JOURNAL OF VIROLOGY, July 2005, p. 9912–9925
Copyright © 2005, American Society for Microbiology. All Rights Reserved.
Vol. 79, No. 15
Kaposi’s Sarcoma-Associated Herpesvirus K-bZIP Represses Gene
Transcription via SUMO Modification
Yoshihiro Izumiya,1Thomas J. Ellison,1Edward T. H. Yeh,2Jae U. Jung,3Paul A. Luciw,4
and Hsing-Jien Kung1*
Department of Biological Chemistry, University of California—Davis (UC Davis) School of Medicine, UC Davis Cancer
Center, Research Building III, Room 2400, 4645 2nd Avenue, Sacramento, California 95817,1and Center for
Comparative Medicine and Department of Pathology, UC Davis, 1 Sheilds Avenue, Davis, California 956164;
Department of Cardiology, The University of Texas M. D. Anderson Cancer Center, 1515 Holcombe 449,
Houston, Texas 770302; and Virology Division, Department of Microbiology
and Molecular Genetics and Tumors, New England Primate
Research Center, Harvard Medical School,
Southborough, Massachusetts 017723
Received 6 January 2005/Accepted 22 April 2005
Kaposi’s sarcoma-associated herpesvirus (KSHV) is a human gammaherpesvirus implicated in AIDS-
related neoplasms. Previously, we demonstrated that the early lytic gene product K-bZIP is a transcriptional
repressor that affects a subset of viral gene transcriptions mediated by the viral transactivator K-Rta (Y.
Izumiya et al. J. Virol. 77:1441–1451, 2003). Sumoylation has emerged as an important posttranslational
modification that affects the location and function of cellular and viral proteins and also plays a significant role
in transcriptional repression along with Ubc9, the E2 SUMO conjugation enzyme. Here, we provide evidence
that K-bZIP is sumoylated at the lysine 158 residue and associates with Ubc9 both in a cell-free system and
in virus-infected BCBL-1 cells. Reporter assays showed that the expression of SUMO-specific protease 1
attenuated the transcriptional repression activity of K-bZIP. The expression of a K-bZIPK158R mutant, which
was no longer sumoylated, exhibited the reduced transcriptional repression activity. This indicates that
sumoylation plays an important part in the transcriptional repression activity of K-bZIP. Finally, chromatin
immunoprecipitation experiments demonstrated that K-bZIP interacts with and recruits Ubc9 to specific
KSHV promoters. Thus, our data indicate that K-bZIP is a SUMO adaptor, which recruits Ubc9 to specific
viral target promoters, thereby exerting its transcriptional repression activity.
Kaposi’s sarcoma-associated herpesvirus (KSHV), also
known as human herpesvirus 8, is a member of the gamma-
herpesvirus family, which includes Epstein-Barr virus (EBV)
and herpesvirus saimiri. KSHV infection is associated with all
types of Kaposi’s sarcoma (KS), including AIDS-associated
KS, endemic forms of KS, and renal transplant-related KS.
This virus has also been implicated in B-cell lymphoprolifera-
tive diseases such as primary effusion lymphoma and multicen-
tric Castleman’s disease (13). Like other herpesviruses, KSHV
encodes both latent and lytic genes (61). Latent genes are
primarily responsible for the maintenance of latency and di-
rectly involved in cell transformation. Lytic genes participate
directly in viral replication (genome replication, transcription,
etc.) or indirectly by providing a cellular environment condu-
cive for viral infection (e.g., B-cell activation, immune modu-
lation of host response, target cell recruitment) or both (7, 61,
70). Products from both latent and lytic genes of KSHV have
been shown to participate in transformation and tumor pro-
gression (7, 49).
We previously reported the identification of K-bZIP/K8 (45;
also see reference 24) as an early lytic cycle gene. Studies by
others found that K-bZIP is among the earliest to be expressed
after acute infection or reactivation of the latent genome (38,
54, 56). K-bZIP is a 237-amino-acid protein containing a basic
and leucine zipper domain and is the positional and structural
analogue of EBV Zta (also called ZEBRA, BZLF1, and EB1)
(45). EBV Zta is a strong transcriptional factor capable of
triggering EBV reactivation and lytic replication (10) and
transactivates a number of viral and cellular genes (50, 78).
Although transactivation is a major role assumed by EBV Zta,
this gene also exhibits trans-repression activity on certain cel-
lular promoters via either a direct or indirect mechanism (14,
51, 52). In addition to being a transcriptional factor, EBV Zta
is involved in viral genome replication as an origin-binding
protein (15, 63), interacts with p53, and blocks the G1/S tran-
sition of the infected cell (16). The multiple functions of EBV
Zta are modulated posttranslationally by phosphorylation and
sumoylation (1, 14).
The overall structure of K-bZIP is similar to EBV Zta, with
the bZIP region located near the C terminus and the presump-
tive regulatory domain at the N terminus. The sequence ho-
mology between these two proteins, however, is limited, with
37% similarity (45). Although there are a number of biochem-
ical properties of K-bZIP which are shared with EBV Zta,
there are also important differences. Like EBV Zta, K-bZIP
forms homodimers (45) and is localized in the nucleus and in
PODs (for “PML [promyelocytic] oncogenic domains”) (36,
79). Attempts to show that K-bZIP directly binds DNA have
not met with success, although chromatin immunoprecipita-
* Corresponding author. Mailing address: UC Davis Cancer Center,
Research Building III, Room 2400B, 4645 2nd Avenue, Sacramento,
CA 95817. Phone: (916) 734-1538. Fax: (916) 734-2589. E-mail:
tion (ChIP) experiments suggest that it is associated with the
KSHV chromosome, including the regions near the replication
origin and the early gene promoters (44 and this report). Un-
like EBV Zta, overexpression of K-bZIP in a KSHV latent cell
line is insufficient to induce lytic replication (57). K-bZIP has
a relatively strong trans-repression activity (30, 31, 33, 55) and
modest trans-activation activity (76) when coexpressed with
other transcriptional factors. Recent evidence suggests that
K-bZIP directly participates in KSHV genome replication (4),
although the exact role is not clear. We and others recently
showed that K-bZIP delayed G1to S progression by at least
two mechanisms: direct inhibition of cyclin-CDK2 (34) and
transactivation of p21 (80).
