Systematic Identification of Cellular Signals
Reactivating Kaposi Sarcoma–Associated
Fuqu Yu1, Josephine N. Harada2¤, Helen J. Brown3, Hongyu Deng4,5, Moon Jung Song1,6, Ting-Ting Wu1,
Juran Kato-Stankiewicz3, Christian G. Nelson2, Jeffrey Vieira7, Fuyuhiko Tamanoi3, Sumit K. Chanda2, Ren Sun1*
1 Department of Molecular and Medical Pharmacology, University of California Los Angeles, Los Angeles, California, United States of America, 2 Genomics Institute of the
Novartis Research Foundation, San Diego, California, United States of America, 3 Department of Microbiology, Immunology and Molecular Genetics, University of California
Los Angeles, Los Angeles, California, United States of America, 4 School of Dentistry, University of California Los Angeles, Los Angeles, California, United States of America,
5 Institute of Biophysics, Chinese Academy of Sciences, Beijing, China, 6 Division of Biotechnology, College of Life Sciences and Biotechnology, Korea University, Seoul,
Republic of Korea, 7 Department of Laboratory Medicine, University of Washington, Seattle, Washington, United States of America
The herpesvirus life cycle has two distinct phases: latency and lytic replication. The balance between these two phases
is critical for viral pathogenesis. It is believed that cellular signals regulate the switch from latency to lytic replication.
To systematically evaluate the cellular signals regulating this reactivation process in Kaposi sarcoma–associated
herpesvirus, the effects of 26,000 full-length cDNA expression constructs on viral reactivation were individually
assessed in primary effusion lymphoma–derived cells that harbor the latent virus. A group of diverse cellular signaling
proteins were identified and validated in their effect of inducing viral lytic gene expression from the latent viral
genome. The results suggest that multiple cellular signaling pathways can reactivate the virus in a genetically
homogeneous cell population. Further analysis revealed that the Raf/MEK/ERK/Ets-1 pathway mediates Ras-induced
reactivation. The same pathway also mediates spontaneous reactivation, which sets the first example to our
knowledge of a specific cellular pathway being studied in the spontaneous reactivation process. Our study provides a
functional genomic approach to systematically identify the cellular signals regulating the herpesvirus life cycle, thus
facilitating better understanding of a fundamental issue in virology and identifying novel therapeutic targets.
Citation: Yu F, Harada JN, Brown HJ, Deng H, Song MJ, et al. (2007) Systematic identification of cellular signals reactivating Kaposi sarcoma–associated herpesvirus. PLoS
Pathog 3(3): e44. doi:10.1371/journal.ppat.0030044
Kaposi sarcoma–associated herpesvirus (KSHV), also
known as human herpesvirus-8 (HHV-8), is a member of the
gamma-herpesvirus family. This virus family also includes the
Epstein–Barr virus (EBV) and murine gamma-herpesvirus 68
[1–4]. Herpesviruses have two distinct phases in their life
cycle: latency and lytic replication. During latency, the viral
genome is replicated by cellular DNA polymerase, and only a
few gene products are expressed. One of the advantages of
latency is the ability of the virus to evade the host immune
responses. After stimulation, the virus can enter the lytic
cycle by a reactivation process. Genes that are induced in the
lytic phase can be classified as immediate-early genes, early
genes, and late genes according to their temporal expression
pattern and sensitivity to viral protein synthesis and DNA
replication inhibitors. Upon replication of the viral genome
by a viral DNA polymerase, viral progeny are produced,
frequently resulting in cell death.
The distinctive features of gamma-herpesviruses include
their ability to establish long-term infections in lymphocytes,
and their oncogenic potential. EBV is associated with
nasopharyngeal carcinoma, Burkitt lymphoma, Hodgkin
disease, and other types of malignancies [5,6]. KSHV is
associated with Kaposi sarcoma, primary effusion lymphoma
(PEL), and some forms of multicentric Castleman disease
[2,7–11]. Viral infection persists predominantly in a latent
form in tumor cells. However, lytic replication is believed to
play a critical role in tumorigenesis. It is likely that
continuous low-level reactivation leads to efficient viral
transmission and spread, and subsequently disease develop-
ment in a subset of the infected cells. Cytokines of both viral
and cellular origin produced during lytic replication may
provide a favorable environment for the proliferation of
infected cells [12–16].
The switch between latency and lytic replication has been
actively investigated. KSHV replication and transcription
activator (RTA), a protein product encoded mainly by open
Editor: Skip Virgin, Washington University School of Medicine, United States of
Received June 22, 2006; Accepted February 8, 2007; Published March 30, 2007
Copyright: ? 2007 Yu et al. This is an open-access article distributed under the
terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author
and source are credited.
Abbreviations: EBV, Epstein–Barr virus; EMSA, electrophoretic mobility shift assay;
ERK, extracellular signal–regulated kinase; FBS, fetal bovine serum; GFP, green
fluorescent protein; KSHV, Kaposi sarcoma–associated herpesvirus; MEK, mitogen-
activated protein extracellular signal–regulated kinase kinase; ORF, open reading
frame; PAN, polyadenylated nuclear RNA; PEL, primary effusion lymphoma; PKA,
protein kinase A; RRE, replication and transcription activator response element;
RTA, replication and transcription activator; RT-Q-PCR, reverse transcription–
quantitative PCR; TPA, 12-O-tetradecanoylphorbol-13-acetate; v-Ki-ras2, Kirsten rat
sarcoma 2 viral oncogene homolog
* To whom correspondence should be addressed. E-mail: email@example.com
¤ Current address: The Wharton School, University of Pennsylvania, Philadelphia,
Pennsylvania, United States of America
PLoS Pathogens | www.plospathogens.orgMarch 2007 | Volume 3 | Issue 3 | e440444
reading frame (ORF) 50, plays a central role in regulating this
switch in KSHV [17–22]. In latently infected cells, the
expression of RTA is necessary and sufficient to disrupt
KSHV latency and trigger the complete lytic replication
process. RTA functions as a transcription factor, activating
expression of multiple downstream target genes as well as its
own gene [23,24]. Among these downstream effector genes is
the early viral transcript polyadenylated nuclear RNA (PAN,
also called nut-1). PAN is the most abundant transcript made
during the lytic cycle, and is directly induced by RTA [25–28].
