JOURNAL OF VIROLOGY, Feb. 2007, p. 1072–1082
Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Vol. 81, No. 3
Kaposi’s Sarcoma-Associated Herpesvirus-Encoded Protein Kinase and
Its Interaction with K-bZIP?
Yoshihiro Izumiya,1Chie Izumiya,1Albert Van Geelen,2Don-Hong Wang,1,3Kit S. Lam,3
Paul A. Luciw,2* and Hsing-Jien Kung1*
Department of Biological Chemistry and Molecular Medicine, University of California-Davis School of Medicine, University of
California-Davis Cancer Center, Research Building III, Room 2400, 4645 2nd Avenue, Sacramento, California 958171;
Center for Comparative Medicine and Department of Pathology, University of California-Davis, 1 Shields Avenue,
Davis, California 956162; and Division of Hematology and Oncology, Department of Internal Medicine,
University of California-Davis Cancer Center, 4501 X Street, Sacramento, California 958173
Received 11 July 2006/Accepted 30 October 2006
The oncogenic herpesvirus, Kaposi’s sarcoma-associated herpesvirus, also identified as human herpesvirus
8, contains genes producing proteins that control transcription and influence cell signaling. Open reading
frame 36 (ORF36) of this virus encodes a serine/threonine protein kinase, which is designated the viral protein
kinase (vPK). Our recent efforts to elucidate the role of vPK in the viral life cycle have focused on identifying
viral protein substrates and determining the effects of vPK-mediated phosphorylation on specific steps in viral
replication. The vPK gene was transcribed into 4.2-kb and 3.6-kb mRNAs during the early and late phases of
viral reactivation. vPK is colocalized with viral DNA replication/transcription compartments as marked by a
polymerase processivity factor, and K-bZIP, a protein known to bind the viral DNA replication origin (Ori-Lyt)
and to regulate viral transcription. The vPK physically associated with and strongly phosphorylated K-bZIP
at threonine 111, a site also recognized by the cyclin-dependent kinase Cdk2. Both K-bZIP and vPK were
corecruited to viral promoters targeted by K-bZIP as well as to the Ori-Lyt region. Phosphorylation of K-bZIP
by vPK had a negative impact on K-bZIP transcription repression activity. The extent of posttranslational
modification of K-bZIP by sumoylation, a process that influences its repression function, was decreased by vPK
phosphorylation at threonine 111. Our data thus identify a new role of vPK as a modulator of viral
Kaposi’s sarcoma-associated herpesvirus (KSHV), also des-
ignated human herpesvirus 8, has been linked to several
malignancies, including Kaposi’s sarcoma, B-cell lympho-
mas, primary effusion lymphomas, and multicentric Castle-
man’s disease in immunocompromised individuals (reviewed
in reference 46). Kaposi’s sarcoma is the most common malig-
nancy associated with AIDS (45). The viral genome is double-
stranded DNA, approximately 165 kbp, and encodes over 81
open reading frames (ORFs) (43). The majority of the ORFs
are essential for viral replication and include genes necessary
for viral DNA replication, transcription, and assembly of in-
fectious particles (51). In addition, the KSHV genome contains
a large number of ORFs with homology to known cellular
genes. Several of these viral ORFs are implicated in modulat-
ing host immune responses, promoting angiogenesis, and dys-
regulating cell growth (14). A model for regulated expression
of KSHV genes has been deduced from numerous genetic and
biochemical studies of viral RNA patterns and activities of
viral transactivators (22, 31, 39, 49). As for other herpesviruses,
KSHV genes in productive infection have been classified into
the following temporally distinct classes: immediate-early,
early, and late. This virus can also establish a latent state that
is characterized by presence of a multicopy circular episome of
viral DNA, which expresses a small subset of viral proteins.
Productive (lytic) viral replication can be induced by treatment
of latently infected cell lines with butyrate, phorbol esters, or
hypoxia (13). After induction, the transcriptional transactiva-
tor K-Rta (ORF50) is activated, and this protein then induces
the K-bZIP (also called K8 or RAP) transcriptional regulator
and ORF57 (posttranscriptional transactivator). These latter
viral regulators activate other early and late phase genes, and
thus ensues the complete viral life cycle.
Alpha-, beta-, and gamma-herpesviruses encode phospho-
transferases that phosphorylate proteins and nucleosides (re-
viewed in reference 24). One group of viral protein kinases is
conserved among all alpha-, beta-, and gamma-herpesviruses.
The conserved protein kinases are UL13 of herpes simplex
virus (HSV), ORF47 of varicella-zoster virus, UL97 of cyto-
megalovirus (CMV), BGLF4 of Epstein-Barr virus (EBV), and
ORF36 of KSHV. Located within the catalytic region of these
kinases are 11 conserved subdomains that are common to
cellular serine/threonine protein kinases. Experimental studies
on members of each of the three major groups of herpesviruses
have identified a wide array of viral and cellular protein sub-
strates of the conserved viral protein kinases. Structural anal-
ysis of purified virions of several herpesviruses, including HSV,
CMV, and EBV, indicate that these protein kinases are asso-
* Corresponding author. Mailing address for Hsing-Jien Kung: Uni-
versity of California-Davis, Cancer Center, Research III Room 2400B,
4645 2nd Ave., Sacramento, CA 95817. Phone: (916) 734-1538. Fax:
(916) 734-2589. E-mail: firstname.lastname@example.org. Mailing address for Paul
A. Luciw: Center for Comparative Medicine, Department of
Pathology and Laboratory Medicine, University of California-Davis, 1
Shields Ave., Davis, CA 95616. Phone: (530) 752-3430. Fax: (530)
752-7914. E-mail: email@example.com.
?Published ahead of print on 15 November 2006.
ciated with virus particles (2, 12, 37, 50, 52); thus, the kinases
are in a position to influence virion assembly and events that
take place after entry of the virion into the cell. A number of
studies using kinase-null viral mutants demonstrated the im-
portance of the kinase for regulating viral gene expression,
replication, tissue tropism, or infection in animal models (7, 11,
26, 35, 41, 48). Various studies have implicated the viral pro-
tein kinase in influencing viral gene expression (6, 58), viral
DNA replication (27, 52, 53), or nucleocapsid egress from the
nucleus during virus assembly (26, 34, 53). The importance of
the viral protein kinase (vPK) for HSV and CMV replication is
supported by studies showing that changes in this gene can
confer resistance to certain antiviral agents (e.g., ganciclovir)
(7, 26). Taken together, these studies demonstrate that the
conserved herpesvirus protein kinases impact multiple steps in
Orf-36 protein (hereafter designated vPK) of KSHV is a
serine protein kinase, which is localized in the nucleus (38). In
vitro protein kinase assays indicated that this viral protein was
autophosphorylated and that the lysine residue in the catalytic
kinase subdomain II was essential for enzymatic activity (38).
Previous analysis on levels of KSHV transcripts in productive
infection indicated that vPK RNA was accumulated in the late
phase of viral replication (22, 39). However, a recent study
detected vPK RNA at early time points and in the presence of
an inhibitor of viral DNA synthesis (31). Detailed analysis of
KSHV RNA revealed two polycistronic transcripts encoding
vPK that are initiated from promoters that are active in the
early stage of the viral life cycle and inducible by hypoxic
conditions (18). With respect to potential therapeutic signifi-
cance, vPK was shown to phosphorylate the antiherpesvirus
drug ganciclovir (10). In a recent study that explored a poten-
tial role for vPK on cell signaling, we established that vPK
phosphorylated components of the Jun N-terminal protein ki-
nase signal transduction pathway, which in turn activated c-Jun
in the activating protein 1 transcription complex (17).