Located upstream from K-bZIP in the KSHV genome is
another early lytic gene, K-Rta/ORF50. K-Rta, the homolog of
EBV BRLF1/Rta, is a potent transactivator with broad pro-
moter specificity (6, 35, 47, 62, 68) capable of activating a large
number of viral early genes, such as ORF6, K8, K12, K14,
ORF57, nut-1/polyadenylated nuclear (PAN) RNA, viral in-
terleukin-6, viral interferon regulatory factor-1, K-Rta, and
thymidine kinase (9, 35, 41, 54, 62, 68, 73, 74). Overexpression
of K-Rta activates a transcriptional cascade leading to lytic
replication (22, 48, 54, 71). The broad promoter specificity of
this viral gene comes from its ability both to bind DNA directly
(69) and to associate with other DNA-binding transcriptional
factors, such as RBP-Jk (42) and C/EBP? (28). Furthermore,
K-Rta interacts with several transcriptional regulators, includ-
ing Oct-1 (62), STAT3 (27), C/EBP? (28), CBP (25), K-RBP
(75), SWI/SNF, and TRAP230 (26). Thus, K-Rta appears to be
a global transcriptional factor which assembles different tran-
scriptional complexes on different promoter sites. Perhaps re-
lated to its transcriptional potency, constitutive expression of
K-Rta often leads to cell death.
We previously showed that K-bZIP and K-Rta physically
associate with each other, and K-bZIP represses K-Rta trans-
activation in a promoter-dependent manner (33, 43) such that
ORF57 and K8 promoters are affected, whereas the nut1/PAN
RNA promoter is immune to this repression. We interpret this
to mean that K-bZIP only targets certain K-Rta transcriptional
complexes for inhibition and suggest that K-bZIP is a feedback
modulator of K-Rta. This pattern of regulation may be critical
to the survival of virus in the infected cell. This model is
consistent with the tightly and temporally regulated activities
of K-Rta and K-bZIP during viral infection (38). EBV Rta and
Zta also have been shown to functionally interact with each
other in a gene-specific manner (14, 59). For some genes, both
viral regulators are synergistic (2, 17, 58), whereas for others,
EBV Zta represses Rta activity (14, 18, 59). Interestingly, the
repression activity is influenced by the phosphorylation status
of Zta (14). EBV Zta trans-repression activity on RAR (reti-
noic acid receptor) and p53, by direct protein-protein interac-
tion, has also been reported (66, 83). Likewise, K-bZIP was
shown to repress p53 and CBP-mediated transactivation (30,
55). Taken together, the data suggest that trans-repression is
an important function of the bZIP proteins of gammaherpes-
In this study, we explore the repression mechanism of K-
bZIP. A major finding is that K-bZIP is sumoylated and binds
SUMO E2-conjugating enzyme Ubc9. Sumoylation plays an
important part in K-bZIP trans-repression of K-Rta. The re-
pression activity of K-bZIP correlates with its ability to be
sumoylated and to recruit Ubc9 to the promoter site, where
K-Rta resides. The data are consistent with the increasingly
recognized role of SUMO in transcriptional repression (64).
The potential mechanisms whereby K-bZIP represses K-Rta
will be discussed.
MATERIALS AND METHODS
Plasmids. Plasmids encoding the full-length K-bZIP (K-bZIP/wt, residues 1 to
273) and a natural spliced variant of K-bZIP, K-bZIP?LZ, which carries a
deletion of the leucine-zipper region, were cloned into pcDNA3.1 (Invitrogen).
This cloning introduced a CpoI site and a Flag tag or T7 tag to the N terminus
as described previously (45). The resulting plasmids were designated pFlag-K-
bZIP, pT7-K-bZIP, or pT7-K-bZIP?LZ. Full-length cDNAs of SUMO-1,
SUMO-2, SUMO-3, and Ubc9 were amplified from BCBL-1 total RNA by
reverse transcription-PCR (RT-PCR) with primers listed in Table 1 and cloned
into the CpoI site of pcDNA-Flag- or pGEX-modified vector, which introduced
a CpoI site. Our SUMO protein sequences are identical with the one previously
reported (3). In order to prepare the active form of SUMO proteins, a stop
codon was generated immediately after two glycines at the C-terminal region of
each SUMO protein by site-directed mutagenesis (Stratagene) with primers
listed in Table 2, using pGEX expression plasmids served as templates. These
plasmids were designated as pGEX-SUMO-1GG, pGEX-SUMO-2GG, or
pGEX-SUMO-3GG. A plasmid encoding an enzymatically inactive mutant of
Ubc9 was prepared by mutating Cysteine 93 to Serine using PCR-based mu-
tagenesis with primers listed in Table 2. pFlag-K-bZIPK158R was generated with
site-directed mutagenesis (Stratagene) with primers listed in Table 2, using
pFlag-K-bZIP as a template. Deletion fragments of K-bZIP were amplified by
PCR using PFU-turbo (Stratagene) with primers previously described (33) and
cloned into the CpoI site of pcDNA-Flag-modified vector. Plasmids containing
SUMO-specific proteinase 1 (SENP1) were previously described (20), and the
inactive mutant (SENP1 R630L, K631M) was generated by PCR-based mutagen-
esis using primers listed in Table 2. pGEX SAE1/SAE2 expression plasmid (72)
was a generous gift from Ronald T. Hay (University of St. Andrews).
Cell culture. Human embryonic kidney epithelial 293 cells and 293T cells were
grown in monolayer cultures in Dulbecco’s modified Eagle medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) in the presence of 5% CO2.
The TREx-K-Rta BCBL-1 cell line had been generated by Nakamura et al. (54)
and was cultured in RPMI 1640 supplemented with 20% FBS, 100 ?g/ml of
blasticidin (Invitrogen), and 100 ?g/ml of hygromycin (Invitrogen).
Immunoprecipitation and immunoblot analyses. TREx-K-Rta BCBL-1 cells
were rinsed in ice-cold phosphate-buffered saline (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 [PMSF])
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) were added to the supernatants and preincubated overnight at 4°C.
Five-hundred micrograms of each of the cleared supernatants was reacted with
3 ?g of anti-Ubc9 (Santa Cruz) or anti-hemagglutinin (HA) tag (Babco) for 3 h
at 4°C with gentle rotation. The immune complex was then captured by the
addition 20 ?l of a protein A and protein G Sepharose bead mixture and was
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% bromphenol blue). Protein samples from total cell lysates (50
TABLE 1. Primers used for cloning
SUMO-1 F.......aacggtccg ATG TCT GAC CAG GAG GCA AAA
SUMO-1 R......aacggaccg CTA AAC TGT TGA ATG ACC CCC
SUMO-2 F.......aacggtccg ATG GCC GAC GAA AAG CCC AAG
SUMO-2 R......aacggaccg TCA GTA GAC ACC TCC CGT CTG
SUMO-3 F.......aacggtccg ATG TCC GAG GAG AAG CCC AAG
SUMO-3 R......aacggaccg CTA GAA ACT GTG CCC TGC CAG
Ubc9 F .............aacggtccg ATG TCG GGG ATC GCC CTC AGC
Ubc9 R.............aacggaccg TTA TGA GGG CGC AAA CTT CTT G
aIn all sequences, the italic lowercase nucleotides represent restriction en-
zyme sites used for cloning the PCR products.
