Latent Kaposi’s Sarcoma-Associated Herpesvirus Infection of
Monocytes Downregulates Expression of Adaptive Immune Response
Costimulatory Receptors and Proinflammatory Cytokines
Sean M. Gregory,a,bLing Wang,aJohn A. West,aDirk P. Dittmer,a,band Blossom Damaniaa,b
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA,aand Department of Microbiology and
Immunology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USAb
herpesvirus subfamily. KSHV is the etiological agent of Kaposi’s
sarcoma (KS) (8), primary effusion lymphoma (PEL), and multi-
centric variant of Castleman’s disease (MCD) (1, 8, 36). KS is a
highly inflammatory and angiogenic vascular tumor defined by
characteristic spindle cells, which are believed to originate from
endothelial cells. PEL and MCD are both B cell lymphoprolifera-
Like other herpesviruses, KSHV establishes latent infection in
its host. A number of PEL cell lines have been established where
of cells undergoing spontaneous lytic reactivation (28, 38). Dur-
ing latency, a limited number of viral proteins are expressed, in-
cluding the latency-associated nuclear antigen (LANA), vFLIP,
vCyclin, kaposin, vIRF3, K1, and vIL-6 (7, 12, 35, 37). Mainte-
nance of the viral genome is absolutely dependent on the LANA
protein, which tethers the latent viral episome to the host cell
to be expressed in latently infected B cells and endothelial cells, as
well as in the KSHV-positive tumors associated with these cell
types (3, 10, 13).
KSHV can successfully infect human monocytes and macro-
phages in vitro and in vivo (4–6, 24). Rappocciolo et al. demon-
strated that KSHV uses the receptor DC-SIGN to enter macro-
in the THP-1 acute monocytic leukemia cell line, KSHV primary
infection was dependent on ?3?1, ?v?3, ?v?5, and ?5?1 integ-
FAK, Src, PI3K, NF-?B, and ERK1/2 signaling (20). Coinfection
of monocytes with KSHV and HIV increased the replication of
have been shown to support viral replication (5). Additionally,
KSHV has been found to infect CD34?stem cell precursors in
vitro, suggesting that stem cells or later-stage committed progen-
itor cells may be infected and subsequently differentiate into B
aposi’s sarcoma-associated herpesvirus (KSHV), also known
as human herpesvirus 8 (HHV8), is a member of the gamma-
infected, differentiated cells (45).
T cell receptor (TCR) cognate interactions with the antigen-
presenting cell (APC) major histocompatibility complex (MHC)
surface molecules displaying KSHV peptide. On inactivated T
cells, CD28 is expressed at low levels. Upon TCR-MHC contact
and APC surface receptor CD40 ligation by CD40 ligand
(CD40L), CD28 expression is upregulated. Concomitantly, the
costimulatory molecules CD80 and CD86 on the APCs, which
lated. Despite the existence of KSHV-specific T cells and immune
control in healthy individuals, latent virus is unable to be elimi-
molecules during latency also contributes to evasion of the host
response (22). Cells involved in immunity that are tropic for
KSHV may facilitate suppression of host immune responses.
Given that KSHV infects monocytes in vivo, we established a
latently infected monocytic cell line by using the monocytic leu-
kemia cell line THP-1 to characterize viral gene expression in la-
tently infected monocytes (2, 39). THP-1 cells are susceptible to
human cytomegalovirus (HCMV) infection and support viral la-
tency (43). Importantly, although we have previously shown that
KSHV can infect primary human monocytes (44), we could not
establish a long-term latent culture in these cells because of the
primary nature of the monocytes. In contrast, THP-1 cells enable
Received 28 September 2011 Accepted 17 January 2012
Published ahead of print 25 January 2012
Address correspondence to Blossom Damania, firstname.lastname@example.org.
S. M. Gregory and L. Wang contributed equally to the work.
Copyright © 2012, American Society for Microbiology. All Rights Reserved.
jvi.asm.org 0022-538X/12/$12.00Journal of Virologyp. 3916–3923
the establishment of a monocytic latent cell line that harbors
KSHV and which can be passaged over a long period.