RTA contains an N-terminal DNA-binding domain and a
C-terminal activation domain. The N-terminal DNA-binding
domain mediates sequence-specific DNA binding. RTA
response elements (RREs) have been identified within several
lytic gene promoters, including the PAN, v-IL-6, ORF57, and
Kpsn promoters [16,29–31]. The RRE within the PAN
promoter was incorporated into a highly sensitive luciferase
reporter construct named pPAN-69Luc. RTA has also been
shown to interact with several transcription modulatory
proteins to maximally facilitate lytic gene expression,
including CREB-binding protein (CBP), the SWI/SNF chro-
matin remodeling complex and the TRAP/Mediator coac-
tivator, and CSL, a target of the Notch signaling pathway [32–
35]. RTA functionally interacts with other viral proteins as
Although the function of RTA in KSHV reactivation has
been extensively studied, the cellular pathways involved in
regulating transcription and expression of RTA have not
been systematically studied. In an attempt to systematically
identify these signals, we carried out a genome-wide cell-
based screen utilizing an arrayed cDNA expression library.
The screen was conducted in KS-1 cells (a PEL cell line
latently infected with KSHV, a twin cell line of BC-3 cell line)
in a 384-well format. The pPAN-69Luc reporter construct is
highly responsive to RTA and was therefore used as an
indicator of KSHV reactivation. The screen assessed the
effect of ectopic expression of 26,000 individual cDNA clones
on RTA-dependent reporter activity and identified a list of
positive cellular genes. We then conducted more extensive
analyses on one of the most potent reactivators, Ras, and
investigated the signaling components downstream of Ras to
elucidate the underlying molecular mechanisms for reactiva-
A Large-Scale Screen with a Reporter System
To systematically identify the cellular signals that induce
RTA activity (and therefore reactivate KSHV), a reporter
system was established (Figure 1A). pPAN-69Luc has low basal
activity in the absence of RTA and is highly sensitive to RTA
in a dose-dependent manner (Figure S1) . The screen
design involved cotransfection of the 26,000 mammalian
cDNA clones individually into KS-1 cells with pPAN-69Luc. A
very large amount of cDNA was used in the high-throughput
screen to increase the sensitivity. Activation of the endoge-
nous RTA promoter on the viral genome by exogenous cDNA
gene products would cause a chain of events culminating in
luciferase expression from the RTA-dependent pPAN-69Luc
The screen results are summarized in Figure 1B. The
majority of the reporter signals cluster within a narrow range
between 0.5- to 2-fold of the median luciferase activity,
whereas a small number of cDNAs induced luciferase
expression activity greater than 5-fold over the median
(indicated by the arrow). There were only a very limited
number of signals detected between these two groups,
indicating the significance of the outliers. The signals in the
outlier group were considered as positive signals.
To validate the ability of these cDNA gene products to
induce reactivation, we transfected each cDNA individually
into KS-1 cells and assessed KSHV lytic gene expression levels
by Western blot and reverse transcription–quantitative PCR
(RT-Q-PCR) analysis. Lower ratios of DNA to cells were used
in the verification experiments (Figure 1C–1E; Table 1). The
verified list contains a number of signaling molecules, either
kinases or transcription factors. Among them was a pre-
viously identified molecule that reactivates KSHV, the
catalytic subunit of protein kinase A (PKA, ‘‘A9’’), which
was one of the molecules that gave the strongest signal in the
screen . Another robust inducer, Kirsten rat sarcoma 2
viral oncogene homolog (v-Ki-ras2, ‘‘A11’’), is an important
upstream signaling molecule that regulates cellular functions
via a number of distinct pathways. In addition to PKA and
Ras, some other positive cellular cDNA clones are: 1) XBP1:
encodes X-box binding protein 1, a B cell differentiation
factor that is involved in late stage of B cell terminal
differentiation and the unfolded protein response [38,39];
2) Zfp64: encodes mouse zinc finger protein 64, a nuclear
protein which has been reported to be involved in tran-
scription regulation ; 3) NR4A1: encodes a member of the
steroid-thyroid hormone-retinoid receptor superfamily and
acts as a nuclear transcription factor. It has been reported to
be related to TPA (12-O-tetradecanoylphorbol-13-acetate)
and VP-16–induced apoptosis ; 4) Pitx1: encodes a mouse
paired-like homeodomain transcription factor 1, which is
found to be a cofactor of AP-1 [42,43]; 5) Nfib: encodes mouse
nuclear factor I/B, an important transcription factor in
development ; 6) Rit1: encodes a Ras-like protein ex-
pressed in many tissues ; 7) mouse Ets-1 and Etv1 (mouse
Ets variant gene 1). The Ets family proteins are among the
transcription factors downstream of Ras and are involved in
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Cellular Signals Reactivating Herpesvirus
Kaposi sarcoma is a cancer that commonly occurs in AIDS patients.
The tumor-associated virus, Kaposi sarcoma–associated herpesvirus,
has two distinct phases in its life cycle: inactive latency and active
lytic replication. The balance between these two phases is critical for
viral pathogenesis. Cellular signals play a role in the switch from
latency to lytic replication, termed reactivation. To systematically
evaluate the cellular signals regulating this reactivation process in
Kaposi sarcoma–associated herpesvirus, a genome-wide cDNA
library screen was conducted. Twenty-six thousand mammalian
genes were individually expressed in cells that harbor the latent
virus, and their effect on reactivation was assessed through a
sensitive reporter system. A group of diverse cellular signaling
proteins were identified and validated. Further analysis revealed that
the activation of the cellular Raf/MEK/ERK/Ets-1 pathway is shared
by multiple upstream inducers to trigger reactivation. This work
provides a functional genomic approach to systematically identify
the cellular signals regulating the herpesvirus life cycle, thus
facilitating better understanding of a fundamental issue in virology
and identifying novel therapeutic targets.
various biological functions, including regulation of cell
proliferation, angiogenesis, and immune response [46,47];
and 8) Gadd45a: encodes mouse growth arrest and DNA-
damage-inducible 45 alpha. GADD45 proteins are induced by
ultraviolet radiation (UV), irradiation, stress-related path-
ways, histone deacetylase (HDAC), and prostaglandin J2
[48,49]. Some of these cellular proteins are functionally
connected to known physical and chemical herpesvirus
reactivation inducers such as UV, stress, and TPA.