We have continued to study the vPK of KSHV by focusing
on viral targets of this protein kinase. The main finding is that
vPK interacts with the transcriptional regulator K-bZIP in pro-
ductively infected cells. We demonstrated that a specific resi-
due of K-bZIP, threonine 111, was phosphorylated by vPK and
that this phosphorylation modulated the transcription function
of K-bZIP. Interestingly, this threonine residue of K-bZIP is
also the target of the cyclin-dependent kinase Cdk2. Chroma-
tin immunoprecipitation experiments showed that vPK and
K-bZIP were recruited to selected viral promoters as well as
the Ori-Lyt region. High-resolution microscopy revealed that
vPK was localized to replication/transcription complexes in
infected cells. Interestingly, vPK is packaged in mature virions;
this finding raises the possibility that vPK may influence events
at viral entry into host cells, including at a very early stage of
viral gene expression. We discuss a model whereby vPK exerts
pleiotropic effects by modulating several steps in the lytic rep-
lication cycle of KSHV, in part by interacting with and influ-
encing K-bZIP function on viral gene expression.
MATERIALS AND METHODS
Cell culture. Human embryonic kidney epithelial 293 cells and 293T cells were
grown in monolayer culture in Dulbecco’s modified Eagle medium supplemented
with 10% fetal bovine serum (FBS) in the presence of 5% CO2. The TREx-K-
Rta BCBL-1 cell line, generated by Nakamura et al. (36), was cultured in RPMI
1640 medium supplemented with 15% FBS, 100 ?g/ml of blasticidin (Invitrogen),
and 100 ?g/ml of hygromycin (Invitrogen).
Plasmids. Plasmids encoding the full-length K-bZIP and K-Rta genes were
described previously (20). This cloning introduced a CpoI site and a Flag tag or
T7 tag at the N terminus of each protein as described previously (20). The
resulting plasmids were designated pFlag-K-bZIP or pT7-K-bZIP. Plasmids en-
coding the full-length vPK wild type and kinase-dead mutant vPK-K108Q were
described previously (17). Deletion fragments of K-bZIP were amplified by PCR
using Pfu Turbo (Stratagene) and cloned into the CpoI site of pGEX-modified
vector (21) using cDNA encoding K-bZIP wild type or phosphor-mutant K-bZIP
as a template. Newly synthesized primers for preparing deletion DNA fragments
are listed in Table 1. Plasmids encoding phosphor-mutant K-bZIP-T111D and
K-bZIP-T111A were prepared by site-directed mutagenesis (Strategene). Full-
length wild-type vPK, kinase-dead mutant vPK-K108Q, ORF6, ORF9, ORF44,
ORF56, and ORF59 were amplified by PCR and cloned into pVL1392 vector
(Invitrogen), which introduced a Flag tag and CpoI site (pVL-Flag). Full-length
primase-associated factor (ORF40 to ORF41) (5) was amplified from cDNA of
tetradecanoyl phorbol acetate (TPA)-treated BCBL-1 and cloned into the CpoI
site of pVL-Flag vector. The CpoI DNA fragments of K-Rta and K-bZIP were
TABLE 1. Primers prepared in this study
ORF6 CpoI-F .....................aaaCGGTCCGACCATGGCGCTAAAGGGACCA
ORF9 CpoI-F .....................aaaCGGTCCGATCATGGATTTT TCAATCCA
ORF40 CpoI-F ...................aaaCGGTCCGTCAATGGCAACGAGCGAAGAA
ORF44 CpoI-F ...................aaaCGGTCCGACCATGGACAGCTCGGAAGG
ORF56 CpoI-F ...................aaaCGGTCCGACCATGGAGACGACATACCGC
ORF59 CpoI-F ...................aaaCGGTCCGATATGCCTGTGGATTTTCAC
vPK CpoI-R ........................aaaCGGACCGTCAGAAAACAAGTCCGCGGGT
K-bZIP aa75 CpoI-R.........aaaCGGACCGAAGGTCAATGACCGTTTCACA
K-bZIP aa76 CpoI-F..........aaaCGGTCCGACAGCGCCTTCCCAAAGTGGC
K-bZIP aa150 CpoI-R.......aaaCGGACCGGCGACCTGCGCCCTGTTTGGC
K-bZIP aa151 CpoI-F........aaaCGGTCCGCCCGTGCCTGCGTCTGTAGTT
K-bZIP aa72 CpoI-F..........aaaCGGTCCGGTCATTGACCTTACAGCGC
K-bZIP aa87 CpoI-F..........aaaCGGTCCGGAACATCTGCCATGCTCAC
K-bZIP aa113 CpoI-F........aaaCGGTCCGCCAAGAGGACCACACATTT
K-bZIP aa128 CpoI-F........aaaCGGTCCGAAGAGGCGACTACATAGAA
K-bZIP aa102 CpoI-R.......aaaCGGACCGTTAGGGGATGTGGAATTTA
K-bZIP aa123 CpoI-R.......aaaCGGACCGTTATGGAAGCTGTTGCGAA
K-bZIP aa154 CpoI-R.......aaaCGGACCGTTACGCAGGCACGGGGCGA
K-bZIP T111A-R ...............TGGTGGTGCGTGAGAGAGCGTCCAGGATC
aBoldfaced nucleotides represent restriction enzyme sites used for cloning the
PCR products; underlining indicates an initiation codon, stop codon, or mutated
codon; lowercase letters indicate nucleotides added to enhance recognition by
the restriction enzyme.
VOL. 81, 2007 CHARACTERIZATION OF KSHV PROTEIN KINASE1073
also cloned in the pVL-Flag vector. Primers used in this study are listed in Table
1. Cloned DNA sequences were confirmed by sequencing.
Purification of KSHV virions. Purification of KSHV virions was performed
essentially with the method previously described by Zhu et al. (59). One litter of
TREx-K-Rta BCBL-1 was cultured in a roller bottle with 15% FBS. For virus
production, KSHV reactivation was induced by adding a final concentration of 1
?g/ml of doxycycline (DOX). After a 96-h induction, the medium was collected
and cleared by centrifugation at 4,000 ? g for 30 min and then at 8,000 ? g for
15 min to remove cells and cell debris. Virions were pelleted at 27,000 rpm for
1 h through a 5% sucrose cushion (5 ml) in a Beckman SW28 rotor and resus-
pended in 1? phosphate-buffered saline (PBS) in 1/100 of the original volume.
The concentrated virus particles were centrifuged through a 10 to 50% sucrose
gradient at 27,000 rpm for 2 h in a Beckman SW55Ti rotor. The virus band at the
gradient junction was collected. This virus band was diluted with 1? PBS and
pelleted at 27,000 rpm for 1 h. Pellets were resuspended in 1? PBS and again
purified through a 10 to 50% continuous sucrose gradient at 27,000 rpm over-
night with a Beckman SW55Ti rotor. Virions were diluted with 1? PBS and
pelleted at 27,000 rpm for 1 h. The pellet was resuspended in 1 ml of 1? PBS.
Purified virions were digested with trypsin for 30 min at 37°C in either the
absence or presence of 1% Triton-X and subjected to immunoblotting analysis
with either anti-K8.1 mouse monoclonal antibody (Advanced Biotechnology
Inc.) or anti-vPK rabbit immunoglobulin G (IgG).
