JOURNAL OF VIROLOGY, Nov. 2005, p. 13538–13547
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 21
?-Adrenoreceptors Reactivate Kaposi’s Sarcoma-Associated
Herpesvirus Lytic Replication via PKA-Dependent
Control of Viral RTA
Margaret Chang,1Helen J. Brown,2Alicia Collado-Hidalgo,3,6Jesusa M. Arevalo,3
Zoran Galic,3Tonia L. Symensma,2,4Lena Tanaka,4Hongyu Deng,2
Jerome A. Zack,1,3,5Ren Sun,2,5and Steve W. Cole3,5,6*
Departments of Microbiology, Immunology and Molecular Genetics,1Molecular and Medical Pharmacology,2and
Medicine,3David E. Geffen School of Medicine, University of California, Los Angeles, California; Department
of Biological Sciences, Mount Saint Mary’s College, Los Angeles, California4; and Jonsson Comprehensive Cancer Center,5
UCLA AIDS Institute, and UCLA Molecular Biology Institute5and Norman Cousins Center,6
University of California, Los Angeles, California
Received 25 April 2005/Accepted 5 August 2005
Reactivation of Kaposi’s sarcoma-associated herpesvirus (KSHV) lytic replication is mediated by the viral
RTA transcription factor, but little is known about the physiological processes controlling its expression or
activity. Links between autonomic nervous system activity and AIDS-associated Kaposi’s sarcoma led us to
examine the potential influence of catecholamine neurotransmitters. Physiological concentrations of epineph-
rine and norepinephrine efficiently reactivated lytic replication of KSHV in latently infected primary effusion
lymphoma cells via ?-adrenergic activation of the cellular cyclic AMP/protein kinase A (PKA) signaling
pathway. Effects were blocked by PKA antagonists and mimicked by pharmacological and physiological PKA
activators (prostaglandin E2and histamine) or overexpression of the PKA catalytic subunit. PKA up-regulated
RTA gene expression, enhanced activity of the RTA promoter, and posttranslationally enhanced RTA’s trans-
activating capacity for its own promoter and heterologous lytic promoters (e.g., the viral PAN gene). Mutation
of predicted phosphorylation targets at RTA serines 525 and 526 inhibited PKA-mediated enhancement of RTA
trans-activating capacity. Given the high catecholamine levels at sites of KSHV latency such as the vasculature
and lymphoid organs, these data suggest that ?-adrenergic control of RTA might constitute a significant
physiological regulator of KSHV lytic replication. These findings also suggest novel therapeutic strategies for
controlling the activity of this oncogenic gammaherpesvirus in vivo.
known as human herpesvirus 8 [HHV-8]) is a lymphotropic
gammaherpesvirus originally identified in the context of Ka-
posi’s sarcoma (8) and subsequently implicated in primary
effusion lymphoma and multicentric Castleman’s disease (6,
66). The KSHV genome most closely resembles herpesvirus
saimiri (60), but the virus also bears more distant structural
and functional similarities to Epstein-Barr virus (48, 49). Like
all herpesviruses, KSHV displays a bifurcating gene expression
program that allows it to defer lytic replication and enter a
protracted state of latency during which only a small minority
of viral genes are expressed (19, 21, 71, 78). KSHV establishes
latency in B lymphocytes and vascular endothelial cells, but it
must resume lytic replication to disseminate or colonize a new
host. Discovery of the physiological signals that control KSHV
reactivation is thus key to controlling its pathogenic potential.
Suppression of such signals could block the spread of infection,
and pharmacological induction of such signals might flush la-
tently infected cells into lytic replication for elimination by
nucleoside analogue drugs (31).
Impaired cellular immunity plays a critical role in allowing
KSHV replication, but the physiological stimuli that positively
induce lytic gene expression are poorly understood. Chemical
agents such as phorbol esters or N-butyrate can reactivate
KSHV in vitro (45, 50, 57), and proinflammatory cytokines
have similar, though weaker effects (7, 44, 46). At the level of
the viral genome, lytic reactivation is mediated by the KSHV-
encoded transcription factor RTA. Overexpression of the RTA
gene is sufficient to trigger lytic replication in latently infected
B-cell lines (37, 70), and RTA mutations can block viral reac-
tivation in vitro (36). RTA protein can also autoactivate the
RTA promoter (16, 25, 62), but the cellular signals that initiate
RTA expression are not well understood.