MATERIALS AND METHODS
Production of recombinant rKSHV.219 virus. Vero cells containing la-
tent rKSHV.219 (KSHV-Vero) and a recombinant baculovirus KSHV
Orf50 (Bac50) were kindly provided by Jeffrey Vieira (40). rKSHV.219
resistance gene as a selectable marker. Insect SF9 cells were grown in
SF900-II serum-free medium at 28°C. SF9 cells were infected with bacu-
lovirus expressing KSHV Orf50 (Bac50) for 3 days, after which time the
rpm for 10 min). KSHV-Vero cells were then infected with Bac50 and
and cells were removed by centrifugation (1,500 rpm for 10 min). Super-
natants were subsequently passed through a 0.45-?m filter.
Establishment of the KSHV-THP-1 cell line. THP-1 cells were cul-
37°C in a 5% CO2environment. The rKSHV.219 produced from KSHV-
Vero cells was used to infect THP-1 cells. We first made an infection
cocktail containing complete RPMI medium and rKSHV.219 superna-
tants (volume ratio of 1:2) with 4 ?g/ml Polybrene. THP-1 cells were
centrifuged at 1,500 rpm for 5 min, and then 2 ? 106THP-1 cells were
resuspended in 3 ml infection cocktail and added to one well of a 6-well
plate. The cells in the 6-well plate were spun for 90 min at 30°C at 2,500
in 3 ml complete RPMI medium. The cells were incubated at 37°C for 72
h and then selected in complete RPMI medium containing 1.0 ?g/ml
puromycin for 3 to 5 weeks to establish stable KSHV-THP-1 cells. Once
THP-1 cells were 100% KSHV positive, KSHV-THP-1 cells were main-
tained in 0.1 ?g/ml puromycin, which is necessary for 12-O-
tetradecanoylphorbol-13-acetate (TPA)-induced reactivation. THP-1
control cells and KSHV-THP-1 cells were synchronously passaged.
hyde for 30 min. Cells were washed twice with phosphate-buffered saline
(PBS) for 5 min each, permeabilized with PBS, 0.1% Triton X-100 for 20
min, and washed twice with PBS, 1% bovine serum albumin (BSA) for 5
min each. Incubation with primary antibody anti-HHV8 open reading
frame 73 (ORF73) (Advanced Biotechnologies) (1:500) was performed
and incubated with anti-rat tetramethyl rhodamine isothiocyanate
(TRITC)-conjugated secondary antibody (Sigma), 1:500, for 1 h at room
temperature. Cells were washed twice with PBS, 1% BSA for 5 min each
and then stained with 4=,6-diamidino-2-phenylindole (DAPI) for 2 min.
Vectashield (Vector Laboratories) mounting medium and visualized us-
ing a Nikon Microphot FXA upright fluorescence microscope.
Quantitative real-time-PCR (qPCR). DNase-treated total cellular
into cDNA using a reverse transcription system from Promega. The real-
green PCR master mix (Applied Biosystems). PCR was carried out with 1
for 15 s and 60°C for 1 min. All fold activations were normalized by
GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression in the
Viral gene profiling. THP-1 or KSHV-THP-1 cells (1 ? 106) were
harvested, and total RNA was isolated with an RNeasy kit (Qiagen) ac-
cording to the manufacturer’s instructions and then DNase treated using
a DNA-free RNA kit (Zymo Research). The cDNA was synthesized using
a high-capacity cDNA reverse transcription kit (Applied Biosystems) as
directed by the manufacturer.