VOL. 79, 2005REPRESSION VIA SUMO MODIFICATION9913
?g/lane) or immunoprecipitates were subjected to SDS-polyacrylamide gel elec-
trophoresis (PAGE) and then transferred to a polyvinylidene fluoride membrane
(Millipore) using a semidry transfer apparatus (Amersham Pharmacia). 293T
cells were cotransfected with 2 ?g of pT7-K-bZIP or pT7-K-bZIP?LZ and 3 ?g
of pFlag-Ubc9 or pFlag-empty expression plasmids using FuGENE 6 (Roche)
according to the supplier’s recommendations. The cells were harvested 48 h after
transfection and lysed in EBC buffer. Five-hundred micrograms of cell lysates
was immunoprecipitated with the addition of 25 ?l of anti-Flag antibody-conju-
gated agarose (Sigma). Beads were washed four times with EBC 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–12%
PAGE and then transferred as described above. After blocking for 1 h at room
temperature with 5% skim milk in TBST (20 mM Tris-HCl [pH 7.5], 137 mM
NaCl, 0.05% Tween 20), the membranes were incubated with primary antibodies
for 2 h at room temperature. The membrane was washed with TBST three times
for 10 min each at room temperature and incubated with horseradish peroxidase-
conjugated antibodies for 1 h at room temperature. Membranes were washed
three times with TBST and visualized with enhanced chemiluminescence reagent
(Amersham-Pharmacia). Final dilutions of the primary antibodies for immuno-
blotting were 1 ?g/ml of anti-K-bZIP immunoglobulin G (IgG), 1 ?g/ml of
anti-K-Rta, 1 ?g/ml of anti-actin (Santa Cruz), 1:3,000 anti-T7 tag antibody
(Novagen), 1 ?g/ml of anti-SUMO-1 (Zymed Laboratories), and 1 ?g/ml of
anti-SUMO-3 (Zymed Laboratories) containing 5% skim milk. The anti-K-bZIP
and anti-K-Rta rabbit sera were raised against purified glutathione S-transferase
(GST)–K-bZIP full-length or GST–K-Rta (residues 400 to 604) protein, respec-
tively. Purification of rabbit IgG was performed with a standard procedure.
Protein amount was examined by bicinchoninic acid (BCA) protein assay, and
purity of IgG (?90%) was confirmed by SDS-PAGE. For SUMO-modified
proteins, the cells were washed twice with PBS and lysed with lysis buffer (50 mM
Tris-Hcl [pH 6.8], 2% SDS, 10% glycerol, 20 ?M N-ethylmaleimide [Sigma])
supplemented with a protease inhibitor cocktail (Roche). The cells were briefly
sonicated and centrifuged, and the supernatant was used for immunoblot anal-
ysis. For immunoprecipitation of SUMO-modified K-bZIP, after lysing the cells
as described above the lysate was incubated at 95°C for 10 min and cleared by
centrifugation at 15,000 ? g for 10 min at room temperature. The lysate was then
diluted 1:10 in dilution buffer (50 mM HEPES, pH 7.0, 250 mM NaCl, 0.1%
NP-40, and protease inhibitors) and immunoprecipitated with 4 ?g of anti-K-
bZIP rabbit IgG or preinoculated rabbit IgG.
Immunofluorescence assay. Forty-eight hours after doxycycline treatment,
TREx-K-Rta BCBL-1 cells were fixed with 3.7% formaldehyde in PBS for 5 min
at room temperature and subsequently treated with 1.0% Triton X-100 followed
by 1.0% NP-40 in PBS for 10 min each at room temperature. After washing twice
with 0.2% Tween 20 in PBS, cells were smeared on a coverslip (Fisher). After
treatment with blocking solution containing 2% bovine serum albumin (BSA;
[Fisher]) in PBS, cells on the coverslip were incubated with anti-K-bZIP rabbit
IgG (1:2,000) in PBS-2% BSA for 1 h at room temperature. After washing four
times with PBS, Alexa Fluor 555-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 at room temperature.
293 cells were grown on coverslips in 6-well plates. Expression vector, pcDNA
Flag-K-bZIP, or pcDNA Flag-K-bZIPK158R was transfected into 293 cells.
Forty-eight hours after transfection, cells were fixed with 3.7% formaldehyde–
PBS for 5 min at room temperature and then rinsed with PBS three times and
subsequently treated with 1.0% Triton X-100 followed by 1.0% NP-40 in PBS for
10 min, each at room temperature. After washing twice with 0.2% Tween 20 in
PBS, coverslips were treated with blocking solution. The cells were incubated
with 0.5 ?g/ml of anti-K-bZIP rabbit serum (1:2,000) and anti-PML mouse
monoclonal antibody (1 ?g/ml; PG-M3 [SantaCruz]) in blocking solution for 1 h
at room temperature. After washing four times with PBS, Alexa Fluor 555-
conjugated goat anti-rabbit IgG F(ab?)2(1:5,000) (Molecular Probes) and Alexa
Fluor 488-conjugated goat anti-mouse IgG F(ab?)2(1:5,000) (Molecular Probes)
in blocking solution were applied and allowed to react for 1 h at room temper-
ature. After washing twice with TBST and once with PBS, coverslips were air
dried and mounted on glass slides (Fisher). Imaging was viewed with an Olympus
BX61. Images were captured with a CCD camera operated with Slide Book
Software (Intelligent Imaging Innovations, Inc.).
Preparation and purification of GST fusion proteins. GST fusion proteins
were expressed in Escherichia coli strain BL21 transformed with pGEX-Ubc9,
pGEX-SUMO-1GG, pGEX-SUMO-2GG, pGEX-SUMO-3GG, pGEX SAE1/
SAE2, or pGEX4T-2. The GST fusion proteins were purified using glutathione-
Sepharose beads (Amersham-Pharmacia) by standard procedures. After induc-
tion, bacterial cells (500 ml) were cultured in Luria broth for each construct.
Protein expression was induced for 3 h with 1 mM (final concentration) isopro-
pylthio-?-D-galactoside. Bacterial cells were washed once in PBS and then lysed
with BugBuster (Novagen) supplemented with a protease inhibitor cocktail
(Roche). After clearing by centrifugation at 8,000 ? g for 10 min at 4°C, gluta-
thione-Sepharose beads (500 ?l of a 1:1 slurry in PBS) were added to the lysates
for affinity purification. After incubation for 1 h at 4°C with rotation, the beads
were washed four times in PBS containing 1% Triton X-100 and 1% sarcosyl.
The GST fusion proteins were cleaved by biotinylated thrombin (Novagen) while
bound on glutathione-Sepharose beads. Biotinylated thrombin was captured by
streptavidin-conjugated agarose beads (Novagen). Purified proteins were dia-
lyzed against HEPES buffer (20 mM HEPES [pH 7.4], 1 mM dithiothreitol
[DTT]), and protein amounts were measured by BCA protein assay (Pierce).