Western blot analysis showed that at the 48-h time point,
the majority of the cDNA clones induced lytic protein K8
expression (Figure 1C). The RT-Q-PCR analysis at 24 h or 48
h post-transfection also showed that the majority of the
cDNA clones induced the transcript levels of immediate-early
and early genes RTA/ORF50 and PAN, strongly suggesting
that they are able to reactivate KSHV. Our results suggest that
the reporter screen system accurately identified cellular gene
products able to reactivate KSHV. Further studies on the
underlying mechanisms of these genes would reveal large
amount of information on regulation of KSHV reactivation
by the cellular signaling network.
The Raf/MEK/ERK Pathway Mediates Ras-Induced KSHV
Reactivation through Activating the RTA Promoter
As one of the strongest inducers in our screen system and
an important signaling molecule, v-Ki-ras2 was selected for
follow-up analysis in this study to further verify the systematic
approach and examine the molecular mechanisms underlying
the strong reactivation effect. v-Ki-ras2 is a member of the ras
gene family, which consists of three members: Ha-ras, Ki-ras,
and N-ras. These genes encode highly related small GTP-
binding proteins, which are important upstream signaling
components of several cellular pathways involved in cell
proliferation, differentiation, stress responses, and apoptosis.
All Ras proteins have an identical Ras effector binding
domain, which is responsible for interacting with all known
downstream signaling molecules, including Raf and PI3K .
To verify and characterize the effect of Ras on KSHV
reactivation, we utilized two different constitutively active
forms of Ras: v-Ki-ras2 and Ha-ras (Q61L).
An RT-Q-PCR analysis was conducted to determine if lytic
transcript levels increased when Ras was ectopically ex-
pressed (Figure 2). The results demonstrated that Ha-Ras
(Q61L) can induce KSHV immediate early (RTA/ORF50) to
Figure 1. Schematic Representation and Results of the Primary Screen
(A) Overview of the screen approach. Individual cDNA clones were
transfected into KSHV latently infected KS-1 cells in 384-well plates.
Positive signals activate the RTA promoter and induce the expression of
RTA protein from the endogenous viral genome. RTA can also activate its
own promoter. In response to the endogenous RTA expression, the PAN
promoter in the cotransfected PAN-69Luc reporter construct will be
activated and the reporter activity can be analyzed.
(B) Histogram of a representative screen result. The x-axis represents the
relative luciferase intensity generated by transfected cDNAs normalized
to the median luciferase intensity; the y-axis represents the frequency of
signals falling in the binned range of luciferase intensity. The arrow
indicates the group of signals that showed greater than a 5-fold increase
in luciferase intensity.
(C–E) Western blot showing the induction of KSHV lytic protein K8
expression; RT-Q-PCR showing the induction of lytic transcripts RTA/
ORF50 (D) and PAN (E) upon transfection of the cDNA clones in the top
hits list into KS-1 cells.
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Cellular Signals Reactivating Herpesvirus
more than 10-fold. The RT-Q-PCR data also showed the
induction of early (PAN and viral thymidine kinase) and late
(ORF65) lytic transcripts 5- to over 10-fold. The induction of
ORF65 indicated the onset of lytic viral DNA replication,
which is consistent with the previous finding that induction
of RTA is necessary and sufficient to trigger the complete
lytic replication process [20,28]. The activation of the KSHV
lytic program by Ras was also confirmed by Western blot and
immunofluorescence assay (Figures 3A and S2).
The signals transduced by Ras diverge into multiple
pathways, including mitogen-activated kinase (MAPK) path-
ways and the PI3K/Akt pathway. To explore which pathways
are directly involved in transducing the reactivation signal,
different MAPK and PI3K inhibitors were applied to cells
transiently transfected with Ras. Western blot analysis
revealed that the early lytic protein K8 expression induced
by the ectopic expression of Ha-Ras (Q61L) was not affected
by p38 inhibitor SB203580, JNK inhibitor SP600125, or PI3K/
Akt inhibitor LY294002 (Figures 3B and S4). In contrast,
when an increasing amount of the inhibitors specific to the
Table 1. Cellular Genes That Activated KSHV
Array ID GenBankaIDSymbol AnnotationLuciferase Intensity
Fold Induction in Original Screening
X-box binding protein 1
ets variant gene 1
Similar to zinc finger protein 64
Protein kinase, cAMP dependent, catalytic, alpha
Similar to v-Ki-ras2 Kirsten rat sarcoma 2 viral oncogene homolog
Homo sapiens nuclear receptor subfamily 4, group A, member 1
Similar to cyclin-dependent kinase inhibitor 1B (P27)
Similar to RAS-like protein expressed in many tissues
Mus musculus, nuclear factor I/B, clone MGC:13959
Similar to paired-like homeo-domain transcription factor 1
Mus musculus, E26 avian leukemia oncogene 1, clone MGC:18571
Similar to growth arrest and DNAdamage-inducible 45 alpha
Mus musculus mRNA for transcriptional coactivator (Taz gene)
Mus musculus upstream transcription factor 2
Mus musculus, clone MGC:36628
RIKEN cDNA 3100004P22 gene
RIKEN cDNA 8430401F14 gene
Figure 2. Effect of Ras on KSHV Lytic Transcript Levels Assessed by RT-Q-PCR
BC-3 cells were transfected with pCMV, RTA, or Ha-Ras (Q61L). At 24, 48, and 72 h post-transfection, levels of immediate-early lytic transcripts RTA/
ORF50 (A), early lytic transcripts PAN (B), viral thymidine kinase (TK) (C), and late lytic transcript ORF65 (D) were measured by RT-Q-PCR. (The high level
of ORF50 transcript level in the RTA transfected cells in [A] is largely due to the transfection of exogenous RTA cDNA expression clone.)