Northern blotting. Total RNA from TREx-K-Rta BCBL-1 cells was prepared
by ISOGEN (Nippon Gene, Tokyo, Japan) as recommended by the manufac-
turer. Where indicated, cells were incubated with 300 ?g/ml phosphonoacetic
acid (PAA) during reactivation to inhibit viral DNA replication. Total RNA (10
?g/lane) was separated on a 1.0% agarose-formaldehyde gel and transferred to
a nylon membrane (Biodyne; Pall BioSuport, New York). The RNA was immo-
bilized on the membrane by drying at room temperature (RT) for 1 h and then
cross-linked by UV light. DNA probes were prepared from total genomic DNA
of BCBL-1 cells by PCR amplification with specific primer sets (Table 1). DNA
fragments purified from agarose gels were radiolabeled with [?32-P]dATP using
a Strip-EZ DNA kit (Ambion) as recommended by the supplier. To detect
low-abundance ORF34 and ORF35 transcripts, RNA probes were generated by
T7 RNA polymerase with Strip-EZ RNA kit (Ambion) and incorporated with
[?32-P]UTP. Hybridization was performed at 42°C for DNA probes and at 64°C
for RNA probes as described previously (20).
Immunoprecipitation and immunoblot analysis. TREx-K-Rta BCBL-1 cells
were rinsed in ice-cold PBS, and 1 ? 107cells were lysed in EBC lysis buffer
(50 mM Tris-HCl [pH 7.5], 120 mM NaCl, 0.5% NP-40, 50 mM NaF, 200 ?M
Na2VO4, 1 mM phenylmethylsulfonyl fluoride) supplemented with a protease
inhibitor cocktail (Roche). After centrifugation (15,000 ? g for 10 min at
4°C), 20 ?l of protein A and protein G Sepharose beads (Upstate) was added
to the supernatants and preincubated overnight at 4°C. Five hundred micro-
grams of each of the cleared supernatants was reacted with 3 ?g of anti-vPK
or preinoculated rabbit IgG for 3 h at 4°C with gentle rotation. The immune
complex was then captured by the addition of 20 ?l of a protein A and protein
G Sepharose bead mixture and rocked for an additional 2 h at 4°C. Beads
were washed four times with EBC buffer and boiled for 5 min in 20 ?l of 2?
sodium dodecyl sulfate (SDS) sample buffer (125 mM Tris-HCl [pH 6.8], 4%
SDS, 10% 2-mercaptoethanol, 20% glycerol, 0.6% bromophenol blue). Pro-
tein samples from total cell lysates (50 ?g/lane) or immunoprecipitates were
subjected to SDS-polyacrylamide gel electrophoresis (PAGE) and trans-
ferred to a polyvinylidene difluoride membrane (Millipore) using a semidry
transfer apparatus (Amersham Pharmacia).
For detecting a molecular weight shift by phosphorylation, cells were lysed
with phosphoprotein-specific lysis buffer (50 mM Tris-HCl [pH 7.4], 1% Tri-
ton-X, 10% glycerol, 50 mM KCl, 50 mM ?-glycerophosphate, 50 mM NaF, 2
mM Na2VO4, 5 mM EDTA, 2 mM benzamide) supplemented with a protease
inhibitor cocktail (Roche). 293T cells were cotransfected with 2 ?g of pT7-K-
bZIP and 2 ?g of pFlag-vPK or pFlag-empty expression plasmids using Fugene
6 (Roche) according to the supplier’s recommendations. The cells were har-
vested 48 h after transfection and lysed in radioimmunoprecipitation assay
(RIPA) buffer (10 mM Tris-HCl [pH 7.5], 1.0% NP-40, 0.1% sodium deoxy-
cholate, 150 mM NaCl, 1 mM EDTA, 10 ?g/ml of aprotinin). Five hundred
micrograms of cell lysate was immunoprecipitated with the addition of 25 ?l of
anti-Flag antibody-conjugated agarose (Sigma). Beads were washed four times
with RIPA buffer and then boiled for 5 min in 20 ?l of 2? SDS sample buffer.
Protein samples from total cell lysates (50 ?g/lane) or immunoprecipitates were
subjected to SDS-PAGE and then transferred to polyvinylidene difluoride mem-
brane as described above and previously (20). For detecting small ubiquitin-like
modifier protein (SUMO)-modified K-bZIP, cells were washed twice with PBS
and lysed with SUMO lysis buffer (50 mM Tris-HCl [pH 6.8], 2% SDS, 10%
glycerol, 20 ?M N-ethylmalemide [Sigma]) supplemented with a protease inhib-
itor cocktail (Roche). Cell lysates were briefly sonicated and cleared by centrif-
ugation (15,000 ? g for 10 min at 4°C), and the supernatant was used for
immunoblot analysis. For immunoprecipitation of proteins modified by sumoy-
lation, cell lysates were boiled for 10 min at 95°C and diluted 10-fold with either
phosphate buffer (for immunoprecipitation with His tag) or RIPA buffer (for
immunoprecipitation with anti-Flag antibody). Final dilutions of the primary
antibodies for immunoblotting were 1 ?g/ml of anti-K-bZIP, 1 ?g/ml of anti-
vPK, 1 ?g/ml of anti-Flag M2 (Sigma), and 1 ?g/ml of anti-SUMO-2/3 (Zymed
ChIP assay. After 48 h with or without induction of viral reactivation with
DOX (1 ?g/ml), 107TREx-K-Rta BCBL-1 cells were fixed with 1% formalde-
hyde at RT for 10 min and washed with ice-cold PBS. Cells were washed in buffer
I (0.25% Triton X-100, 10 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 6.5).
Cell pellets were collected by centrifugation and washed in buffer II (200 mM
NaCl, 1 mM EDTA, 0.5 mM EGTA, 10 mM HEPES, pH 6.5). A total of 200 ?l
of cell pellets was resuspended in 1 ml of lysis buffer (0.5% SDS, 10 mM EDTA,
50 mM Tris-HCl [pH 8.1], 1? protease inhibitor cocktail [Roche]) and sonicated
four times for 30 s with 0.5-s pulses (Fisher 550 Sonic Dismembrator). Cell debris
was removed by centrifugation, and the chromatin solutions were diluted five
times in dilution buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM
Tris-HCl [pH 8.1], 1? protease inhibitor cocktail). A sample of total chromatin
was collected to serve as a total input DNA control. Chromatin fragments were
immunoprecipitated with 5 ?g of anti-K-bZIP IgG, preinoculated rabbit IgG, or
anti-vPK rabbit IgG overnight at 4°C with gentle rotation. Immunocomplexes
were recovered and eluted as described before (19). After reverse cross-linking
at 65°C overnight, the DNA fragments were purified with a QIAquick PCR
Purification Kit (QIAGEN) after pH was adjusted with 3 M sodium acetate (pH
7.0) and eluted with 100 ?l of 1? TE buffer (10 mM Tris-HCl [pH 8.0], 1 mM
EDTA [pH 8.0]). The chromatin-immunoprecipitation (ChIP) DNA was ana-
lyzed by PCR for Fig. 5 with primer sets described previously (19).