Purity of the proteins was confirmed by standard SDS-PAGE. For GST pull-
down assay, the proteins immobilized on the glutathione-agarose beads were
measured by Coomassie blue staining, using BSA as a protein standard.
In vitro SUMO conjugation assays. SUMO conjugation assays were per-
formed in 40-?l volumes. The substrates were in vitro translated (IVT) by using
the TNT quick-coupled reticulocyte lysate system (Promega). Two microliters of
labeled translation product was incubated for 2 h at 37°C in an ATP-regenerating
buffer (50 mM Tris [pH 7.5], 5 mM MgCl2, 2 mM ATP, 10 mM creatine
phosphate [USB, Cleveland Ohio], 3.5 U/ml of creatine kinase [USB], 0.6 U/ml
of inorganic pyrophosphatase [USB]) containing 100 ng/ml of E1 (SAE1/SAE2),
50 ?g/ml of E2 (Ubc9), and 200 ?g/ml of SUMO-GG. After termination of the
reaction with SDS sample buffer containing ?-mercaptoethanol, reaction prod-
ucts were fractionated by SDS-PAGE. The gel was dried and analyzed by Phos-
Pulse-chase analysis. 293 cells were seeded in a 6-cm2dish and transfected
with 4 ?g of pFlag-K-bZIPwt or pFlag-K-bZIPK158R. Transfected cells were
metabolically labeled for 4 h with 50 ?Ci of [35S]methionine and [35S]cysteine
(Amersham) after 24 h posttransfection in DMEM supplemented with 10%
PBS-dialyzed FBS. After labeling, medium was removed and cells were washed
three times with 1 ml of DMEM containing unlabeled methionine and cysteine.
Cells were harvested in 1 ml of lysis buffer (50 mM Tris-HCl [pH 7.5], 500 mM
NaCl, 1% Triton X-100, 1 mM NaF, 1 mM Na2VO4, 1 mM PMSF) supple-
mented with a protease inhibitor cocktail (Roche) and immunoprecipitated with
3 ?g of an anti-K-bZIP IgG. Immunoprecipitates were subjected to SDS-PAGE
TABLE 2. Primers used for mutagenesis
Primer Sequence (5?–3?)a
SUMO-1GG-F...........................................................................................................ACG GGA GGT TAA TCA ACA GTT TAC GGT CCG TGA
SUMO-1GG-R..........................................................................................................AAC TGT TGA TTA ACC TCC CGT TTG TTC CTG ATA
SUMO-2GG-F...........................................................................................................CG GGA GGT TAA TAC TGA CGG TCC GTG AAT TCA TC
SUMO-2GG-R..........................................................................................................CCG TCA GTA TTA ACC TCC CGT CTG CTG TTG GAA C
SUMO-3GG-F...........................................................................................................ACG GGA GGT TAG CCG GAG AGC AGC CTG GCA GG
SUMO-3GG-R..........................................................................................................GCT CTC CGG CTA ACC TCC CGT CTG CTG CTG GAA C
SENP1 RK/LM..........................................................................................................CCA TAC TTC CTG ATG CGG ATG GTC TGG GAG
SENP1 RK/LM..........................................................................................................CTC CCA GAC CAT CCG CAT CAG GAA GTA TGG
K-bZIP158K/R-F.......................................................................................................TCT GTA GTT AGG GCC GAA GTA TGT GAT CAG TCA
K-bZIP 158K/R-R.....................................................................................................TTC GGC CCT AAC TAC AGA CGC AGG CAC G
Ubc9 C93S-F..............................................................................................................GGG ACA GTG TCC CTG TCC ATC TTA GAG GAG GA
Ubc9 C93S-R.............................................................................................................GGA CAG GGA CAC TGT CCC CGA AGG GTA CAC AT
aA mutation was introduced in the underlined sequence.
9914 IZUMIYA ET AL.J. VIROL.
and transferred to a membrane as described above. Transferred membrane was
quantified by PhosphorImager, and signal intensity was measured by using Quan-
tityOne software (Bio-Rad).
In vitro interaction assay. GST-protein beads, containing approximately 2.0
?g of proteins, were resuspended in binding buffer (20 mM HEPES [pH 7.9], 150
mM NaCl, 1 mM EDTA, 4 mM MgCl2, 1 mM DTT, 0.1% NP-40, 10% glycerol;
supplemented before use with 1 mg/ml BSA, 0.5 mM PMSF, and 1? protease
inhibitor cocktail), and incubated for 30 min at 4°C with 10 ?l of IVT proteins,
which were labeled with [35S]methionine using the TNT coupled transcription
and translation system (Promega). The beads were washed four times with the
binding buffer and resuspended and boiled in 2? SDS sample buffer. After
proteins were separated by SDS-PAGE, radiolabeled polypeptides retained on
the beads were visualized by autoradiography.
Chromatin-immunoprecipitation assay. After 48 h with or without induction
of viral reactivation with doxycyclin (1 ?g/ml), 107of TREx-K-Rta BCBL-1 cells
were fixed with 1% formaldehyde at room temperature 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). Two-hundred-microliter cell pellets were re-
suspended 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, 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 anti-K-bZIP rabbit serum (1:100), preinoculated rabbit serum (1:100), or
anti-Ubc9 antibody (1:100 [Santa Cruz]) overnight at 4°C with gentle rotation.
Immunocomplexes were recovered and eluted as described before (8). After
reverse cross-linking at 65°C overnight, the DNA fragments were purified with a
QIAquick PCR Purification kit (QIAGEN), after adjusting pH with 3 M sodium
acetate (pH 7.0), and eluted with 100 ?l of 1? Tris-EDTA buffer, pH 8.0.
Southern blotting was performed using KSHV cosmid clones GB11, GA29,
Not39, Not33, and GA2. The cosmid clones GB11, GA29, and GA2 are generous
gifts from Ren Sun, University of California at Los Angeles. Two NotI fragments
(nucleotides 43172 to 76999 and 77000 to 116203; GenBank accession number
U75698) were cloned into the SuperCosI vector (Stratagene) after digestion of
the KSHV genomic DNA with NotI (New England Biolabs). Each cosmid clone
was digested with three different restriction enzymes overnight at 37°C. DNA
fragments were separated on a 0.8% agarose gel. The gel was depurinated by
incubation in depurination buffer (0.25 M HCl), followed by denaturation in the
buffer (1.5 M NaOH, 0.5 M NaCl) for 20 min each. After denaturation, the
restriction fragments were transferred to a nylon membrane (Biodyne; Pall
Gelman Laboratory) by standard procedures. The DNA was immobilized on the
membrane by drying at room temperature for 1 h and UV cross-linking. Immu-
noprecipitated DNA fragments were radiolabeled with [?32-P]dATP using a
Strip-EZ DNA kit (Ambion), and hybridization was performed in ULTRAhyb
buffer (Ambion) as recommended by the supplier. Equal amounts of probe (1 ?