Several recent studies have shown that high levels of auto-
nomic nervous system activity can accelerate the onset of
AIDS-defining conditions during human immunodeficiency vi-
rus type 1 infection (11, 12, 14). These effects have been at-
tributed to autonomic nervous system regulation of human
immunodeficiency virus type 1 replication (10, 13, 14), but it is
also conceivable that autonomic nervous system activity might
directly activate opportunistic pathogens such as KSHV. The
present studies examined that hypothesis, with an emphasis on
the cellular signal transduction pathways that might allow cat-
echolamine neurotransmitters from the autonomic nervous
system to regulate key molecular events in KSHV reactivation.
Results show that physiological concentrations of epinephrine
and norepinephrine can induce lytic replication of KSHV in
* Corresponding author. Mailing address: Department of Medicine,
Division of Hematology-Oncology, 11-934, Factor Building, David
Geffen School of Medicine at UCLA, Los Angeles, CA 90095-1678.
Phone: 310-267-4243. Fax: 310-206-1318. E-mail: email@example.com.
latently infected lymphoid cells via ?-adrenergic activation of
the cellular protein kinase A (PKA) signaling pathway. These
effects are mediated by increased expression of RTA and post-
translational enhancement of its trans-activating capacity. Re-
sults provide an endocrinological perspective on the control of
KSHV replication and suggest novel strategies for therapeutic
MATERIALS AND METHODS
Cell culture. KSHV reactivation was analyzed in the primary effusion lym-
phoma (PEL) cell lines KS-1, BC3, and BCBL-1. DG75 and Ramos served as B
lymphoid cells uninfected with KSHV or Epstein-Barr virus. Cells were cultured
under standard conditions in RPMI plus 10% fetal bovine serum or in serum-
free X-VIVO 15 (Cambrex, East Rutherford, NJ) supplemented by a defined mix
of growth factors (MITO?, BD Biosciences, Bedford, MA). Reactivation exper-
iments were performed as described (70), using the PKC inducer phorbol-12-
myristate-13-acetate (PMA, also known as TPA; Calbiochem, San Diego CA),
the PKA inducer dibutyrl cyclic AMP (db-cAMP), norepinephrine ([?]-artere-
nol; Sigma-Aldrich, St. Louis, MO), or other indicated agents acting on the
?-adrenoreceptor (?AR)/cAMP/PKA signaling pathway (all from Sigma-Aldrich
or Calbiochem). Norepinephrine and PKA modulating agents were diluted in
phosphate-buffered saline and added to cultures in volumes ?1% of total culture
volume. Unstimulated controls received an equivalent volume of vehicle.
Lytic protein expression. Intracellular expression of KSHV ORF59 was as-
sayed by flow cytometric quantification of indirect immunofluorescence in per-
meabilized KS1, BC3, or BCBL1 cells; 2 ? 106cells were stained for 30 min at
4°C with 1 ?g of mouse-anti KSHV ORF59 monoclonal antibody (Advanced
Biotechnology Incorporated, Colombia, MD) in 50 ?l of BD Cytofix/Cytoperm
(BD Immunocytometry Systems, San Jose, CA), washed in 450 ?l of permeabi-
lization buffer, stained for 30 min with 0.5 ?g of fluorescein isothiocyanate-
conjugated rat anti-mouse immunoglobulin G2b Ab (BD Immunocytometry
Systems), and washed again in 450 ?l of permeabilization buffer. Fluorescence
intensity data were acquired on a FACScan flow cytometer (BD Immunocytom-
etry) with dead cells and debris excluded on the basis of forward- versus side-
scatter gating using CellQuest software. KSHV lytic proteins and cellular ?-actin
were also assayed by Western blot with enhanced chemiluminescence using
KSHV patient serum, as previously described (70).