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Cellular Signals Reactivating Herpesvirus
Raf/mitogen-activated protein (MAP)/extracellular signal–
regulated kinase (ERK) kinase (MEK)/ERK (Raf/MEK/ERK)
pathway (PD98059 and U0126) was applied, the induction of
K8 expression by Ras was inhibited in a dose-dependent
manner (Figure 3C). Moreover, constitutively active mutants
of Raf (Raf22W and Raf22W/DDED) induced K8 expres-
sion (Figure 3A). Though it is difficult to compare the
robustness of induction by Ras and Raf expression due to
different expression levels and induction kinetics of different
cDNA constructs, our results clearly showed that the Ras/Raf/
MEK/ERK pathway is the main pathway through which Ras
exerts its effect on KSHV reactivation.
To explore the mechanism by which the Ras/Raf/MEK/ERK
pathway reactivates KSHV, reporter assays were conducted
on the RTA promoter in 293T cells. The results showed that
v-Ki-Ras2 activated the RTA promoter, and this effect was
inhibited by U0126 in a dose-dependent manner (Figure 4A).
It has also been shown that the RTA protein can activate its
own promoter . In 293T cells where there is no
endogenous RTA protein, the cotransfection of v-Ki-ras2
with RTA resulted in an enhanced activation of the RTA
promoter. This effect was inhibited by U0126 to a level
similar to that induced by the RTA protein alone (Figure 4B),
suggesting that RTA and v-Ki-Ras2 activate expression from
the RTA promoter by two distinct mechanisms. To examine
the effect of Ras on other downstream lytic viral promoter
activation, reporter assays were conducted in 293T cells using
the reporter constructs pE4T-PAN, pE4T-Kpsn, pE4T-
ORF57, and pE4T-v-IL-6, which contain the RRE sequences
in the respective lytic promoters. The results showed that
RTA significantly induced their luciferase activity [16,26,29–
31]. However, v-Ki-Ras2 did not have significant effect on any
of these promoters, either by its own expression or by co-
expression with RTA (Figure 4C). Collectively, our reporter
assays suggested that the Ras/Raf/MEK/ERK pathway can
activate the RTA promoter, which further leads to the
activation of downstream lytic genes. The delayed induction
of the levels of viral transcripts by Ha-Ras (Q61L) compared
to RTA was also consistent with this hypothesis (Figure 2). We
have further determined that the DNA sequences mediating
the Ras and RTA responses are located in different regions on
the RTA promoter (unpublished data).
The Raf/MEK/ERK Pathway Mediates TPA-Induced KSHV
Reactivation and Spontaneous Reactivation in PEL Cells
The phorbol ester TPA is a commonly used chemical
inducer of herpesvirus reactivation . We examined the
mechanism of TPA-induced KSHV reactivation in JSC-1 cells
that are latently infected with a KSHV reporter virus,
rKSHV.219. This recombinant virus expresses the red
fluorescent protein from the KSHV lytic PAN promoter as
an indicator of reactivation, and the green fluorescent
protein (GFP) from the EF-1alpha promoter as an internal
control . TPA treatment activated the transcription of the
lytic gene PAN, and this induction was blocked by U0126
pretreatment in a dose-dependent manner (Figure 5A).
Western blot of KS-1/BC-3 cells also showed that the TPA-
induced expression of K8 was blocked by U0126 treatment,
consistent with previous RT-PCR results (Figure 5B) . In
contrast, reactivation induced by another commonly used
chemical, sodium butyrate, was not affected by U0126 (Figure
S3A), suggesting that the role of the Raf/MEK/ERK pathway is
specific to the effect of TPA. Consistent with our hypothesis
that the Raf/MEK/ERK pathway reactivates virus in an RTA-
dependent manner, an RTA-null virus failed to express lytic
genes and produce infectious viral particles upon TPA
treatment . The Raf/MEK/ERK pathway can be activated
by multiple upstream signals . We demonstrated that Ha-
Ras (S17N), a dominant-negative mutant of Ras, was not able
to inhibit TPA-induced K8 expression, which suggests that
TPA induces KSHV reactivation by a Ras-independent
mechanism (Figure 5C). By examining the mechanisms of
Ras- and TPA-induced KSHV reactivation, we revealed that
different upstream signals can converge into one pathway to
mediate herpesvirus reactivation.
We further investigated whether the Raf/MEK/ERK pathway
also contributes to KSHV spontaneous reactivation. Although
KSHV persists predominantly in the latent form in Kaposi
sarcoma tumors and PEL, low frequency spontaneous
reactivation can be detected in both Kaposi sarcoma tumor
cells [10,57,58]and B cells [10,28,59–62]. The cause of
spontaneous reactivation is not yet clear for any herpesvirus,
but it is believed to be important for virus-associated disease
pathology and transmission. To more sensitively measure
spontaneous reactivation rate, we established a BC-3-based
reporter cell line, BC-3-G, with GFP expression driven by the
Figure 3. Effect of Chemical Inhibitors on KSHV Reactivation in PEL Cells
(A) Expression of KSHV lytic protein RTA or K8 24 h after transfection with pCMV, RTA, Ha-Ras (Q61L), Raf22W, and Raf22W/DDED.
(B) The effects of the p38 pathway inhibitor SB203580 (20 uM), the JNK pathway inhibitor SP600125 (20 uM), and the PI3K/Akt pathway inhibitor
LY294002 (20 uM) on Ras-induced K8 expression.
(C) Dose-dependent inhibition of lytic protein K8 expression upon treatment with the Raf/MEK/ERK pathway–specific inhibitors PD98059 and U0126.
PLoS Pathogens | www.plospathogens.org March 2007 | Volume 3 | Issue 3 | e440448
Cellular Signals Reactivating Herpesvirus
PAN promoter. The PAN promoter is very sensitive and
specific to the presence of viral RTA protein, but is not
activated at all by other cellular factors, as indicated by a lack
of regulation by any of the 26,000 cDNA clones in the
genome-wide cDNA screen in the absence of viral RTA
activation. Thus, the activation of the PAN promoter in BC-3-
G cells serves as a specific indicator of KSHV spontaneous
reactivation. KSHV spontaneous reactivation in these cells
was assessed by flow cytometry (Figure 6). We found that
upon U0126 treatment, the percentage of GFP-positive cells
(gated in the R2 region) was significantly reduced. The total
cell population had a clearly ‘‘shrunken tail’’ of GFP
expression. The percentage of GFP-positive cells in the total
cell population is consistent with the spontaneous and
induced reactivation rate that was previously reported in
PEL cells . The ratio of inhibition by U0126 remained
similar even if we set the fluorescence-activated cell sorting
(FACS) gate value up or down 2-fold. This result indicated
that the Raf/MEK/ERK pathway is an important factor
regulating KSHV spontaneous reactivation in lymphoma
cells. To our knowledge, our result is the first example that
has revealed a specific cellular signaling pathway regulating
spontaneous reactivation of a herpesvirus.