106cpm/ml) were used for each hybridization.
In order to confirm the results of Southern blotting, low-cycle-number PCR
amplification was performed with recombinant Taq polymerase (Invitrogen).
DNA input was either of total-input DNA (control), preimmune-, or K-bZIP-
immunoprecipitated DNA fractions. All primer sequences are provided in Table 3.
Dual luciferase reporter assays. Reporter plasmids were constructed by in-
serting promoter regions (74) upstream of the firefly luciferase coding region
(Luc) in the pGL3-Basic vector (Promega). 293 cells were seeded in 12-well
plates at 1 ? 105/well in 1.5 ml of DMEM 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/expression plasmid, were trans-
fected using the Fugene6 reagent following the manufacturer’s protocol (Roche).
All wells were cotransfected with a control reporter, pRL-cytomegalovirus
(CMV) Renilla luciferase plasmid (Promega), which served as an internal con-
trol to normalize for variation in transfection efficiency. Cell lysates were pre-
pared 48 h after transfection with 1? Passive Lysis Buffer (Promega). Dual
luciferase assay was performed according to the manufacturer’s protocol using a
Lumat LB 9501 Luminometer (Wallac Inc.). At least three independent exper-
iments were performed in each setting.
K-bZIP is sumoylated in vitro. The observation that K-bZIP
possesses transcriptional repression activity and is localized in
PML bodies or PODs (a nuclear structure replete with sumoy-
lated molecules) prompted us to investigate the possibility that
K-bZIP is sumoylated. First, the potential of K-bZIP to be
sumoylated was tested in a cell-free system using in vitro-
translated (IVT)-K-bZIP and purified components of SUMO
activating enzymes (SAE1/SAE2) and SUMO-conjugating en-
zyme (Ubc9) (21). This reaction was supplemented with
Sumo1-GG, Sumo2-GG, or Sumo3-GG, the activated forms of
SUMO proteins, and ATP was provided. As shown in Fig. 1A,
K-bZIP was efficiently modified by either of the three SUMO
peptides to the extent that the majority of K-bZIP were in the
conjugated form. By contrast, K-Rta, another transcriptional
factor encoded by KSHV, exhibited very little, if any, sumoy-
lation (Fig. 1A). To identify the domain(s) responsible for
sumoylation, the sumoylation potential of a series of K-bZIP
mutants was tested. The K-bZIP mutant with the basic region
deleted (?Basic) and the mutant without bZIP domain
(?bZIP) were not sumoylated (Fig. 1B). This finding suggests
that the major sumoylation site(s) resides in the basic domain.
Interestingly, K-bZIP?LZ, with the leucine zipper domain de-
leted, or K-bZIP?N, the N-terminal deletion mutant, was
sumoylated at much lower efficiency than full-length K-bZIP;
TABLE 3. Primers used for ChIP assay
Primer Sequence (5?–3?)
ORF9 pro.-F ......................................................................................................................................................ATCGGAAAAACGGTGGTGAAC
ORF57 pro.-F ....................................................................................................................................................CCTCCTCTGAGTTTGACGAATCG
K-bZIP pro.-R ...................................................................................................................................................CCTTGCGAACACTTCAGTCTCG
ORF36 pro.-F ....................................................................................................................................................CGCCATTCGCTACTTCTCGG
PAN RNA pro.-F..............................................................................................................................................GGGTTTGACCACGGTTACTGATAGG
PAN RNA pro.-R..............................................................................................................................................CCATTTTTGGAAGCCACGCC
ORF57 coding region-F....................................................................................................................................GCTTTCGTGGAGGAACAAATGAC
ORF57 coding region-R...................................................................................................................................CGTTTAGTAGCCCCATCACATCC
VOL. 79, 2005REPRESSION VIA SUMO MODIFICATION 9915
this finding suggests that the native, or dimer, conformation of
K-bZIP may be important for the sumoylation (Fig. 1B).
K-bZIP is sumoylated in vivo. Sumoylation of K-bZIP was
studied in vivo using 293T cells by transfection of K-bZIP
expression vector (Fig. 1C), which utilized the endogenous
SUMO ligation system. High-molecular-weight K-bZIP, with
an increased size expected for monosumoylation, was readily
detected (lanes 1 and 4). To demonstrate that this modification
was due to sumoylation, SENP1, a SUMO-specific proteinase
1, was cotransfected with K-bZIP at a dose of 0.5 (lane 2) or 1.0
?g (lane 3). The putative sumoylated bands gradually disap-
peared (Fig. 1C, lanes 2 and 3). In contrast, cotransfection with
a plasmid carrying inactive SENP1 (SENPmut) (lanes 5 and 6)
did not affect the intensity of the high-molecular-weight bands
(Fig. 1C). These data suggest that the modification of K-bZIP
in transfected cells was largely, if not completely, due to
Sumoylation of endogenous K-bZIP was examined during
the reactivation of the latent KSHV genome. In order to ar-
chive higher specificity, we employed the cell line, TREx-K-
Rta BCBL-1, developed by Nakamura et al. (54), where the
K-Rta gene is under the control of a tetracycline (Tet)-induc-
ible promoter. Induction of K-Rta with the Tet analog, doxy-
cycline (Dox), activated K-bZIP expression, and the level of
K-bZIP reached a peak at about 48 h after induction. At that
time, cells were harvested, and K-bZIP was probed with spe-
cific antibody (Fig. 1D). Bands corresponding to full-length
and the type II splice variant of K-bZIP were identified, to-
gether with the mono- and disumoylated forms of K-bZIP. A
significant fraction of K-bZIP in BCBL-1 was sumoylated,
FIG. 1. (A) SUMO conjugation in vitro. K-bZIP but not K-Rta is efficiently modified by SUMO. In vitro-translated (IVT)–K-bZIP was
incubated with purified recombinant proteins E1 (SAE1/SAE2), E2 (Ubc9), and each activated form of SUMO protein (SUMO-1GG, SUMO-
2GG, or SUMO-3GG). The reaction was carried out in the presence of the ATP regenerating system. Reactions without Ubc9 served as negative
controls. (B) Mapping of SUMO-modified region. Deletion mutants of IVT–K-bZIP were incubated in the SUMO reaction. Basic region (amino
acids 122 to 189) of K-bZIP is important for the sumoylation (?). (C) Sumoylation of K-bZIP in vivo. K-bZIP transfected into 293T cells displays
a high-molecular-weight band when probed with anti-K-bZIP antibody, and which disappeared upon cotransfection with increasing amounts of
SUMO-specific protease (SENP) but not an SENP mutant (SENPmut) lacking protease activity. (D) Sumoylated K-bZIP was detected in
TREx-K-Rta BCBL-1 activated by K-Rta expression. Protein extract (100 ?g/lane) was loaded. K-bZIP was detected with anti-K-bZIP antibody.