Detailed methods for real-time qPCR arrays and primer names were
described previously (11, 12). The primer name is as published before or
indicates the orf name in KSHV followed by position of the forward
primer. In addition, the original primer set was replaced with improved
orf50 (kshv10052-1), orf53 (kshv10055-1), orf63 (kshv10069-1), orf65
(kshv10070-1), orf71 (kshv10076-1), orf K5 (kshv10014-1), and orf K15
The threshold was set to five times the standard deviation (SD) of the
analyzed to verify that identical primer-specific, single reaction products
independent primer sets. Samples were normalized to GAPDH levels,
centered by median of gene, and ordered by hierarchical clustering using
dard correlation metrics (11, 12). Further statistical analysis was con-
ducted using the R programming environment (v 2.5.1).
Flow cytometry. To assess the expression of cell surface receptors,
THP-1 and KSHV-THP-1 monocytes were suspended at 1 ? 107cells/ml
in staining buffer (1? PBS, 2% BSA) and labeled with 20 ?l of mouse
allophycocyanin-conjugated anti-human CD86 (BD Pharmingen),
mouse allophycocyanin-conjugated anti-human CD83 (BD Pharmin-
gen), or isotype control allophycocyanin-conjugated IgG1 kappa (BD
then thoroughly washed twice with staining buffer by centrifugation at
30 min at 4°C, followed by washing with staining buffer. Samples were
resuspended in 250 ?l of staining buffer and analyzed using a BD FACs
version 4.3 (Dako). Data were acquired for a minimum of 25,000 total
CD86, and results were analyzed by the UNC Flow Cytometry Core facil-
ity using iCyt/Sony Reflection under sterile conditions.
Western blotting. THP-1 or KSHV-THP-1 cells were washed in ice-
cold 1? PBS and lysed by 3? dry ice-ethanol fractionations in radioim-
at 13,000 rpm, 4°C for 10 min. Protein quantifications were performed
was resolved by SDS-polyacrylamide gel electrophoresis and transferred
to nitrocellulose membranes by wet transfer at 30 V overnight at 4°C.
nonfat dry milk (NFDM), 1? PBS, 0.2% Tween 20. Following three
washes in 1? PBS, 0.2% Tween 20 for 10 min each at room temperature,
membranes were incubated with primary antibody overnight at 4°C.
Rabbit monoclonal anti-human CD86 (Novus Biologicals) or rabbit
monoclonal anti-human CD80 (Novus Biologicals) was diluted in 5%
NFDM at 1:5,000 or 1:10,000, respectively. Following incubations,
membranes were washed three times and incubated with peroxidase-
conjugated, anti-rabbit IgG (Cell Signaling) in 5% NFDM at 1:2,000
for 1 h at room temperature. Following the three washes, membranes
were developed using Amersham ECL Plus Western blotting detection
reagents (GE Healthcare). Viral protein K8.1 was detected using
mouse K8.1 monoclonal antibody (ABI Biologicals) diluted 1:1,000 in
5% NFDM overnight at 4°C, followed by peroxidase-conjugated, anti-
mouse IgG as described above.
Cytokine expression analysis. THP-1 or KSHV-THP-1 cells (1 ?
106) were plated in 12-well plates containing complete medium. After
incubation at 37°C for 24 h, supernatants were harvested and cleared of
cell debris by centrifugation at 1,250 rpm for 10 min. Supernatants were
analyzed for cytokine expression using Millipore Luminex multianalyte
technology according to the manufacturer’s instructions.
KSHV reactivation. KSHV-THP-1 cells (1 ? 106) were plated in 12-
well plates in growth medium lacking puromycin and treated with either
dimethyl sulfoxide (DMSO) or 20 ng/ml TPA for up to 120 h. After 48 h
Effect of Latent KSHV Infection in Monocytes
April 2012 Volume 86 Number 7jvi.asm.org 3917
viral interleukin-6 (IL-6) primers and GAPDH endogenous controls.
PCR products were analyzed by 1.2% agarose gel electrophoresis. Viral
K8.1 protein expression was analyzed 120 h after TPA treatment as de-
KSHV can successfully infect human monocytes and macro-
the CMV promoter, red fluorescent protein (RFP) under a lytic
viral promoter, and the puromycin resistance gene (40). Seventy-
two hours postinfection, cells were added to puromycin-
containing selection medium to maintain KSHV infection and to
achieve a 100% KSHV-infected monocytic cell line as monitored
by GFP expression (Fig. 1A).