Ets-1 Mediates Ras- and TPA-Induced Reactivation
Ets family transcription factors are potential downstream
effectors of Raf/MEK/ERK pathway. There are two major
groups of proteins in the Ets family: the Ets group, which
includes Ets-1, Ets-2, and Pointed; and the ternary complex
factors (TCFs), which include Elk1, Sap1a, Sap1b, Fli1, and
Net (reviewed in ). The identification of mouse Ets-1 and
Etv1 from the cDNA screen led us to examine the effect of
Ets-1 protein in KSHV reactivation. We found that K8
expression was induced by ectopic expression of Ets-1 in KS-
1/BC-3 lymphoma cells, but not by a dominant negative Ets-1
mutant (DN-Ets-1), which contains the DNA-binding domain
but lacks the transactivation domain (Figure 7A) . Ets-1,
but not DN-Ets-1, was also able to activate the RTA promoter
(Figure 7B). Furthermore, DN-Ets-1 inhibited Ras-induced
RTA promoter activation and KSHV lytic transcript levels
(Figure 7B–7D). Western blot analysis also showed that DN-
Ets-1 expression reduced TPA-induced K8 expression (Figure
S3B), suggesting that both Ras- and TPA-initiated KSHV
reactivation is mainly mediated by Ets-1.
To examine whether activation of the Raf/MEK/ERK path-
way leads to endogenous Ets-1 activation in PEL cells,
electrophoretic mobility shift assay (EMSA) was conducted
with nuclear extracts from BC-3 cells. The results showed that
Ets-1 DNA binding ability was increased more than 2-fold as
early as 2 h after TPA treatment. It was a significant increase,
considering that viral lytic replication was only induced in a
subset of the cell population. In comparison, the Oct-1
binding activity did not change at all (Figure 7E). The
specificity of the Ets-1 binding was verified by competition
experiments with specific and mutant probes and cold
oligonucleotides (Figure 7F). The data provided additional
evidence for the involvement of Ets-1 in reactivation induced
by Raf/MEK/ERK pathway activation.
It is generally hypothesized that multiple cellular signaling
pathways regulate herpesvirus reactivation in latently in-
fected cells. In this study, we address this issue directly,
utilizing a genome-scale analysis to identify cellular factors
that can reactivate endogenous herpesvirus in latently
infected B cells. By using this approach, we were able to
identify diverse signaling molecules as activators of KSHV
reactivation. The effectiveness of this genome-wide analysis
was validated by revealing the critical role of the Ras/Raf/
MEK/ERK/Ets-1 pathway in mediating signals from the cell
surface to the viral genome in the nucleus to reactivate
Figure 4. Effect of Ras on KSHV Lytic Promoter Activity Assessed by
(A and B) The effect of increasing doses of U0126 (5 uM, 10 uM, 20uM) on
Ras-induced RTA promoter activity in the absence (A) or presence (B) of
endogenous RTA protein.
(C) The effect of Ras on the promoter activity of four KSHV lytic genes
PAN, Kpsn, ORF57, and v-IL-6 in the absence or presence of RTA protein.
The fold induction represents the increase in luciferase intensity
corrected over the background induction on pE4T basic construct. The
data presented here are the average of three independent experiments.
The promoter activity is the relative luciferase intensity compared to
pcDNA3-transfected cells whose luciferase intensity was set as 1.
PLoS Pathogens | www.plospathogens.orgMarch 2007 | Volume 3 | Issue 3 | e440449
Cellular Signals Reactivating Herpesvirus
KSHV. This pathway is also crucial for TPA-induced
reactivation and spontaneous reactivation.
Our primary screen was designed to measure the activation
of the endogenous RTA promoter, using a reporter generated
from a downstream transcriptional target of RTA, PAN. The
PAN promoter provides a robust measure for RTA function,
and further enabled us to capitalize on three rounds of signal
amplification in our screen output: the direct activation of
the RTA promoter by the transfected gene product,
autoactivation of the RTA promoter by RTA itself, and
subsequent transactivation of the PAN promoter by RTA.
This substantially increased the sensitivity of our approach,
and enabled us to overcome typically low transfection
efficiencies observed in the lipid-mediated transfection of
lymphoid cells. At the same time, the PAN reporter construct
pPAN-69Luc contains only a minimum promoter region (69
base pair) that is very sensitive to RTA expression. This short
sequence makes it less likely for other transcription factors to
bind to and activate the PAN promoter. Indeed, the cDNA
clones identified in our screen did not activate PAN
promoter in 293T cells in the absence of the viral genome,
as shown for v-Ki-Ras2 in Figure 4C. Our screen identified a
wide range of cellular gene products that were able to
increase RTA expression and thereby initiate viral reactiva-
tion. Defining the consequences of multiple inputs is often
too difficult to address using traditional methodologies, but
has become a new exciting topic in systems biology. The
multiple signals that have been identified in the high-
throughput screen, in combination with our sensitive
fluorescent/luminescent reporter cell systems, can be utilized
to further characterize their combinatory effect. This also
forms a unique platform, to which other highly efficient
interdisciplinary approaches, such as microfluidic optical and
algorithm-based control systems, can be applied to explore
viral processes at single cell and population levels.
In latently infected cells, virus reactivation is thought to be
the consequence of a disruption of the ‘‘balance’’ between
signaling pathways that induce or suppress virus lytic
replication. Our screen system utilizes an ectopic expression
strategy to achieve the disruption of the balance of the
signaling network, thus exposing the virus to certain path-
ways that are involved in virus reactivation. One potential
limitation of this strategy is that the activation of certain
cellular signaling pathways might require post-translational
modification of their component molecules, such as phos-
phorylation or methylation, rather than mere overexpression.
Thus, our screen system may not have revealed some of these
genes and pathways, or those involved in repressing reac-
tivation. The latter might be discovered by assays such as an
siRNA library screen that specifically aims to inhibit
endogenous gene expression.