KSHV negative B-cell line (GA10) was the negative control. An alternatively spliced K-bZIP (K-bZIP?LZ) and sumoylated form of K-bZIP was
also observed. Actin protein served as an internal control for the amount of protein on membrane and was detected by anti-actin goat serum.
(E) K-bZIP is modified by both SUMO-1 and SUMO-2/3. BCBL-1 cells (1 mg) induced viral reactivation by K-Rta expression and lysed in SDS
and were immunoprecipitated (IP) with 4 ?g of preinoculated rabbit IgG (Pre) or anti-K-bZIP rabbit IgG (Kz). Immunoprecipitates were
separated on a SDS–9% PAGE and immunoblotted (W.B.) with indicated antibodies. Anti-SUMO-3 polyclonal antibody detects both SUMO-2
and SUMO-3 proteins because of significant identity.
9916IZUMIYA ET AL.J. VIROL.
reaching a level higher than reported for most cellular factors
without cotransfection of exogenous SUMO (46, 53).
We also investigated the type(s) of SUMO peptide conju-
gated to the K-bZIP in naturally infected cells. The BCBL-1
cell lysates, after Dox induction, were immunoprecipitated
with anti-K-bZIP antibody, followed by blotting with either
anti-SUMO-1 monoclonal antibody or anti-SUMO-3 rabbit
polyclonal antibody (which recognizes both SUMO-2 and
SUMO-3 moieties, due to their high degree of homology).
Immunoprecipitation with preimmune rabbit IgG served as a
negative control. Figure 1E showed that both SUMO-1 and
SUMO-2/3 were covalently attached to K-bZIP during reacti-
vation. Because of the dual specificity of the antibody, we were
unable to determine whether the modification is due to
SUMO-2, SUMO-3, or both. Our data are also consistent with
the structures of the different SUMO species, in that SUMO-1
usually conjugates with the substrate in the monomer form,
whereas SUMO-2 and -3 are themselves sumoylated and thus
can form dimers and trimers (72).
Taken together, these results indicate that K-bZIP is sumoy-
lated in vitro, in vivo, and during viral reactivation. Our data
also showed that K-bZIP is able to conjugate with both
SUMO-1 and SUMO-2/3.
Mapping the sumoylation site of K-bZIP. Inspection of se-
quences within the basic domain reveals that amino acids 157
to 160 (VKAE) of K-bZIP matches the SUMO consensus
(?KXE, with ? being a hydrophobic residue). The putative
conjugation site is lysine-158, and this motif is located in the
basic region of the molecule, consistent with the sumoylation
mapping data described above. Using PCR-based site-directed
mutagenesis, the lysine-158 of K-bZIP was mutated to argi-
nine, and the K158R mutant was transfected into 293T cells.
The K158R mutant protein synthesized in transfected 293T
cells was not sumoylated (Fig. 2), indicating that lysine 158 was
the predominant, if not the sole, in vivo target site of sumoy-
lation. Interestingly, a natural splice variant of K-bZIP,
K-bZIP?LZ lacking the leucine-zipper domain (45), was not
sumoylated in vivo, despite the presence of the VKAE motif,
suggesting that the consensus motif is not the sole structural
determinant of the SUMO reaction.
Subcellular distribution of the K-bZIP sumoylation mutant.
One of the major functions of sumoylation is to change the
subcellular distribution. Sumoylation is important for nuclear
and PML translocation of certain proteins (12, 32, 84). Previ-
ous studies showed that K-bZIP is colocalized with PML in
POD, a structure enriched with sumoylated molecules (36, 79).
In this study, the ability of the K-bZIP mutant to colocalize
with PML was examined. Wild-type K-bZIP and K158R mutant
were individually transfected into 293 cells, and expressed pro-
teins were visualized by Alexa Fluor 555-conjugated antibodies
against anti-K-bZIP rabbit IgG. As shown in Fig. 3, the overall
subcellular distribution of K-bZIP and K158R were similar. Fur-
thermore, K158R mutant and PML colocalized in POD, like
the wild type, indicating that sumoylation was not essential for
K-bZIP translocation into PML; this finding is similar to the
analyses reported for EBV Zta (1) and CMV IE2 (3).
K-bZIP protein stability is unaffected by sumoylation.
SUMO modification has been implicated in the modulation of
protein stability (64). To explore whether SUMO modification
affects K-bZIP stability, we conducted a pulse-chase experi-
ment. 293 cells transfected with K-bZIP wild type or the
K-bZIPK158R were labeled with [35S]methionine and [35S]cys-
teine for a 4-h pulse from 24 to 28 h posttransfection, and cells
were chased for 4, 18, 28, or 40 h. No appreciable difference in
the synthesis and reduction in the amount of labeled protein
during the time course was observed (Fig. 4). This result sug-
gested that sumoylation does not affect the overall stability of
Sumoylation and K-bZIP mediated transcriptional repres-
sion. Another major function of sumoylation is transcriptional
repression. Increasing evidence suggests that transcriptional
factors, when modified by sumoylation, often become repres-
sors (29, 60). Our previous work showed that K-bZIP is a
transcriptional repressor of K-Rta by physical interaction with
K-Rta (33, 43). Accordingly, repression activity of K-bZIP was
examined by assessing K-Rta-mediated activation of the
ORF57 promoter (33). When cotransfected with K-bZIP, the
transactivation of the ORF57 promoter by K-Rta was drasti-
cally reduced to about 25%, a result consistent with our pre-
vious work (33). If the K158R mutant was used, transactivation
activity of K-Rta remained high (60% of the full K-Rta activ-
ity). This suggests that sumoylation of K158 is important for
K-bZIP’s repression activity. To ensure that sumoylation, but
not other modifications of lysine-158, were responsible,
SENP1, a SUMO-specific protease, was transfected at two
different doses (0.5 and 1 ?g) with K-Rta and wild-type K-
bZIP. The addition of SENP1 also relieved the repression
activity of K-bZIP, while SENP1 alone had little effect on
K-Rta. As an additional control, a SENP mutant with impaired
protease activity was used; K-bZIP repression activity was re-
FIG. 2. Identification of sumoylation site. K-bZIP wild type, K-
bZIP K158R, or K-bZIP?LZ was transfected into 293T cells. Protein
extract (50 ?g/lane) lysed in SDS was loaded. K-bZIP was detected
with anti-K-bZIP antibody. W.B., Western blotting.
VOL. 79, 2005 REPRESSION VIA SUMO MODIFICATION 9917
stored (Fig. 5A). That these variations in activity are not due to
changes in the amount of proteins was shown by immunoblots
of the reaction mixtures with K-bZIP and K-Rta antibodies.
These data, taken together, indicate that sumoylation of K-
bZIP is largely, but not exclusively, responsible for the repres-
sion activity on K-Rta.