To confirm KSHV infection of THP-1 cells, we performed im-
munofluorescence assays on KSHV-infected and uninfected
THP-1 cells for LANA protein expression (Fig. 1B). Briefly,
KSHV-THP-1 or THP-1 cells suspended in PBS were spotted on
slides, air dried, and fixed with paraformaldehyde. Cells were
stained with an antibody directed against LANA followed by a
TRITC-conjugated secondary antibody. Cells were also stained
with DAPI to demarcate the nucleus. As can be seen in Fig. 1B,
KSHV-THP-1 cells showed characteristic nuclear speckled stain-
ing for LANA protein (19).
THP-1 cells and uninfected THP-1 cells. qPCR primer pairs were
designed for each KSHV ORF as previously described (11). Equal
amounts of total poly(A) mRNA from THP-1 and KSHV-THP-1
FIG 1 KSHV infection in THP-1 cells. (A) Fluorescence microscopy of GFP expression in KSHV-THP-1 cells compared to that in uninfected THP-1 control
cells. (B) Immunofluorescence staining of KSHV LANA expression in KSHV-THP-1 cells compared to that in uninfected THP-1 cells. Images were obtained at
Gregory et al.
jvi.asm.org Journal of Virology
cells were used as starting material. Figure 2A shows cycle thresh-
old (CT) values for each KSHV gene versus the relative log fold
gene expression in KSHV-THP-1 cells compared to that in unin-
genes, including vCyclin and vIRF-3 genes, were detected at high
the positive controls, were present at a ratio of 1, i.e., at equal
the density distributions, i.e., the number of genes with a given
expression level above that for uninfected cells. Fewer than 10
transcripts were expressed at levels significantly greater than 100
FIG 2 KSHV gene expression profile of KSHV-THP-1 cells. (A) Analysis of log KSHV gene expression versus qPCR cycle threshold (CT) value. Dotted line
represents limit of detection for viral genes. (B) Density distribution (on the vertical axis) of relative log fold change in gene expression (on the horizontal axis)
in KSHV-THP-1 cells compared to that in uninfected THP-1 cells for each KSHV-specific primer pair. Also shown is the 95% CI (104.99. . . 103.64) between the
conditions and the difference between these two groups based on nonparametric test. (C) Heat map depicting KSHV transcription in KSHV-THP-1 cells
undetectable. Each condition depicts two biological replicates. Rows indicate the primers, which were labeled using the name of the KSHV orf followed by a
position of the forward primer.
Effect of Latent KSHV Infection in Monocytes
April 2012 Volume 86 Number 7jvi.asm.org 3919
remainder of genes were undetectable or present at low levels
(?100-fold of level for mock-infected cells). The difference be-
tween these two groups of genes was significant to a P value of
?2.4 ? 10?9, with a 95% confidence interval (95% CI) between
the means of expression of 104.99and 103.64. Relative expression
levels are depicted by heat maps comparing KSHV-THP-1 and
THP-1 cells (Fig. 2C). Lytic transcripts detected at high to mod-
erate expression levels likely reflect the low number of cells spon-
taneously reactivating, similar to results for other KSHV-infected
cell lines in culture (33). Importantly, KSHV-THP-1 cells were
capable of reactivation after treatment with 20 ng/ml TPA (Fig.
3A), as evidenced by measuring viral IL-6 mRNA levels after 48 h
and 72 h compared to GAPDH mRNA levels as the endogenous
control. No signal was obtained in the absence of reverse tran-
expression of KSHV K8.1 could also be detected in the TPA-
reactivated cells (Fig. 3B).