Immunofluorescence assay. BCBL-1 cells were treated with TPA for 48 h,
collected by low-speed centrifugation, fixed with methanol-acetone (1:1) for 15
min at RT, and then washed three times with PBS. After treatment with blocking
solution, 2% bovine serum albumin (BSA; Fisher) in PBS, cells on the coverslip
were incubated with anti-vPK rabbit IgG (1:1,000) in PBS–2% BSA for 1 h at
RT. For the colocalization study, the coverslip was incubated with anti-ORF59
mouse monoclonal antibody (1:3,000) (Advanced Biotechnology Inc.) together
with anti-vPK rabbit IgG. After four washes with PBS, Alexa Fluor 555-conju-
gated goat anti-mouse IgG F(ab?)2(1:5,000) (Molecular Probes) and/or Alexa
Fluor 488-conjugated goat anti-rabbit IgG F(ab?)2(1:5,000) (Molecular Probes)
in blocking solution was applied and allowed to react for 1 h. Purified anti-vPK
IgG and anti-K-bZIP IgG were labeled with either Alexa Fluor 488 or Alexa
Fluor 647 with a protein labeling kit (Molecular Probes) and were applied and
allowed to react for 1 h at concentration of 1 ?g/ml each. After four washes with
PBS, coverslips were mounted on glass slides. Imaging was performed by using
a confocal microscope (LSM 510-MicroSystem; Carl Zeiss Co., Ltd).
Generation of recombinant baculoviruses and protein purification. Spodopt-
era fugiperda Sf9 cells were maintained in EX-CELL 420 medium (JRH Bio-
sciences). The pVL Flag-ORF36 wild type, pVL Flag-ORF36 K108Q, pVL
Flag-ORF6, pVL Flag-ORF9, pVL Flag-ORF40-41, pVL Flag-ORF44, pVL
Flag-K-Rta, pVL Flag-K-bZIP, pVL Flag-ORF56, or pVL Flag-ORF59 was
cotransfected with linearized BaculoGold viral DNA (Pharmingen) into Sf9 cells
by using Fugene 6 (Roche), and recombinant viruses were subsequently ampli-
fied. Expression of the proteins in Sf9 cells was confirmed by immunoblotting
with anti-Flag monoclonal antibody (Sigma). A large-scale culture of Sf9 cells
(100 ml) was infected with recombinant baculovirus, and cells were harvested
48 h postinfection. Cells were lysed with Flag lysis buffer (50 mM Tris-HCl [pH
7.5], 500 mM NaCl, 1 mM EDTA, 1% Triton X-100) supplemented with a
protease inhibitor cocktail (Roche). Cell lysates were cleared by centrifugation
and immunoprecipitated with the addition of 100 ?l of anti-Flag antibody-
conjugated agarose (Sigma). Beads were washed three times with Flag lysis
buffer and twice with TBS buffer (50 mM Tris-HCl [pH 7.4], 150 mM NaCl).
Flag-tagged protein was eluted with 100 ?g/ml of 3? Flag peptide (Sigma) in
TBS buffer. Purification of protein was confirmed by immunoblotting with anti-
Flag antibody, and the amount of purified protein was measured by SDS-PAGE
with BSA as a standard.
Preparation and purification of GST fusion proteins. Glutathione transferase
(GST) fusion proteins were expressed in Escherichia coli strain BL21 trans-
formed with pGEX-ORF36 N-151, pGEX-K-bZIP N-75, pGEX-K-bZIP 76-150
pGEX-K-bZIP 151-C, pGEX-K-bZIP 72-102, pGEX-K-bZIP 87-123, pGEX-K-
bZIP 87-123 (T111A), pGEX-K-bZIP 113-154, pGEX-K-bZIP 128-154, or
pGEX4T-2 (N and C correspond to termini; number represents amino acid
1074 IZUMIYA ET AL.J. VIROL.
position). The procedure of purification of GST fusion proteins was described
In vitro kinase assay. Purified proteins were dialyzed to Tris buffer (50 mM
Tris-HCl [pH 7.5], 1 mM dithiothreitol). Purified wild-type kinase or kinase-dead
mutant (100 ng) was incubated with 2 ?g of purified substrates in kinase buffer
(20 mM HEPES [pH 7.5], 5 mM MnCl2, 10 mM ?-mercaptoethanol) supple-
mented with 10 ?Ci [?-32P]ATP at 37°C for 30 min. The reaction was stopped by
adding 2? sample buffer, protein was separated by 10% SDS-PAGE, and the gel
was subjected to autoradiography.
Reporter assays in transient transfection experiments. Reporter plasmid was
constructed by inserting the promoter region (29) upstream of the firefly lucif-
erase coding region (Luc) in the pGL3-Basic vector (Promega). 293 cells were
seeded in 12-well plates at 1 ? 105cells/well in 1.0 ml of Dulbecco’s modified
Eagle medium supplemented with 10% FBS and incubated at 37°C with 5% CO2.
For each well, an equal amount of plasmid DNA, including the reporter and the
control or expression plasmid, was transfected using the Fugene 6 reagent fol-
lowing the manufacturer’s protocol (Roche). Cell lysates were prepared 48 h
after transfection with 1? passive lysis buffer (Promega). A luciferase assay was
performed according to the manufacturer’s protocol using a Lumat LB 9501
Luminometer (Wallac Inc., California). At least three independent determina-
tions were performed at each setting.
vPK transcription during lytic replication. In previous re-
ports, the vPK gene was shown to be expressed as either an
early or a late gene during lytic replication, depending on the
conditions used to reactivate virus in latently infected B-lym-
phoid tumor cells (22, 31, 38, 39). Because the mode of action
of the inducers (e.g., phorbol ester or butyrate) is complex and
the fraction of cells affected is relatively small, we chose to
measure the expression kinetics of vPK by K-Rta-mediated
reactivation. In the TREx-K-Rta BCBL-1 cell line (36), a de-
rivative of the BCBL cell line, the expression of K-Rta is
induced by DOX, resulting in a well-ordered KSHV gene ex-
pression program and a relatively synchronized cycle of viral
replication in nearly all cells (19, 36). RNA from induced cells
was analyzed by Northern blot analysis, which showed that vPK
is encoded in two transcripts (4.2 and 3.6 kb), together with
ORF37 and ORF38. These transcripts were detected as early
as 24 h after induction and maintained essentially the same
intensity thereafter. ORF34 probe detected the 4.2-kb tran-
script but not the 3.6-kb transcript; this result indicates that the
4.2-kb mRNA initiated from ORF34 and that the 3.6-kb
mRNA is transcribed from ORF35. In addition to the 4.2- and
3.6-kb transcripts, ORF37 probe detected a 2.0-kb transcript,
and ORF38 probe detected 2.0- and 0.6-kb transcripts. The
transcript data are consistent with a transcriptional map of
these ORFs shown in Fig. 1A, where all transcripts are co-
terminal at the strong poly(A) site at nucleotide (nt) positions
58852 to 58858 (accession no. U75698).
The kinetics of vPK expression suggests that it is an early or
early-late gene. To define this further, the expression of vPK
was examined by using the DNA polymerase inhibitor, PAA, a
treatment for temporal classification of viral genes. The TREx-
K-Rta BCBL-1 cells were induced with DOX in the presence
of PAA. Expression of the 4.2-kb mRNA was reduced to 31%
in the presence of PAA, but the 3.6-kb mRNA was relatively
resistant to PAA treatment, and, in fact, the level of the 3.6-kb
transcript increased from 48 h to 72 h in the presence of PAA
(Fig. 1C). These results indicated that vPK was expressed as
both an early (3.6 kb) and an early-late (4.2 kb) gene.
Identification of vPK protein in naturally infected cells.
Previously, we showed that vPK activates the Jun N-terminal
protein kinase cellular signal transduction pathway (17). Other
investigators reported the localization of vPK in the nucleus of
transiently transfected cells (38). These studies, while informa-
tive, were carried out with ectopically expressed, epitope-
tagged vPK in transfected cells. To characterize vPK during
lytic replication, we generated rabbit serum, using a GST-vPK
(residues 1 to 150) fusion protein as an antigen. The anti-vPK
antibody reacted with the cell lysates from KSHV-reactivated
BCBL-1 (Fig. 2A, lane TPA, showing the lytic phase) but not
with nonreactivated BCBL-1 (Fig. 2A, lane No TPA, showing
the latent phase); the size, 48 kDa, corresponds well with the
size calculated from the amino acid sequence of ORF36. Fur-
thermore, this antibody interacts with Flag-tagged vPK protein
FIG. 1. Transcription of vPK (ORF36). (A) Schematic diagram of
the KSHV genome structure and gene organization of ORF36 region.