Requirement of Ubc9 activity in K-bZIP-mediated repres-
sion. To demonstrate the role of Ubc9 in K-bZIP-mediated
repression, we took advantage of a catalytically inactive mutant
Ubc9C93S, shown previously to behave in a dominant-negative
manner against endogenous Ubc9 (19). If Ubc9 is involved in
K-bZIP-mediated repression of K-Rta, coexpression of
Ubc9C93S should reduce the repression effect. When cotrans-
fected with K-Rta and K-bZIP, Ubc9C93S, but not Ubc9 wild
type, largely restored the ability of K-Rta to transactivate the
ORF57 promoter, indicating that Ubc9 activity is required to
achieve the full repression activity of K-bZIP (Fig. 5B). It is
noteworthy that expression of Ubc9 (or UbcC93C), in the
absence of K-bZIP, has no effect on K-Rta’s transactivation
activity. This reinforces the notion that Ubc9 is recruited to the
K-Rta complex by K-bZIP.
Genome-wide scanning for K-bZIP binding sites on the
KSHV chromatin. Previously, we showed that K-bZIP sup-
presses the transcriptional activation of a subset of K-Rta
target genes. For instance, the transcription of ORF57 and
K-bZIP, but not nut-1/PAN RNA, was suppressed by over-
expressed K-bZIP (33). If K-bZIP association with K-Rta
was important for this process, K-bZIP should be recruited
to ORF57 and K-bZIP promoters but not the nut-1/PAN
RNA promoter. Taking advantage of a chromatin immuno-
precipitation-based genome-wide scanning approach we de-
veloped (40), the major binding sites of K-bZIP on the
KSHV chromatin were analyzed during reactivation of the
lytic replication cycle. The K-Rta inducible cell line, TREx-
K-Rta BCBL-1, was used (54). As shown previously, upon
FIG. 3. Localization of K-bZIP wt (a) and K-bZIPK158R (b) with PML in 293 cells. Immunofluorescence analysis was performed by using
anti-K-bZIP rabbit serum and anti-PML mouse monoclonal antibody. K-bZIP (red) and PML (green) were detected with Alexa Fluor 555-conjugated
anti-rabbit IgG and Alexa Fluor 488-conjugated anti-mouse IgG. Fluorescence images were collected separately and overlaid by a computer. The
rightmost panel shows enlarged images of indicated cells. These panels are representative of 10 different fields. (magnification, ?600).
FIG. 4. Determination of the stability of the K-bZIP wild type and
the K-bZIPK158R mutant. (A) K-bZIP wild type and K-bZIPK158R
were transfected into 293 cells and cells were labeled with [35S]methi-
onine and [35S]cysteine for a 4-h pulse from 24 to 28 h posttransfection.
Cells were chased with medium containing nonlabeled amino acids for
the indicated time periods. K-bZIP proteins were immunoprecipitated
with anti-K-bZIP antibody and subjected to SDS-PAGE followed by
autoradiography. (B) Quantification of the labeled protein. Protein
amount was quantified by PhosphorImager analysis of the dried SDS-
PAGE gel with Quantity One (Bio-Rad). Black indicates the K-bZIP
wild type, while gray refers to K-bZIPK158R.
9918 IZUMIYA ET AL.J. VIROL.
K-Rta induction by Dox, a complete cycle of KSHV repli-
cation was triggered, following a well-ordered KSHV gene
expression program, including the immediate induction of
K-bZIP expression. A significant advantage of this system
over the 12-O-tetradecanoyl phorbol-13-acetate induction
protocol is that almost every cell is induced (based on im-
munostaining of K-bZIP protein; Fig. 6A). This approach
greatly increased the sensitivity of ChIP analysis, which uses
radiolabeled DNA fragments, associated with K-bZIP chro-
matin, to probe a Southern blot of restriction enzyme-di-
gested Cosmid clones that span almost the entire KSHV
genome (Fig. 6B). Based on relative hybridization intensi-
ties of restriction fragments (compare to those of the
ethidium bromide-stained gel), the major K-bZIP binding
sites can be mapped. K-bZIP antibody (Kz) was used to
generate K-bZIP ChIP. Preinoculation rabbit serum (Pre)
served as a negative control. Without Dox induction, little
K-bZIP was present and only basal hybridization was de-
tected, attesting to the specificity of the antibodies. Forty-
eight hours after Dox induction, bands with strong hybrid-
ization intensity, above the general background, were
identified (Fig. 6C). This result suggests low-affinity binding
of K-bZIP at numerous sites along the KSHV genome, in
addition to its high-affinity binding sites. The high-intensity
hybridization regions were mapped to six sites: K1-ORF6
promoter, ORF9 promoter, K4 promoter/KSHV-Ori lytic
DNA replication, ORF26-ORF27 region, K-bZIP promoter,
and ORF57 promoter. As predicted, ORF57 and K-bZIP
promoters, but not the nut-1/PAN RNA promoter, were
among the hybridized regions. In addition, the two origins of
lytic-phase DNA replication, shown by others to be K-bZIP
binding sites (4, 44), were also identified (Fig. 6D). These
results were confirmed by analysis of PCR products gener-
ated with specific primer sets corresponding to these regions
(Fig. 6E). Primer sets for the ORF57 coding regions and
other promoters such as nut-1/PAN RNA promoter were
also included as negative controls. The PCR results are in
complete agreement with the Southern blot results, thus
substantiating the mapping data and indicating that K-bZIP
exerts its repression activity by direct association with the
K-bZIP association with Ubc9 in vitro and in vivo. Mech-
anistically, there are two ways to explain how sumoylation of
K-bZIP results in the repression of the K-Rta transcrip-
tional complex. The first is that the SUMO moieties directly
hinder assembly of the productive transcription machinery,
as proposed by Holmstrom et al. (29). The second is that the
sumoylation site of K-bZIP recruits E2 conjugating enzyme
Ubc9 to the K-Rta complex. Recently, Ubc9 was identified
as a strong transcriptional corepressor (37). These two
mechanisms are not mutually exclusive, and most likely K-
bZIP utilizes both mechanisms for transcriptional repres-
sion. In the first case, K-bZIP is a corepressor itself, whereas
in the second, K-bZIP serves as an adaptor, more akin to the
role of an E3 SUMO ligase. To test the second mechanism,
the association of endogenous K-bZIP with Ubc9 was ex-
amined in BCBL-1 cells. TREx-K-Rta-BCBL-1 was treated
with Dox to induce K-bZIP expression, and cell extracts
FIG. 5. (A) Repression via sumoylation. K-Rta activation of the ORF57 promoter (leftmost bar) was repressed by K-bZIP wild type but had
less repression by K-bZIP K158R. Cotransfection with the SENP1 wild type but not an inactive mutant diminished the repression. 293 cells were
cotransfected with K-Rta, ORF57 promoter, and the indicated plasmids. K-Rta and K-bZIP amounts were examined by immunoblotting with
anti-K-Rta or anti-K-bZIP antibody. (B) Ubc9 dominant-negative relived K-bZIP repression. 293 cells were cotransfected with indicated plasmids.