Primary infection of dendritic cells and macrophages with
KSHV has been shown to downregulate DC-SIGN, and in B cells
whether latently infected monocytes show downregulation of
monocyte activation markers. We found that surface expression
of CD86 was reduced from approximately 31% in THP-1 cells to
4% for KSHV-THP-1 cells (average fold change, 5.02 ? 2.4) (Fig.
downregulated from 12% to 1.20% in KSHV-THP-1 cells com-
pared with that in uninfected control cells (average fold change,
5.1 ? 4.0). Since KSHV-infected monocytes are prevalent in KS
lesions, reduced expression of costimulatory molecules on the
surface of antigen-presenting cells, such as monocytes, during la-
tency may dampen host immune responses against KSHV-
reverse transcription-qPCR (RT-qPCR) for these markers as well
FIG 3 TPA treatment of KSHV-THP-1 cells results in lytic replication. (A)
at 48 h and 72 h after TPA treatment. (B) KSHV-THP-1 or BCBL-1 control
cells (1 ? 106) were treated with 20 ng/ml TPA (T) or mock treated with
DMSO (M). One hundred twenty hours later, K8.1 protein expression was
analyzed by immunoblotting.
FIG 4 KSHV-THP-1 cells exhibit reduced CD86 and CD83 costimulatory molecule expression. THP-1 (A) or KSHV-THP-1 (B) cells were incubated with
compared to that seen with the isotype control was analyzed by flow cytometry. Representative data are shown.
FIG 5 Latent KSHV inhibits expression of costimulatory molecules. (A)
qPCR analysis for expression of CD86, CD80, CD83, and CD1a in KSHV-
THP-1 cells (K) compared to that in uninfected THP-1 control cells (T). (B)
Expression of CD86 and CD80 was determined by immunoblot analysis of
KSHV-THP-1 cells compared to uninfected THP-1 control cells.
Gregory et al.
jvi.asm.org Journal of Virology
found that several genes associated with macrophage/dendritic
cell activation were downregulated compared to levels in unin-
fected THP-1 control cells (Fig. 5A). By densitometry, we found
that genes for costimulatory molecules CD80, CD86, CD1a, and
CD83 were downregulated 2.9-, 8.1-, 2.7-, and 4.1-fold, respec-
tively. The downregulation of CD86 and CD80 basal protein ex-
pression levels in KSHV-THP-1 cells was confirmed by Western
blot analysis (Fig. 5B).
There is a heterogeneous level of CD86 coreceptor expression
on THP-1 cells. Our findings may allow for the alternative possi-
and that KSHV-infected latent THP-1 cells display a low CD86
were stained for CD86, and the cells that expressed the highest
levels of CD86 coreceptor were isolated using fluorescence-
activated cell sorting (FACS). This population is denoted as
CD86hi THP-1 (Fig. 6A). Sorted CD86hi THP-1 cells were then
infected with KSHV and analyzed by flow cytometry for GFP ex-
pression (Fig. 6B). CD86hi THP-1 cells were 68.6% GFP positive
THP-1 cells with the highest level of CD86 surface expression
prior to establishing latency. Hence, it is more likely that CD86 is
downregulated by KSHV and that this effect is not an artifact of
amount of CD86 on their surface.
compared to that in THP-1 controls by Luminex multianalyte
analysis. THP-1 or KSHV-THP-1 cells were incubated for 24 h
and cell-free supernatants collected for analysis. Overall, we ana-
a significant difference in tumor necrosis factor alpha (TNF-?)
and interleukin-1? (IL-1?) was observed (Fig. 7A). TNF-? and
IL-1? expression levels were 7.73 ? 0.56 pg/ml and 4.90 ? 0.59
pg/ml, respectively, in THP-1 cells, and these cytokines were un-
detectable in KSHV-THP-1 cells. In order to confirm that these
cytokines were downregulated at the transcription level, RT-PCR
analysis was performed. Figure 7B shows that transcription of
both genes is suppressed in KSHV-infected THP-1 cells. TNF-?