Lytic gene expression. mRNA for KSHV gene products and human ?-adre-
noreceptor subtypes were quantified relative to cellular housekeeping genes
using real-time reverse transcription (RT)-PCR. Reactions utilized 1/10 of the
total DNase-treated (QIAGEN, Valencia, CA) RNA extracted from 106cells in
a one-step thermal cycling protocol (QIAGEN One-Step RT-PCR) with 30 min
of reverse transcription at 50°C, 15 min. of RT denaturation at 95°C, and 40
cycles of DNA amplification (15 s at 95°C, 60 s at 60°C). Reactions utilized
established primers and fluorescent detection probes for human glyceraldehyde-
3-phosphate dehydrogenase (GAPDH) and KSHV K8.1, ORF29, ORF50,
ORF72, and ORF57 (21). Primers for human ?-adrenoreceptor subtypes were:
?1(forward: TCG GAA TCC AAG GTG TAG GG, reverse:TGG CTT TTC
TCT TTG CCT CG), ?2(forward: CAT GTC TCT CAT CGT CCT GGC CA,
reverse:CAC GAT GGA AGA GGC AAT GGC A), and ?3(forward: GGC
TTC TTG GGG AGT TTC TTA GG, reverse: TTC TGG AGG GTA GAG
TGT CAC AGC), derived from GenBank sequences ADRB1: J03019, ADRB2:
M15169, and ADRB3: X70811, respectively. mRNA expression was normalized
to GAPDH by subtraction of threshold cycles (Ct; normalized target Ct? target
Ct? GAPDH Ct) and quantified as a fold change relative to an unstimulated
baseline (fold change ? 2[stimulated normalized target Ct– baseline normalized
DNA replication. Replicating KSHV DNA was assayed by Gardella gel anal-
ysis as previously described (23). Briefly, 2 ? 107KS1 cells were lysed during
electrophoresis through a 0.75% agarose gel for 3 h at 0.8 V/cm followed by 17 h
at 4.5 V/cm. Resolved DNA was transferred to a Hybond N? membrane (Am-
ersham-Pharmacia, Buckinghamshire England), UV cross-linked, and probed
with a 3-kb32P-labeled PCR product spanning the majority of the KSHV ORF50
locus. Supernatant particle-associated KSHV DNA was assayed by treating 1 ml
of 0.45-?m-filtered PEL cell supernatants with 2 ?g DNase (Worthington Bio-
chemical Corporation, Lakewood, NJ) and 10 mM MgCl for 15 min, followed by
extraction of particle-protected DNA (QIAGEN MiniElute virus spin kit) and
real-time PCR amplification of KSHV K8.1 DNA (45 cycles of 95°C for 15
seconds and 60°C for 60 seconds) with resolution of products on an 3% agarose
Infectious virus. PEL cell production of infectious KSHV was assayed by
suspending peripheral blood mononuclear cells (PBMC) from healthy donors in
PEL cell supernatants that had been filtered at 0.45 ?m and treated with DNase
as described above; 2 ? 106PBMC were stimulated with PHA for 72 h, washed,
and incubated for 1 h. in 2 ml of cell culture supernatant from PEL cells treated
with phorbol myristate acetate (PMA), norepinephrine, db-cAMP, or vehicle for
48 h. After extensive washing, PBMC were cultured for another 24 h and
establishment of KSHV genomic DNA in PBMC was assayed by PCR detection
of ORF50 (forward: AAC CAG AAG CCT CGG GCG AAG, reverse: GTG
CAC GCC ACG GAT GTC) or K8.1 (forward: AAA GCG TCC AGG CCA
CCA CAG A, reverse: GGC AGA AAA TGG CAC ACG GTT AC) in cellular
DNA (45 cycles of 95°C for 15 seconds and 60°C for 60 seconds, with SYBR
green real-time detection). Cellular RNA was also extracted from 8 ? 106
PBMC, treated with RNase-free DNase (QIAGEN RNEasy), and assayed for
expression of the latent gene product ORF72 by real-time RT-PCR as described
(21). Results were normalized to GAPDH DNA amplified in parallel and visu-
alized on a 2% agarose gel.