Positions of probes for Northern blotting are indicated by ORF num-
ber: 34 (nt 55042 to 55346), 35 (nt 55639 to 55872), 36 (nt 56415 to
56812), 37 (nt 58022 to 58392), and 38 (nt 58688 to 58843). The
nucleotide number corresponds to the sequence of accession no.
U75698. Putative direction and coding regions are shown as arrows.
The direction of transcripts and their coding regions were determined
from the size and position of the polyadenylation site. (B) Northern
blot analysis. After induction of K-Rta expression, total RNA was
prepared at the indicated time points. Ten micrograms was loaded per
lane, electrophoresed, and transferred to nylon membranes. The re-
sults of Northern hybridization with the indicated probes are shown.
(C) Classification of ORF36 transcripts. The TREx-K-Rta BCBL-1 cell
line was induced with DOX in the presence of 300 ?g/ml of PAA. Ten
micrograms of total RNA was loaded per lane. The results of Northern
hybridization with the ORF36 probe are shown (left). Transcripts were
quantified (right) by using Quantity-One (Bio-Rad). The highest tran-
script level was normalized to a value of 100%.
VOL. 81, 2007 CHARACTERIZATION OF KSHV PROTEIN KINASE1075
expressed in transiently transfected 293T cells (Fig. 2A, lane
vPK) but not with proteins from vector-transfected cells (lane
Vec), attesting to the specificity of the antibody.
With the specific antibody in hand, we examined the local-
ization of vPK in BCBL-1 cells 48 h after induction by TPA. As
shown in Fig. 2B, during the replicative phase of the lytic cycle,
vPK is localized primarily in the nucleus, exhibiting a punctate
pattern in the midst of the diffuse staining; this pattern resem-
bles KSHV DNA replication/transcription compartments (54).
Antibody to polymerase processivity factor (PPF)/ORF59,
known to be part of the replication/transcriptional complex
(54), was used to “mark” such a structure, and, indeed, the
merged picture showed colocalization of vPK and PPF (Fig.
vPK is packaged in the virion. After establishing the expres-
sion kinetics and subcellular localization of vPK in BCBL-1
cells, we determined whether vPK is packaged in the virion.
Viruses released from BCBL-1 cells at 96 h after K-Rta induc-
tion were purified through a sucrose gradient, and the presence
of vPK protein was probed with anti-vPK antibody with or
without trypsin digestion. The K8.1 virion membrane associ-
ated glycoprotein was used as a control. As shown in Fig. 2C,
vPK but not K8.1 was protected from trypsin digestion, sug-
gesting that vPK is primarily packaged inside the viral mem-
brane. Addition of the detergent Triton X, which solubilized
the membrane, rendered vPK susceptible to trypsin digestion.
These results demonstrate that a fraction of vPK is packaged in
the virion and suggest that vPK function may not be limited to
just the early/late phase. Through phosphorylation, vPK may
regulate the functions of virion proteins, as well as viral regu-
latory proteins, upon entry into the host cell.
vPK phosphorylates K-bZIP in vitro. Given the close prox-
imity of vPK with the KSHV replication complex, we determined
whether the replication proteins of this virus are potential sub-
strates for vPK. Eight KSHV gene products, shown to be es-
sential for viral DNA replication and localized in the replica-
tion complex (3, 30, 54), were tagged with Flag epitope and
expressed by baculovirus vectors. These tagged proteins were
purified with Flag antibody conjugated to agarose beads and
subsequently eluted with Flag peptide. The eight viral proteins
are single-strand DNA binding protein (SSB), DNA polymer-
ase (POL), primase-associated factor (PAF) (5), helicase
(HER), primase (PRI), PPF, K-bZIP, and K-Rta. The vPK
wild-type and kinase-dead mutant proteins were similarly pre-
pared. An in vitro protein kinase assay was performed by
incubating vPK and the individual replication-associated pro-
teins in the presence of ATP and 5 mM MnCl2(38). The
wild-type vPK exhibited significant autokinase activity com-
pared to the kinase-dead (K108Q) mutant (Fig. 3A). Among
the eight proteins tested, K-bZIP was the most efficiently phos-
phorylated by vPK. In addition, SSB, POL, PAF, K-Rta, and
PPF were phosphorylated but to a much smaller extent. Phos-
phorylation of K-bZIP by vPK was further verified by using
GST-K-bZIP as a substrate (Fig. 3B). Strong phosphorylation
of this K-bZIP by vPK wild type but not the vPK-K108Q
mutant strongly suggests that the observed phosphorylation
was due to intrinsic kinase activity of vPK.
In vivo phosphorylation was examined by mobility shift of
K-bZIP in transfected cells. K-bZIP was coexpressed with ei-
ther wild-type vPK or the kinase-dead K108Q mutant in 293
cells. Cell lysates were subjected to SDS-PAGE, and K-bZIP
protein was probed with anti-K-bZIP antibody. We observed a
mobility shift of K-bZIP only in cells transfected with wild-type
vPK and not with the K108Q kinase-dead mutant (Fig. 3C
upper blot), suggesting that K-bZIP is phosphorylated by vPK
in vivo. To show that this mobility shift was indeed due to
phosphorylation, the K-bZIP immunoprecipitates were treated
with lambda protein phosphatase before Western blotting, and
as shown in Fig. 3C (lower blot), K-bZIP mobility was restored
to the fast-migrating form. This finding is consistent with K-
bZIP phosphorylation by vPK in vivo.
vPK physically associates with K-bZIP in vivo. The data
shown above suggest that vPK phosphorylates and colocalizes
with K-bZIP. We determined whether vPK and K-bZIP phys-
ically associate with each other in vivo. This was first done by
a coimmunoprecipitation assay in cotransfected 293T cells.
Flag-vPK was cotransfected with T7-K-bZIP. Flag-empty vec-
tor served as a negative control in a separate transfection.
Twenty-four hours after transfection, cells were lysed with
RIPA buffer and immunoprecipitated with anti-Flag mouse
monoclonal antibody. To avoid detecting mouse IgG heavy
chain, we performed immunoblotting with rabbit IgG against
either vPK or K-bZIP to detect coprecipitants. As shown in
Fig. 4A (upper blot), K-bZIP was efficiently coprecipitated
with vPK. The reciprocal experiment, shown in the lower blot
in Fig. 4A, confirmed the association of these two viral pro-
FIG. 2. Characterization of vPK in infected cells. (A) Immunoblot
analyses of vPK. Cell lysates were prepared from 293T cells transfected
with the vPK expression plasmid or control vector (Vec) or from
TPA-treated or nontreated (No TPA) BCBL-1 cells. Total cell lysate
(50 ?g/lane) from each cell culture was subjected to immunoblot
analysis. (B) Colocalization of vPK with Orf59 protein. Confocal anal-
ysis was performed by using affinity-purified anti-vPK IgG and anti-
ORF59 mouse monoclonal antibody. vPK (green) and ORF59 (red)
were detected with Alexa Fluor 488-conjugated anti-rabbit IgG and
Alexa Fluor 555-conjugated anti-mouse IgG. (C) vPK localization in-
side virions. KSHV virions were prepared as described in Materials
and Methods. Virion protein was digested with trypsin in either the
presence or absence of 1% Triton-X. The indicated protein was
probed with specific antibodies.