To validate the biological relevance of our genome-wide
Figure 5. Effect of TPA on KSHV Reactivation in PEL Cells
(A) Fluorescence microscopy of rKSHV.219 latently infected JSC-1 cells after 48 h incubation with TPA (20 ng/ml) and increasing amount of U0126
(B) The expression of KSHV lytic protein K8 after 24 h incubation with TPA (20 ng/ml) and increasing amount of U0126 pretreatment.
(C) BC-3 cells were cotransfected with MACS4.1 plasmid Ha-Ras (S17N) (Flag-tagged) 24 h before incubation with TPA (20 ng/ml). Sixteen hours later, the
successfully transfected cells were then enriched by a MACSelection system asþve portion, and untransfected cells were indicated as?ve portion. Ha-
Ras (S17N) and K8 expression was assessed by Western blot analysis with anti-Flag and anti-K8 antibody, respectively.
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Cellular Signals Reactivating Herpesvirus
screen, we performed detailed analysis of the signaling
pathway from Ras to Ets-1. It is known that Ras-related
pathways can be activated by a number of extracellular
stimuli including growth factors vascular endothelial growth
factor (VEGF) and platelet-derived growth factor (PDGF), as
well as immunological cytokines and chemokines. Studies on
KSHV have associated some of these stimuli with KSHV
infection and Kaposi sarcoma pathogenesis. For example, it
has been reported that VEGF-induced Raf activation pro-
motes KSHV entry into cells  and that inflammatory
cytokines can directly modulate KSHV replication [12,66].
Furthermore, it was shown that ERK1/2 and MEK1/2 induced
by KSHV early during de novo infection of target cells are
essential for expression of viral genes and for establishment
of infection . Our study directly revealed how Ras-related
pathways play a role in herpesvirus reactivation. In both
cases, the Raf/MEK/ERK pathway enhances the transcription
of the RTA gene. It is biologically rational that the same
cellular signaling pathways can induce viral reactivation as
well as facilitate herpesvirus de novo infection.
In the in vitro culturing system, the cells are usually
maintained in 10%–15% fetal bovine serum (FBS), where
various growth factors and cytokines are present. Some of
them are natural activators of certain cellular signaling
pathways. Our study reveals an interesting phenomenon in
that the intracellular processes can be triggered by further
Figure 6. Effect of U0126 on KSHV Spontaneous Reactivation in BC-3-G Cells by Flow Cytometry
Three independent experiments were conducted at 18 h (A) and 42 h (B) time points; a representative experiment result for the 18-h time point is
shown here (C): (a) untreated parental cell line BC-3; (b) BC-3-G untreated; (c) BC-3-Gþ0.1% dimethyl sulfoxide (DMSO); (d) BC-3-Gþ5 uM U0126; (e) BC-
3-Gþ10 uM U0126; (f) BC-3-Gþ20 uM U0126; (g). BC-3-Gþ20 ng/ml TPA; (h) BC-3-Gþ20 ng/ml TPAþ5 uM U0126; (i) BC-3-Gþ20 ng/ml TPAþ10 uM
U0126; (j) BC-3-G þ 20 ng/ml TPA þ 20 uM U0126.
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Cellular Signals Reactivating Herpesvirus
activation of these signaling pathways, and the induction level
does not necessarily correlate with the different preexisting
activation level of the related signaling components. KSHV-
infected cell lines were shown to express a higher level of
VEGF-A and B-Raf compared to uninfected cells, but the rate
of spontaneous reactivation of latently infected cells is low,
with variable levels of B-Raf in different cell lines [66,68]. This
suggests that KSHV reactivation does not necessarily corre-
late with the endogenous Ras/Raf activation level, but is most
likely balanced by other signaling pathways. We showed that
additional activation of Ras could overcome a threshold to
induce virus reactivation. Meanwhile, the preexisting Ras/Raf/
Figure 7. Effect of Ets-1 on KSHV Reactivation by Western Blot, Reporter Assays, and RT-Q-PCR
(A) Expression of K8 protein in BC-3 cells 48 h after transfection of ets-1 or DN-ets-1. Expression of Ets-1 and DN-Ets-1 was detected using an anti-Flag
(B) The activation of the RTA promoter upon expression of increasing amount of Ets-1 or DN-Ets-1 (10 ng, 50 ng, 100 ng, and 250 ng) in 293T cells by
(C) The effect of increasing amount of DN-Ets-1 on RTA promoter activity induced by Ets-1 (left), Ha-Ras (Q61L), or v-Ki-Ras2 (right). The firefly luciferase
readings were normalized to Renilla luciferase reading, and the relative luciferase induction compared to vector alone was presented here.
(D) Levels of lytic transcripts RTA/ORF50, PAN, and viral thymidine kinase (TK) upon transfection of Ha-Ras (Q61), DN-Ets-1, or a combination of both.
(E) EMSA assessing Ets-1 DNA binding ability using BC-3 cell nuclear extract after incubation with TPA (20 ng/ml) for indicated time. The binding of Oct-
1 probe was used as loading control. NS, non-specific binding.
(F) Competition experiments confirming the specificity of binding to Ets-1 probe. NS, non-specific binding.
