium,
and
surprisinglytwo cell lines of nonhuman origin, owl monkey kidney
cells (OMK), a line used for the growth of the related herpesvirus
saimiri, and BHK-21 cells (Fig. 3 and Table 1). In summary, these data
show that several cell lines are partially susceptible to KSHV
infection. However, none of the 11 cell lines or primary cells
seemingly permissive for KSHV were more susceptible than 293 cells.
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|
FIG. 5.
Screening for KSHV-susceptible cell lines. ORF29 RT-PCR
after inoculation of cell-free virus concentrated from supernatants of
TPA-induced BCBL-1 cells. A summary of all lines tested is given in
Table 1. Assays were performed as described in the legend to Fig. 3.
Endo, endothelial cells.
|
|
| |
DISCUSSION |
We have established a reliable and sensitive RT-PCR assay to
screen for KSHV-susceptible cell lines and/or primary cell populations. The selection of a spliced gene (ORF29) as the RT-PCR target avoids possible confusion from contaminating input DNA, since the PCR product
from the spliced gene will be 3.2 kb smaller than that derived from the
genomic KSHV DNA (Fig. 1). The sensitivity of the assay allows
detection of this presumably late mRNA in solid KS tumors where we have
previously demonstrated that at least 1 to 5% of the cells are
expressing lytic genes (27). We used this assay (i) to
evaluate the infectivity of BCBL-1-derived KSHV virion preparations and
(ii) to identify cell lines which are susceptible to KSHV. Using
supernatants from TPA-induced BCBL-1 cells, we could readily infect 293 cells, as judged by our RT-PCR assay. Clearly then, 293 cells must
possess at least one functional receptor or entry mechanism for KSHV,
though the possibility that other, perhaps more-efficient, entry
pathways exist is by no means excluded. In herpes simplex virus, for
example, multiple entry pathways, based in part upon different viral
envelope proteins, are known to exist (25). Analysis of the
viral transcription pattern in infected 293 cells revealed major
differences compared to those of TPA-induced BCBL-1 cells and KS tumors
(Fig. 3); nut-1, for example, a transcript which is highly
expressed in BCBL-1 and BC-1 cells and in KS tumors, was not detected
in 293 cells (Fig. 2, lanes 1 and 9) (32). Although it is
formally possible that the transcription patterns in cells of
epithelial origin differ from that in either endothelial or
lymphoblastoid origins, the observed transcription pattern in 293 cells
is more suggestive of an abortive infection. This interpretation is in
agreement with the lack of serial passaging of the virus in 293 cells
and the absence of observable cytopathic effects that had been reported by others (12).
Our findings confirm that 293 cells are at least semipermissive for
KSHV infection in that they support the entry of virions and limited
transcription of viral DNA. Although infection of 293 is inefficient,
both with BCBL-1 virus and with primary isolates, it is highly
reproducible and could serve as the basis for assays of infectivity and
neutralization with materials from clinical specimens. In fact, one
such use has already been reported: KSHV-infected subjects frequently
display virions in their saliva, and these virions can infect 293 cells
with efficiencies roughly similar to those found here (29).
Importantly, these primary salivary isolates also could not be serially
propagated on 293 cells and showed no cytopathic effect on these cells
(29), again suggesting that 293 cells are not fully
permissive to the KSHV lytic cycle. Although our results and those of
Vieira et al. (29) differ from those of Foreman et al.
(12), who reported serial transmission of KSHV in 293 cultures, we note that some of these differences may relate to the use
of different viral isolates, different lots of 293 cell lines, and
possibly to subtle differences in in vitro culture conditions.
Testing BCBL-1-derived virus preparations on a variety of cell lines
and/or primary cells of different origin (Table 1 and Fig. 3) indicates
that cells of fibroblastic, epithelial, endothelial, or lymphoid origin
appear to support limited KSHV infection; notably, even cells of
hamster or simian origin were sensitive to low-level infection. In
agreement with the fact that the mass of KS tumor cells consists of
spindle cells believed to be of endothelial origin, it is also
noteworthy that two different endothelial cell lines (BB19 and
181GB1-4) as well as primary neonatal capillary endothelial cells were
infectible. In addition, we confirm with our assay that
CD19+ primary B cells of healthy donors can be infected
(5); this cell population has been shown to be infected in
KS patients in vivo by DNA-based PCR assays (1). However, we
emphasize that all of these in vitro infections proceed extremely
inefficiently, in general at or below the level seen in 293 cells, and
it seems likely that many of these are abortive infections as well,
though we have not tested supernatants from most of these cells for
serial transmission. Clearly, there remains a need for the
identification of cell lines supporting more robust replication of this
emerging pathogen. Such cell culture systems will be crucial for a
better understanding of the life cycle and pathogenesis of KSHV.
| |
ACKNOWLEDGMENTS |
We thank Simon Hayward, Scott Heron, Eliott Kieff, E. Lenette,
Ashlee Moses, Jay Nelson, and Marc Shuman for providing cell lines and
primary cell cultures.
D.B. was supported in part by the University of California
Universitywide AIDS Research Program (UARP). R.R. is a fellow of the
Leukemia Society of America. This work was supported by the Howard
Hughes Medical Institute. This work was also supported by UARP grant
R95-SF-088 and by the Medical Research Council.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Microbiology, UCSF, 513 Parnassus Ave., San Francisco, CA 94143-0414. Phone: (415) 476-2826. Fax: (415) 476-0939. E-mail:
ganem{at}socrates.ucsf.edu.
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Abstract
Introduction