1076 IZUMIYA ET AL.J. VIROL.
teins. We further extended the studies to the naturally infected
BCBL-1 cell line. Forty-eight hours after activation of KSHV
lytic replication by TPA, vPK was immunoprecipitated with
affinity-purified anti-vPK antibody. As a negative control, pre-
inoculated rabbit IgG was used. This experiment showed that
K-bZIP was coprecipitated with vPK (Fig. 4B), demonstrating
the association of these two viral proteins in the natural setting
of viral reactivation. This association was verified by additional
microscopy studies revealing colocalization of K-bZIP and vPK
after anti-K-bZIP IgG and anti-vPK IgG were conjugated with
Alexa Fluor 647 and Alexa Fluor 488, respectively. In addition
to PPF, K-bZIP showed colocalization with vPK (Fig. 4C). It is
noteworthy that the vPK-K-bZIP colocalization pattern is not
restricted to KSHV DNA replication/transcription foci, sug-
gesting a complex association between vPK and K-bZIP, in-
volved in both replication and transcriptional regulation. The
data also revealed that the subcellular locations of vPK are not
static, perhaps depending on the different stages of replication
and different viral proteins associated with vPK.
vPK and K-bZIP are corecruited to viral promoters. Previ-
ously, we showed that K-bZIP is a strong transcriptional re-
pressor, which modulates the transcription of K-bZIP and
ORF57 promoters mediated by K-Rta (20, 29). Based on ChIP
assays, K-bZIP is recruited to these two promoters and Ori-Lyt
DNA during the early phase of KSHV lytic replication (19).
Given the close association between vPK and K-bZIP, we
determined whether vPK is also recruited to these sites on the
viral genome by the ChIP assay on lysates of reactivated TREx-
K-Rta-BCBL-1 cells. As shown in Fig. 5, vPK and K-bZIP are
corecruited to both the K-bZIP and ORF57 promoter regions
as well as Ori-Lyt DNA but not to the ORF57 coding region.
These data, together with results described in the previous
section, indicate that vPK and K-bZIP are likely to be func-
tional partners in vivo.
FIG. 3. In vitro protein kinase assay. (A) Viral substrates. Sub-
strates and the protein kinases (wild-type vPK and kinase-dead vPK)
were expressed by using the baculovirus vector and purified with Flag-
agarose. SypoRuby-stained gel is shown at the bottom of the panel,
and autoradiography of the same gel after drying is shown at top.
Proteins and the associated ORFs are the following: SSB (ORF6),
POL (ORF9), PAF (ORF40 to ORF41), HER (ORF44), K-Rta
(ORF50), K-bZIP (K8), PRI (ORF56), PPF (ORF59), and vPK
(ORF36). (B) vPK wild type phosphorylates GST-K-bZIP. The in vitro
kinase assay was performed by using either GST-K-bZIP or GST as a
substrate. Purified GST and GST-K-bZIP are shown at the far left.
The vPK wild type but not kinase-dead mutant significantly phosphor-
ylates GST-K-bZIP. (C) In vivo phosphorylation. The K-bZIP expres-
sion plasmid was cotransfected with indicated plasmids into 293 cells.
K-bZIP was probed with anti-K-bZIP antibody. (a) Molecular mass of
K-bZIP was increased when K-bZIP was cotransfected with vPK wild-
type plasmid but not mutant vPK plasmid or vector control. (b) K-
bZIP was treated with 100 U of lambda phosphate (PP) for 30 min at
37°C or carrier (?) after immunoprecipitation (IP). After phosphate
treatment, K-bZIP was separated by PAGE, and probed with anti-K-
bZIP. WB, Western blotting; vPK KQ, vPK with the mutation K108Q
FIG. 4. Association between K-bZIP and vPK. (A) Association be-
tween K-bZIP and vPK in cotransfected 293T cells. 293T cells were
cotransfected with the indicated plasmids. Cell lysates were precipi-
tated with Flag-antibody conjugated to agarose, and coimmunoprecipi-
tation of K-bZIP (a) or vPK (b) was detected by using anti-K-bZIP
rabbit IgG or anti-vPK rabbit IgG. The expression of K-bZIP or vPK
in total cell lysates is shown in the same blot as a control. (B) Coim-
munoprecipitation assay with KSHV-positive BCBL-1. BCBL-1 cells
induced for KSHV lytic replication for 48 h with TPA were harvested,
and 500 ?g of cell lysate was immunoprecipitated with either preim-
mune rabbit IgG or anti-vPK rabbit IgG. The same membrane was
stripped and reprobed with anti-vPK rabbit IgG. (C) Colocalization of
vPK with K-bZIP. vPK (green) and K-bZIP (red) were detected with
Alexa Fluor 488-conjugated anti-vPK rabbit IgG (green) and Alexa
Fluor 647-conjugated anti-K-bZIP rabbit IgG (red). IP, immunopre-
cipitation; WB, Western blotting; Vec, vector.
VOL. 81, 2007 CHARACTERIZATION OF KSHV PROTEIN KINASE1077
The major vPK phosphorylation site of K-bZIP. To map the
phosphorylation site(s) of K-bZIP by vPK, we first generated a
series of GST-K-bZIP deletion mutants, roughly dividing the
protein into three sections. Bacterially expressed proteins, pu-
rified as above, were incubated with either wild-type or kinase-
dead vPK. As shown in Fig. 6A, strong phosphorylation was
detected on the middle segment encoding amino acid residues
76 to 150 of K-bZIP. Additional deletion constructs spanning
this region were made for additional tests of phosphorylation
by vPK (Fig. 6B). This approach narrowed the region of major
phosphorylation to residues 87 to 123. Threonine 111 within
this region was determined to be a major phosphorylation site,
because its mutation to alanine significantly diminished the
phosphorylation level of K-bZIP (Fig. 6B). The fact that the
K-bZIP-T111A mutant still retains some phosphorylation sug-
gests that there are additional vPK phosphorylation site(s) yet
to be identified. K-bZIP-T111A was cotransfected with vPK in
293 cells, and the proteins were separated in SDS-PAGE and
probed with anti-K-bZIP antibody; K-bZIP-T111A, unlike the
wild type, showed little mobility shift, reinforcing the notion
that the mobility shift of the wild-type K-bZIP is due to phos-
FIG. 5. Recruitment of vPK to the KSHV genome. ChIP assay was
performed on cell lysates of the TREx-K-Rta BCBL-1 cell line by using
the indicated rabbit IgG. Cell lysates were prepared before (No Dox)
and after 48 h of reactivation (Dox-48 h). For each primer set, a PCR
amplification with the total input DNA (Input) before immunoprecipi-
tation was carried out. ChIP fragments of preimmune IgG (IP-Pre),
anti-vPK IgG (IP-vPK), or anti-K-bZIP IgG (IP-K-bZIP) were sub-
jected to PCR amplification analysis. Prom, promoter.
FIG. 6. Mapping of vPK phosphorylation site of K-bZIP. (A) The domains of K-bZIP and the GST-K-bZIP mutants are indicated in the left
panel. TA, transactivation domain; BR, basic domain; LZ, leucine zipper domain. Results of the in vitro protein kinase assay and SyproRuby
staining of the same gel are shown at right. (B) The coding region of recombinant K-bZIP is indicated at the top of the panels. Results of the in
vitro protein kinase assay and SyproRuby staining of the same gels are shown. Asterisks show phosphorylated protein originated from E. coli. (C) In
vivo phosphorylation. The K-bZIP-wild type (WT) or K-bZIP-T111A expression plasmid was cotransfected with the indicated plasmids into 293
cells. K-bZIP or vPK was probed with specific antibody. The molecular weight of K-bZIP wild type but not K-bZIP-T111A was increased when
K-bZIP was cotransfected with vPK. WB, Western blotting; Vec, vector; vPK KQ, vPK with the mutation K108Q.