and IL-? are involved in upregulating the transcription of genes
involved in inflammation, hematopoiesis, and immune re-
sponses, including costimulatory molecules, and therefore their
downregulation may contribute to inhibition of adaptive immu-
KSHV establishes a lifelong persistent infection in the human
host. The virus can establish latency in a number of different cell
sion in lytically and latently infected B cells and endothelial cells
(7, 12, 27, 29). Here we report viral gene profiling of a KSHV
latently infected monocytic cell line. We observed that KSHV-
THP-1 cells predominantly expressed a latent viral gene profile
with low levels of lytic replication, which we attribute to sponta-
neous reactivation. We found that KSHV-THP-1 cells display re-
duced expression of several cellular proteins involved in inflam-
mation and immunity, compared to expression in uninfected
cells. This suggests that viral proteins may downregulate expres-
sion of genes that could lead to detection of the virus by the host
FIG 6 KSHV does not require low surface coreceptor expression to infect THP-1 monocytes. (A) THP-1 cells (1 ? 107) were stained with CD86 antibody and
FIG 7 Expression of select cytokines in KSHV-THP-1 cells. (A) TNF-? and
IL-1? expression in THP-1 compared to KSHV-THP-1 cells was determined
by Luminex multianalyte assay. Units shown are average pg/ml. (B) KSHV-
PCR, samples were subjected to gel electrophoresis using 1% agarose gels.
Effect of Latent KSHV Infection in Monocytes
April 2012 Volume 86 Number 7jvi.asm.org 3921
KSHV-specific cytotoxic T lymphocyte (CTL) responses are
reduced in KS patients compared to responses in KSHV-positive
asymptomatic individuals, suggesting that failure to mount a
CTL-mediated immune response contributes to development of
disease (17). After recognition of a T cell’s cognate antigen in the
context of major histocompatibility complex (MHC) on the sur-
face of APCs, engagement of CD86 or CD80 provides a necessary
costimulatory signal for CTL activation (16, 34). Monocytes help
dictate CTL action, since they contribute to their stimulation by
upregulating costimulatory molecules, such as CD80 and CD86,
upon pathogen detection. Furthermore, monocytes are recruited
to sites of T cell activation and are capable of differentiating into
macrophages and dendritic cells, which are potent antigen-
presenting cells necessary for an effective cell-mediated immune
Previous studies have shown that viral infection reduces ex-
virus (VZV) infection of mature DCs results in selective down-
ing MHC I, CD80, CD83, and CD86 (25). Herpes simplex virus 2
(HSV-2) infection causes the downregulation of MHC I and II,
CD40, CD80, and CD86 on mature DCs (25), although HSV-1
infection of mature DCs results in downregulation of only CD83
regulate MHC I and II, CD40, CD80, CD86, and CD83 on
polysaccharide (LPS) and TNF-? (26, 30); at the same time, pro-
ductive infection in THP-1 cells induces IL-1? and TNF-? (15,
18). In addition, during acute HIV-1 infection, lymphoid tissue
has reduced CD80 and CD86 expression (23). KSHV infection of
myeloid-derived macrophages and dendritic cells results in a re-
duction in differentiation and antigen presentation to CTLs (9,
32). Similarly, our study shows that KSHV latent infection of
monocytic THP-1 cells leads to significant downregulation of
CD1A, CD80, CD83, and CD86. Thus, it appears that human
herpesviruses have multiple strategies to interfere with detection
tion of monocytes and suppression of cellular mechanisms of im-
mune recognition during latency suggest that KSHV specifically
targets APCs to prevent activation of antiviral responses in order
to support long-term dormant infection.
We thank Jeffrey Vieira for providing rKSHV.219 and Stuart Krall for
tissue culture assistance.
This work was supported by NIH grants DE018281 to B.D. and
T32-AI007419 and T32-AI007001. B.D. is a Leukemia & Lymphoma So-
ciety Scholar and Burroughs Wellcome Fund Investigator in Infectious
Disease. Viral expression profiling was conducted by the UNC Vironom-
ics core and supported by grant POI-CA019014.
notechnologies core, supported by grant P30 DK34987.
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Effect of Latent KSHV Infection in Monocytes
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