PLoS Pathogens | www.plospathogens.org March 2007 | Volume 3 | Issue 3 | e440452
Cellular Signals Reactivating Herpesvirus
MEK/ERK activity in the regular culturing condition could be
an important factor that contributes to virus spontaneous
reactivation, as indicated by a reduced spontaneous reac-
tivation rate when this endogenous activity was inhibited by
U0126. The spontaneous reactivation in a small percentage of
cells represents cellular variation among ‘‘genetically identi-
cal cells.’’ There is no easy way to identify and isolate the cells
that will permit virus reactivation before the reactivation
actually takes place; thus, it is difficult to define the cellular
variation that leads to spontaneous reactivation. It is
intriguing that the Ras/Raf/MEK/ERK/Ets-1 signaling pathway
identified via the genome-wide screen is critical for sponta-
neous reactivation as well. The role of this pathway in de novo
infection, induced reactivation, and spontaneous reactivation
implies that this pathway is an essential pathway selected by
the virus to sense environmental cues at different stages of its
The genome-wide screen approach utilized in this study
provides an avenue by which the molecular signals regulating
herpesvirus reactivation may be systematically identified and
studied. The information revealed by the in vitro screen
systems could greatly help identify potential physiological
stimuli that can be further tested in in vivo studies. For
example, the discovery of the role of PKA on KSHV
reactivation led to the finding that physiological concen-
trations of epinephrine/norepinephrine effectively reactivate
KSHV . We also revealed that dopamine and dopamine
derivatives can reactivate KSHV through dopamine recep-
tors, which may function upstream of PKA and Ras
(unpublished data). Thus, identification of the potential
physiological stimuli, facilitated by the large scale screenings,
provides a foundation for further verifications in in vivo
The balance between latency and lytic replication is a
fundamental virology question, particularly in the herpesvi-
rus field. A better understanding of the mechanisms control-
ling the balance between latency and lytic replication may in
turn enable the development of a more effective therapeutic
strategy to treat KSHV and EBV-associated malignancies. For
example, one potential approach to treat KSHV and EBV
infection is to reactivate latent virus, followed by gancyclovir
or acyclovir treatment to kill cells expressing lytic genes [69–
73]. Based on the comprehensive knowledge of herpesvirus
reactivation and the multiple signaling pathways that mediate
this process, clinically applicable approaches to optimally
reactivate herpesvirus in these malignancies could be
developed, which in turn can facilitate the development of
new therapeutic strategies.
Materials and Methods
Cell culture. KS-1 cells are a gift from J. Said, and BC-3 cells are a
gift from E. Cesarman. The construction of JSC-1 cells latently
infected by rKSHV.219 reporter virus was described previously .
These B cells were cultured in RPMI 1640 medium containing 15%
FBS. 293T cells were cultured in DMEM (Dulbecco’s modified Eagle’s
medium) containing 10% FBS.
Plasmids and reagents. KSHV RTA cDNA was expressed in
pFLAG-CMV2 vector . Ha-Ras (Q61L) is a constitutively active
form of Ha-Ras. Ha-Ras (S17N) is a dominant negative form of Ha-
Ras. Raf22W and Raf22W/DDED are constitutively active forms of c-
Raf . The reporter construct pPAN-69Luc contains the PAN
promoter region spanning nucleotides (nt)?69 to þ14 in pGL3-basic
vector , and the reporter pRpluc contains the 3-kb region
upstream of the KSHV RTA coding sequence in pGL2-basic vector
. Reporter constructs pE4T and pE4T-RRE were generated as
previously described . The full-length ets-1 and truncated DN-ets-
1 were cloned from cDNAs from BC-3 cells using primers 59- CCC
AAG CTT ATG AAG GCG GC ?39 (ets-1 forward primer), 59- CCC
AAG CTT ATG CCT GTC ATT C?39 (DN-ets-1 forward primer) and
59- GAA GAT CTT CAC TCG TCG GC ?39 (ets-1/DN-ets-1 reverse
primer). Restriction enzyme sites Hind III and Bgl II were engineered
into the 59 and 39 end of the sequence respectively, and both
sequences were cloned into pFlag-CMV2 vector. Chemicals PD98059,
U0126, SB203580, SP600125, and LY294002 were purchased from
The phospho-p38 MAPK (Thr180/Tyr182) (28B10) antibody, phos-
pho-cJun (Ser63) antibody, and phospho-Akt (Ser473) antibody were
purchased from Cell Signaling Technology (http://www.cellsignal.
Establishment of the BC-3-G cell line. The pPAN-122-d2EGFP
construct was generated by replacing the promoter region in the
pNFkB-d2EGFP vector (Clontech, http://www.clontech.com) with the
PAN promoter region spanning nt ?122 to þ14 from the pGL3-8b
construct . pTW40 that contains the puromycin-resistant gene in
pcDNA3 vector was generated previously in our lab. The BC-3-G cell
line was established by cotransfecting pPAN-122-d2EGFP and pTW40
plasmids into BC-3 cells and selection with 1.5 ug/ml puromycin.
High-throughput screen. High-throughput (retro) transfections of
the 26,000 mammalian cDNA library containing 15,000 full-length
human clones (OriGene, http://www.origene.com), 4,000 additional
full-length human cDNA clones (Mammalian Gene Collection; Open
Biosystems, http://www.openbiosystems.com), and 7,000 full-length
mouse cDNA clones (Mammalian Gene Collection, Open Biosystems)
were carried out in a similar fashion as previously described . In
brief, 20 ng of reporter construct pPAN-69Luc, together with 62.5 ng
of individual cDNAs, were incubated with 20 ul of serum-free
medium containing FuGENE 6 (Roche, http://www.roche.com) in each
well of 384-well plates. After 20 min of incubation, 10,000 KS-1 cells
were delivered into each well. Luciferase activity was measured
approximately 40 h post-transfection using Bright-Glo luciferase
assay reagent (Promega, http://www.promega.com) on the Acquest
multi-mode reader (Molecular Devices, http://www.moleculardevices.
com). Control wells contained KS-1 cells cotransfected with pPAN-
69Luc, and pcDNA6 vector treated with sodium butyrate and TPA.
Reporter assays. Transfections of 293T cells for Dual-Luciferase
assays were conducted as follows: cells were seeded into 24-well plates
20 h prior to transfection so that they were ;90% confluent at the
time of transfection. Then, 50 ng of the firefly reporter construct, 2
ng of Renilla luciferase construct pRL-SV40, and various amounts of
cDNA expression constructs (supplemented with pcDNA3 vector
DNA to a total DNA amount of 0.8–1 ug/well) were transfected using
Lipofectamine 2000 (Invitrogen, http://www.invitrogen.com). Cells
were incubated in DMEM containing 0.5% FBS for 24 h followed by
Dual-Luciferase assays according to the manufacturer’s instructions
RT-Q-PCR analysis. KS-1/BC-3 cells were transfected by electro-
poration or using Lipofectamine 2000 (Invitrogen). For electro-
poration, 107cells were mixed with 9 ug of expression plasmid and 1
ug of pEGFP-C1 (BD Biosciences, http://www.bdbiosciences.com) as a
transfection efficiency marker in a cuvette with a 0.4-cm gap (ISC
BioExpress, http://www.bioexpress.com). Electroporation was per-
formed using the Gene-Pulser II (Bio-Rad, http://www.bio-rad.com)
with capacitance extender, 960 uF, 0.25 kV. At the indicated time
points, cellular RNA was isolated using the RNeasy kit with on-
column DNA digestion (Qiagen, http://www.qiagen.com). The mRNA
was subsequently reverse transcribed into cDNA using SuperScript II
RNase H-Reverse Transcriptase (Invitrogen). The primers used for
RT-Q-PCR were: PAN primers (59-GCCGCTTCTGGTTTTCATTG-39
as the forward primer and 59-TTGCCAAAAGCGACGCA-39 as the
reverse primer), viral thymidine kinase primers (59-CGTAGCC-
GACGCGGATAA-39 as the forward primer and 59-TGCCTGTA-
GATTTCGGTCCAC-39 as the reverse primer) and ORF65 primers
(59-GGCGGCCGTTTCCG-39 as the forward primer and 59-
TCATTGTCGCCGGCG-39 as the reverse primer). The PCR product
amplified by GAPDH primers (59-GAAGGTGAAGGTCGGAGTC-39 as
the forward primer and 59-GAAGATGGTGATGGGATTTC-39 as the
reverse primer) was used as an internal control.