1078 IZUMIYA ET AL.J. VIROL.
phorylation by vPK (Fig. 6C). We thus identified K-bZIP
threonine 111 as the major phosphorylation target of vPK.
Phosphorylation modulates K-bZIP repression function. To
study the functional significance of vPK phosphorylation on
K-bZIP, in addition to the mutant T111A, we mutated threo-
nine 111 to aspartic acid, thereby mimicking the phosphory-
lated form. Our previous work showed that one of the K-bZIP
functions is to serve as a transcriptional repressor of K-Rta (20,
29); this function depends on the sumoylation of lysine 158 of
K-bZIP (19). To determine whether phosphorylation of K-
bZIP at threonine 111 impacts the repression function, wild-
type K-bZIP or its phosphor-acceptor mutants were cotrans-
fected with K-Rta and the reporter plasmid carrying the
K-bZIP promoter, which directs luciferase expression. As ex-
pected, wild type K-bZIP strongly represses K-Rta-mediated
activation of the K-bZIP promoter to about the 30% level (Fig.
7A). The K-bZIP-T111A mutant was even more potent in its
repression function, whereas K-bZIP-T111D displayed a re-
pression level similar to wild-type K-bZIP (Fig. 7A). These
data suggest that phosphorylation of threonine 111 has a neg-
ative impact on the repression activity of K-bZIP. Because
K-bZIP repression activity largely depends on its sumoylation,
we compared the sumoylation patterns of the wild-type K-
bZIP and K-bZIP-T111A mutant. Consistent with its higher
potency of repression activity, K-bZIP-T111A is more heavily
sumoylated compared to K-bZIP wild type and K-bZIP-
T111D, particularly in the formation of SUMO multimers.
Inclusion of vPK in the transfection mix significantly reduced
the sumoylation of K-bZIP wild type (Fig. 7B, left). The dif-
ferential sumoylation of the wild type and T111A is even more
pronounced when the conjugation enzyme Ubc9 for SUMO
was cotransfected to further catalyze the reactions (Fig. 7B,
right). To demonstrate that the high-molecular-weight species
are sumoylated K-bZIP, His-tagged SUMO-2 and Ubc9 were
cotransfected with K-bZIP, and SUMO-modified proteins
were affinity purified with nickel-charged resin. K-bZIP was
probed with anti-K-bZIP antibody. Monomer, dimer, and tri-
mer forms of sumoylated K-bZIP were detected (Fig. 7B,
right). Consistent with immunoblotting results (Fig. 7B, left),
K-bZIP-T111A was more heavily sumoylated. The reciprocal
experiment further confirmed this result (Fig. 7B, right). These
data again demonstrated the potential of K-bZIP to be sumoy-
lated and suggest that phosphorylation by vPK may modulate
the ability of K-bZIP to be modified by sumoylation.
This report focuses on the characterization of vPK of KSHV,
with the goal of building knowledge of the role of this protein
in the viral life cycle. Basic features of vPK were examined
including the transcriptional pattern of the vPK gene, incor-
poration of vPK protein into virions, and localization within
infected cells. Importantly, viral proteins that interact with
vPK, and viral targets of its phosphorylation activity, were
identified. The results of these studies were used to propose a
model of vPK function in KSHV replication.
The transcriptional pattern of the vPK gene was defined in
the BCBL-1 cell line harboring latent virus. During reactiva-
tion, two vPK transcripts, 4.2 and 3.6 kb, are produced with
different kinetics. The result of kinetics study indicated that
vPK was expressed as both an early (3.6 kb) and an early-late
(4.2 kb) gene, and maximum expression required DNA repli-
cation. This kinetics of expression was closely related to EBV
BGLF4, a homolog of vPK (15). Other investigators using
microarray analysis, coupled with the use of an inhibitor of
viral DNA synthesis, also showed that the vPK gene was ex-
pressed early in the viral lytic cycle (31). In K-Rta-inducible
BCBL-1 cells, the 4.2-kb transcript, encoding ORF34 to
ORF38, was sensitive to PAA; this finding differs from an earlier
report (18). We also detected two additional transcripts (2.0 kb
and 0.6 kb) that had not previously been reported (18). These
differences in transcription patterns might be caused by the dif-
ferent method for reactivation, i.e., overexpression of the trans-
activator, K-Rta. The method used for viral reactivation may
partially overcome the defect in late gene expression. In addi-
tion, we cannot rule out that the 0.6-kb transcript is derived
from the opposite strand, as double-stranded DNA probe was
used in that experiment. The two vPK encoding transcripts
were coterminal with transcripts encoding ORF37 and ORF38
(see also reference 18). Precise transcription initiation sites
were recently reported at position 54566 for the 4.2-kb tran-
script and at position 55567 for the 3.6 kb transcript (18).
FIG. 7. Phosphorylation and sumoylation of K-bZIP. (A) Phos-
phorylation alters K-bZIP transcription function. Transient reporter
assays were performed with 293 cells cotransfected with reporter plas-
mid containing K-bZIP promoter. The amount of K-Rta expression
vector was kept at 0.5 ?g. The amounts of K-bZIP wild type, phosphor
mutants, or vPK expression plasmid are shown at the bottom of the
panel. Luciferase activity of K-Rta alone is normalized to a value of
100%. (B) Sumoylation of the wild type and phosphoacceptor mutant
of K-bZIP. Indicated expression plasmids were cotransfected with
SUMO-2 expression vector into 293 cells. (b) Identification of sumoy-
lation of K-bZIP. 293 cells were cotransfected with the indicated plas-
mids. After isolation of SUMO-2-modified protein (IP-His) or K-bZIP
(IP-Flag), immunoblotting was performed with the indicated antibody.
IP, immunoprecipitation; WB, Western blotting; Vec; vector control.
VOL. 81, 2007 CHARACTERIZATION OF KSHV PROTEIN KINASE1079
Potential functional implications of such multicistronic viral
transcripts remain to be determined.
To aid in the study of vPK, a highly specific antibody was
produced by immunizing rabbits with purified recombinant
vPK protein. This monospecific antibody identified vPK as a
48-kDa protein in cells producing virus (i.e., reactivated
BCBL-1 cells). Interestingly, the antibody readily detected vPK
in purified virions. Structural analysis of purified virions of
other herpesviruses, including HSV, CMV, EBV, and rhesus
rhadinovirus, indicate that the conserved protein kinases of
these viruses are also incorporated into mature virus particles
(2, 12, 37, 50, 52). This finding suggests that this kinase may
play a role in phosphorylating viral proteins during virion as-
sembly and/or regulate the functions of viral and cellular pro-
teins upon entry into host cells and during the early stage of de
novo infection (47).