Overexpression of PKA catalytic subunit. PEL cells were transduced with a
self-inactivating lentiviral vector expressing a constitutively active form of PKA
(43) and an enhanced green fluorescent protein (EGFP) reporter gene under
control of a recombinant Rh-MLV promoter (34). Both sequences were trans-
lated from a single transcript bearing the PS3 internal ribosome entry site (IRES)
(73). cDNA for the human immunodeficiency virus type 1 central polypyrimidine
tract (77) was introduced into pSIN-18-Rh (34) at the XhoI site upstream of the
Rh murine leukemia virus promoter to produce pSIN18RhMLV-E-CPPT.
cDNA of the PKA catalytic subunit ? (PRKACA: X07767) was amplified with
primers bearing AgeI and SalI restriction sites and subcloned into pCR-Blunt
TOPO (Invitrogen, Carlsbad, CA), sequenced, released by digestion, purified,
and subcloned into AgeI and SalI sites of pSIN18RhMLV-E-CPPT to produce
pSIN18RhMLV-E-CPPT-PKA. The PS3 IRES-EGFP sequence was excised
from pDF-PS3 (73) and subcloned into the NotI site of a circularized pCRII-
TOPO vector (Invitrogen). The EcoRV site upstream of the subcloned fragment
was changed into a SalI site, and a SalI/XhoI fragment was subcloned into
pSIN18RhMLV-E-CPPT-PKA to produce pSIN18RhMLV-E-CPPT-PKA-PS3-
EGFP. The negative control vector pSIN18RhMLV-E-CPPT-EGFP included
EGFP sequences in the absence of upstream PRKACA-PS3 sequences. Vectors
were constructed by transfection into 293T cells (34), and target cells were
transduced by 1 h of incubation in vector-containing supernatants supplemented
with 10 ?g/ml Polybrene.
RTA promoter activity. Luciferase reporter assays utilized a 3-kb sequence
upstream of the RTA translation start site (pRpluc) (16), and three truncation
variants generated by restriction enzyme digestion of pRpluc (pRp1 cut at PstI,
pRp2 at NdeI, and pRp8 at KpnI) to delete six potential cAMP response
elements (CREs) detected by sequence-based bioinformatics (76); 4 ?g of each
reporter construct was electroporated along with 50 ng of the control pRLCMV
(Renilla luciferase driven by the cytomegalovirus [CMV] promoter) (Promega,
Madison, WI) and 6 ?g of empty pcDNA3 vector (10 ?g total) into 107KS-1,
BC3, BCBL-1, or DG75 cells (240 V, 125 ?, 950 ?F) or 5 ? 107Ramos cells
supplemented with 26 ?g/ml DEAE dextran (2 cycles of 320 V, 0 ?, 950 ?F),
using a BTX ECM 630 pulse generator. Dual luciferase assays (Promega) were
performed in triplicate, with firefly luciferase light units normalized to Renilla
luciferase light units prior to analysis of log fold change by Student’s t test.
RTA trans-activating capacity. RTA protein was expressed in the context of
RTA promoter assays by replacing 4 ?g of pcDNA3 with pcDNA3/RTA (a
pcDNA3-based vector expressing genomic KSHV RTA under control of the
CMV promoter) (16) or pFLAG/RTA (C-terminally FLAG-tagged KSHV RTA)
(3, 65). To control for any effect of PKA on the CMV promoter (56), data were
normalized to Renilla luciferase levels driven by the CMV promoter (pRLCMV).
To assess RTA-mediated trans-activation of a heterologous KSHV promoter,
luciferase reporter assays were conducted as above after replacing pRpluc with
pLUC/-69, a pGL3-Basic vector expressing firefly luciferase from a 69-nucleotide
fragment of the KSHV PAN RNA promoter (64).
Expressed RTA protein levels were assessed in parallel using flow cytometry to
detect C-terminal EGFP-tagged ORF50 expressed under control of the same
promoter (a kind gift from Joonho Choe, Department of Biological Sciences,
Korean Advanced Institute of Science and Technology) (26). Fluorescence in-
tensity data were acquired on a FACScan flow cytometer (BD Immunocytom-
etry) with dead cells and debris excluded on the basis of forward versus side
scatter gating using CellQuest software. In each of three replicate experiments,
GFP expression was quantified as a percent change above background fluores-
cence levels, with statistical significance of differences assessed by paired t test.
RTA phosphorylation targets. The predicted amino acid sequence of RTA
(AF091348) was scanned by Phosphobase 2.0 (32) to identify consensus PKA
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