EMSA. Nuclear extract was prepared using a previously described
method . Ets-1 and Oct-1 DNA binding ability was assessed by
the Ets-1, mutated Ets-1, or Oct-1 consensus sequences with 59 GGG
overhang (Ets-1: 59-GGGGTCAGTTAAGCAGGAAGTGACTAAC-39,
mutated Ets-1: 59- GGGGTCAGTTAAGCAGGCAGTGACTAAC-39,
32P-labeled double-strand oligonucleotides containing
PLoS Pathogens | www.plospathogens.orgMarch 2007 | Volume 3 | Issue 3 | e440453
Cellular Signals Reactivating Herpesvirus
TATG-39) [77,78]. Then, 2 lg of nuclear proteins were incubated with
2 3 104cpm of radiolabeled oligonucleotides in a binding buffer
containing 1 lg poly (dI-dC), 0.25% NP-40, 5% glycerol, 10 mmol/L
Tris HCl (pH 7.5), 50 mmol/L KCl, 1 mmol/L DTT, and 1 mmol/L
EDTA for 20 min at room temperature. For competition experi-
ments, excess amounts of cold Ets-1 or mutated Ets-1 oligo were
added 10 min before the addition of labeled probe. Samples were
separated on 4.5% acrylamide gels and assessed by autoradiography.
Figure S1. Effect of RTA on PAN Promoter Activity
PAN-69Luc reporter and an increasing amount of RTA were
transfected into 293T cells, and luciferase intensity was assessed.
Found at doi:10.1371/journal.ppat.0030044.sg001 (254 KB PDF).
Figure S2. Ras-Induced KSHV Lytic Protein Expression Assessed by
KS-1 cells were transfected with pcDNA3 or Ha-Ras (Q61L) by
electroporation. Then, 72 h after transfection, cells were harvested,
fixed, and subjected to immunofluorescence analysis. The total
numbers of cells and cells expressing the lytic proteins v-IL-6,
ORF59, and K8.1A were counted in three independent fields. v-IL-6
expression increased from 0.98% to 4.52%, ORF59 expression from
0.30% to 5.71%, and K8.1A expression from 0.20% to 1.80%.
Transfection efficiency of KS-1 cells by electroporation ranges
between 10% and 15%. One representative field for each lytic
protein is shown. The total number of cells in each field is indicated
by DAPI (49,6-diamidino-2-phenylindole) staining. The percentage of
cells that express the corresponding lytic proteins is shown below
each pair of fluorescence pictures.
Found at doi:10.1371/journal.ppat.0030044.sg002 (686 KB PDF).
Figure S3. TPA-Induced KSHV Reactivation
(A) BC-3 cells were pretreated with U0126 for 1 h before incubation
with TPA (20 ng/ml) or sodium butyrate (1.5 uM) for 20 h, and K8
expression was assessed by Western blot analysis.
(B) BC-3 cells were cotransfected with MACS4.1 plasmid and DN-ets-1
24 h before incubation with TPA (20 ng/ml). Sixteen hours later, the
successfully transfected cells were then enriched by a MACSelection
system as þve portion, and untransfected cells were indicated as ?ve
portion. K8 expression was assessed by Western blot analysis.
Found at doi:10.1371/journal.ppat.0030044.sg003 (348 KB PDF).
Figure S4. Effect of Specific Signaling Pathway Inhibitors
The effect of SB203580 in the p38 pathway represented by an
increased level of p-p38 (A), SP600125 in the JNK pathway
represented by a reduced level of p-cJun (B), and LY294002 in the
PI3K/Akt pathway represented by a reduced level of p-Akt (C) was
shown by Western blots. BC-3 cells (A and C) or 3T3 cells (B) were
serum starved overnight and pretreated with individual inhibitors for
1 h, followed by UV (400 uCi) and PDGF (50 ng/ml) treatment for 30
min. Protein lysates were prepared in RIPA buffer for Western blots
to detect the phosphorylated proteins.
Found at doi:10.1371/journal.ppat.0030044.sg004 (347 KB PDF).
We thank J. Said, E. Cesarman, J. Jung, O. Marinez-Maza, T. Satoh, and
K.-L. Guan for generously providing reagents; the Janis V. Giorgi
Flow Cytometry Laboratory at UCLA for help on the FACS
experiments; Giri Sulur and Eric Bortz for critical reading and
editing; and all members in the Sun lab for helpful discussions.
Author contributions. FY, HJB, and RS conceived and designed the
experiments. FY, JNH, CGN, and SKC performed the experiments. FY
and JNH analyzed the data. HD, MJS, TTW, JKS, JV, FT, and RS
contributed reagents/materials/analysis tools. FY wrote the paper.
Funding. This study was supported by US National Institutes of
Health (NIH) grants CA83525, CA91791, CA 32737 and DE14153, the
Stop Cancer Foundation, the California Cancer Research Committee,
the Novartis Research Foundation and Burroughs Wellcome Fund.
Competing interests. The authors have declared that no competing
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PLoS Pathogens | www.plospathogens.orgMarch 2007 | Volume 3 | Issue 3 | e440455
Cellular Signals Reactivating Herpesvirus