Analysis of subcellular localization by immunofluorescence
experiments in induced BCBL-1 cells revealed that vPK is
primarily accumulated in the nucleus (Fig. 2C), consistent with
an earlier study based on transient expression of genetically
engineered vPK in transfected 293 cells (38). Interestingly, we
also found that the subcellular localization pattern of vPK, in
the context of viral infection, was more varied than shown in
this previous report. In some cells, staining for vPK displayed
a discrete dot-like pattern. This pattern of vPK compartmen-
talization coincided with viral replication foci marked by the
KSHV DNA PPF (ORF59) (54). In other cells, staining for
vPK exhibited a more diffuse arrangement (Fig. 4C). These
patterns could reflect different phases of viral replication, pre-
sumably where vPK is assembled into different protein com-
plexes. This diverse pattern, coupled with the presence of vPK
transcripts both early and late, implies that vPK could influ-
ence multiple steps in viral replication. The pattern of vPK
localization is similar to the EBV vPK homolog BGLF4, which
is found in the viral replication compartment, with variation in
subcellular localization at different times after reactivation of
virus (2, 52). Likewise, CMV UL97, the vPK homolog, is also
colocalized with UL44 (the PPF homolog) in the viral DNA
replication compartment; UL97 phosphorylates UL44 (33).
Additionally, the viral protein kinase of EBV, BGLF4, was
shown to phosphorylate the processivity factor, EA-D, of this
virus (16). Thus, herpesviruses encode conserved protein ki-
nases that appear to have a common role in viral DNA repli-
The subcellular localization studies, suggesting that vPK is
associated with the DNA replication apparatus, prompted fur-
ther investigation on a role for vPK in the KSHV life cycle.
Accordingly, several recombinant viral proteins involved in
viral DNA replication were tested as substrates of vPK activity.
This included a group of six proteins that directly participate in
viral DNA synthesis (SSB, POL, PAF, HER, PRI, and PPF) as
well as two regulatory proteins, K-Rta and K-bZIP, which have
been shown to physically associate with the lytic origin of DNA
replication (Ori-Lyt) (3, 30). In the cell-free system with puri-
fied vPK and purified substrates, K-bZIP was the most highly
phosphorylated target. Although the vPK kinase-dead mutant
failed to phosphorylate these substrates, it is still possible that
the phosphorylation observed by wild-type vPK could be due to
an associated protein kinase that was activated by wild-type
vPK. Either way, it is clear that vPK alone, or a vPK complex
with an additional protein(s), strongly phosphorylates K-bZIP
in vitro. Additionally, ChIP analysis of infected cells showed
that both vPK and K-bZIP were corecruited to Ori-Lyt DNA.
The potential role of vPK in viral DNA synthesis can be in-
vestigated in the future in an in vitro replication system, which
is built from the aforementioned viral DNA proteins, as de-
scribed for several herpesviruses including KSHV (3, 4).
Additional studies were done to define the site of phosphor-
ylation of K-bZIP and assess the effects of this posttransla-
tional modification on K-bZIP function in regulating viral gene
expression. The threonine residue at position 111 was deter-
mined to be the major site of vPK phosphorylation. Mutation
of this residue to alanine (T111A) largely diminished but did
not completely eliminate the phosphorylation by vPK. Our
previous studies showed that K-bZIP strongly repressed the
transcriptional activation of K-Rta and that this repression
depended to a large extent on sumoylation of K-bZIP (19).
The K-bZIP-T111A mutant exhibited an even stronger repres-
sion function that correlated with its ability to be significantly
sumoylated. In contrast, a phosphor-mimetic mutant, K-bZIP-
T111D, retained the repression activity like wild-type K-bZIP.
Taken together, these data suggest that phosphorylation of
threonine 111 of K-bZIP has a negative effect on its repression
activity and a positive effect on its transactivation function
(data not shown). Thus, K-bZIP joins a growing list of tran-
scriptional factors whose activities are modulated by sumoyla-
tion and phosphorylation in an antagonistic manner (8, 56).
Accordingly, we propose a model whereby vPK switches K-
bZIP from being a strong repressor of K-Rta, which targets
immediate-early genes, to a transactivator that synergizes with
K-Rta to activate early and late viral gene expression (data not
shown). A key feature of this model is that vPK phosphoryla-
tion may play a role as the molecular switch for regulating (i.e.,
balancing) these opposing activities of K-bZIP. Interestingly,
Zta of EBV, the homologue of K-bZIP, is also modified by
sumoylation (1); however, the significance of sumoylation of
Zta on EBV transcription has not been reported.
The studies in this report also revealed functional similarity
of KSHV vPK with the homologous protein kinases of other
herpesviruses that modulates the transactivation function of
viral genes; for example EBV BGLF4 modulates EBNA2 (58),
and HSV UL13 modulates ICP22 (42). Going beyond these
previous studies, the present report utilized ChIP analysis to
show that vPK of KSHV was corecruited with K-bZIP to viral
The phosphorylation target of the conserved protein kinases
of herpesviruses resembles that of the cyclin dependent protein
kinase. Intriguingly, threonine 111 [H-T(111)-P-P-R] was also
identified as a major site of phosphorylation when K-bZIP was
tested as a substrate for the Cdk2 and Cdc2 (40). Furthermore,
a test of vPK phosphorylation on a library of synthetic peptide
substrates implicated the consensus motif T-P-X-X-R, R-R-?-
S-P or R-R-P-T-? (where ? indicates a hydrophobic residue),
which resembles the consensus motif of the cyclin dependent
protein kinase (data not shown). Interestingly, the EBV
BGLF4 and HSV UL13 protein kinases were shown to phos-
phorylate some of the Cdk2 target motifs (23, 25, 57, 58).
Taken together, these findings suggest that the conserved pro-
tein kinases of herpesviruses in some cases can substitute for
cellular protein kinases, perhaps to advance the cell cycle in
1080 IZUMIYA ET AL. J. VIROL.
situations where cells are growth arrested. It has been sug-
gested that during the early stage of herpesvirus infection, cells
are often blocked at G1stage to allow viral mRNA and pro-
teins to be synthesized before the onset of cellular DNA rep-
lication. In previous studies, K-bZIP was shown to be a key cell
cycle regulator that binds Cdk2 and activates p21 to slow down
the cell cycle at the immediate-early stage of lytic infection (21,
55). Accordingly, we suggest that, at the later stages of viral
replication, vPK restores the Cdk2 function by phosphorylating
Cdk2 targets, and vPK may compete with Cdk2, thereby re-
leasing Cdk2 from its complex with K-bZIP. Cdk2 plays an
important role in DNA replication by phosphorylating the
licensing factor MCM (32). Additionally, Cdk2 was shown to
be required for herpesvirus replication (9, 28, 44). Thus, it is
conceivable that vPK may serve a similar role, i.e., as a surro-
gate for Cdk2, during the replication of the KSHV genome.
In summary, our model shows that vPK switches K-bZIP
from being a strong repressor of K-Rta transactivation of im-
mediate-early genes to a protein that synergizes with K-Rta to
activate early and late viral gene expression. Additionally, vPK
may modulate the activity of K-bZIP and perhaps other viral
proteins that play a role in replication of the KSHV genome.
Interestingly, the incorporation of vPK into virions and the
diverse patterns of vPK subcellular localization are findings
which indicate that the kinase is assembled into different viral
protein complexes; this differential compartmentalization im-
plies that vPK functions at multiple steps in viral replication.
The information presented here sets the stage for future stud-
ies to fully define the role of vPK in the KSHV life cycle and
also to explore the effects of vPK on the host cell. Detailed
knowledge of vPK targets and function(s) in the virion and
infected cell will establish a basis for novel therapies against
KSHV and the pathology associated with infection with this
This work was supported by grants from the National Institutes of
Health (CA111185 to H.J.K.) and the U.S. Department of Agriculture
(2004-35204-14207 to H.J.K. and Y.I.). Y.I. was supported by the
California university-wide AIDS Research Program (F03-D-206). Ad-
ditional funding was provided by the University of California-Davis
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