Protein amounts of K-Rta and K-bZIP were examined by immunoblotting with indicated antibodies.
VOL. 79, 2005 REPRESSION VIA SUMO MODIFICATION9919
FIG. 6. Identification of the K-bZIP binding sites on the KSHV chromosome by the ChIP method. (A) Reactivation by K-Rta induction.
K-bZIP expression was analyzed by immunofluorescence assay using anti-K-bZIP antibody (red). DNA was stained with TOPRO-3 (Molecular
Probes; blue). (B) Schematic diagram of the KHSV genome. The five cosmids (GB11, GA29, Not33, Not39, and GA2) spanning the KSHV genome
are indicated. (C) Scanning of K-bZIP binding sites. Cosmids were digested with the restriction enzymes and separated on agarose gel (0.8%). The
9920 IZUMIYA ET AL.J. VIROL.
were isolated at 48 h. Ubc9 was immunoprecipitated with
specific antibody, followed by immunoblotting with antibody
against K-bZIP. Additional unrelated antibodies were used
as controls. As shown in Fig. 7A, K-bZIP coprecipitated
with Ubc9 in vivo. As an independent test of the association
between K-bZIP and Ubc9 and to rule out the possibility
that the observed association in BCBL-1 cells was due to
nonspecificity of the polyclonal antibody, we constructed a
T7-tagged K-bZIP and a Flag-tagged Ubc9 expression vec-
tor. We also included a T7-tagged construct of the natural,
spliced variant of K-bZIP (K-bZIP?LZ), which as shown
above is not sumoylated and lacks repression ability (43).
These constructs were cotransfected into 293T cells. At 48 h
after transfection, the cells were harvested and lysed. Im-
munoprecipitation was carried out with Flag-antibody-con-
jugated agarose. As shown in Fig. 7B, Flag-Ubc9, but not
Flag-empty alone (as encoded by Flag-empty, which bears
only the Flag epitope), pulled down T7-K-bZIP. Further-
more, Flag-Ubc9 did not coprecipitate with T7-K-bZIP?LZ,
demonstrating the specificity of its association. The data
also suggest that the leucine-zipper domain is required for
this association, which may explain why K-bZIP?LZ is not
sumoylated in vivo (Fig. 2).
Finally, to show that the association between K-bZIP and
Ubc9 is direct, purified GST-Ubc9 was incubated with in vitro-
translated K-bZIP (IVT–K-bZIP). As shown in Fig. 7C, GST-
Ubc9 but not GST alone efficiently pulled down IVT–K-bZIP.
Thus, by three different approaches, we have demonstrated
here that K-bZIP associates with Ubc9.
Ubc9 recruitment to the ORF57 and K-bZIP promoters
by K-bZIP. The studies described above have shown that Ubc9
was associated with K-bZIP. Recruitment of Ubc9 to the
ORF57 and K-bZIP promoters was also analyzed using anti-
Ubc9 antibody. Using the ChIP protocol described earlier,
Ubc9 was recruited to the ORF57 promoter and to the K-bZIP
promoter but not to the nut-1/PAN RNA promoter (Fig. 8).
FIG. 7. K-bZIP association with Ubc9. K-bZIP expressed in BCBL-1 was coprecipitated with Ubc9 by Ubc9 antibodies. Anti-HA antibody was
used as a negative control. (B) Association between K-bZIP and Ubc9 in cotransfected 293T cells. 293T cells were cotransfected with the indicated
plasmids. Cell lysates were precipitated with Flag antibody-conjugated agarose, and coimmunoprecipitation of K-bZIP was detected by using
anti-T7 tag antibody. The expression of T7-tagged K-bZIP in one-tenth of total cell lysates used in coimmunoprecipitation is shown in the same
blot as a control. (C) K-bZIP associated with Ubc9 in vitro. GST-Ubc9 but not GST precipitates in vitro-translated (IVT)–K-bZIP. The inputs are
one-tenth of the lysates used for the binding study.
gel was stained with ethidium bromide (EtBr; right panel) and Southern blotted with radiolabeled probes derived from ChIP before (Dox, 0 h)
and after 48 h of KSHV reactivation (Dox 48 h). The DNA associated with K-bZIP chromatin was radiolabeled as described in Materials and
Methods. (D) Schematic diagram of K-bZIP binding sites. Putative major K-bZIP binding sites are summarized in the diagram. (E) PCR
verification of K-bZIP binding sites. PCR primers were designed for the K-bZIP associated regions. Other KSHV promoters and coding region
served as controls (ORF36 promoter, PAN promoter, ORF57 coding region). For each primer set, a PCR with the total input DNA (In) before
immunoprecipitation was carried out. ChIP fragments of preimmune serum (Pre) or anti-K-bZIP (Kz) after 48 h of KSHV reactivation were
subjected to PCR analyses to confirm Southern blotting results.
VOL. 79, 2005 REPRESSION VIA SUMO MODIFICATION9921
A growing list of transcriptional factors has been shown to
be sumoylated, including glucocorticoid receptor, Sp3, Smad4,
p53, and c-jun (29, 46, 53, 60). Although the effects of sumoy-
lation on transcriptional factors are multiple, this posttransla-
tional modification usually causes a reduction of the transcrip-
tional activity of a transactivator or an increase of the
repression activity of a trans-repressor. Previously, we demon-
strated that K-bZIP is a strong transcriptional repressor for
K-Rta (33). In this study, we present data suggesting that a
major mechanism of K-bZIP mediated repression is via sumoy-
lation. These include that K-bZIP is sumoylated in vitro and in
vivo and that the in vivo sumoylation site maps to the lysine at
158. Importantly, the sumoylation of K-bZIP was detected not
only in transient transfected 293T cells but also in naturally
infected BCBL-1 cells during reactivation. Both types of
SUMO peptides (SUMO-1 and SUMO-2/3) were found to be
conjugated to K-bZIP endogenously in naturally infected
BCBL-1 cells. The fraction of sumoylated K-bZIP in BCBL-1
cells was close to 10%, a figure significantly higher than most
other transcriptional factors (46, 53). K-bZIP thus joins the
growing list of sumoylated herpesvirus immediate early pro-
teins, which include human cytomegalovirus IE1, IE2, (3, 81),
EBV Zta (1) and Rta (5), and human herpesvirus-6 IE1 (23).
We showed that mutation of lysine-158 to arginine virtually
eliminated SUMO modification of K-bZIP in vivo, with a con-
comitant loss of much of the repression activity. Because lysine
is a potential site for other posttranslational modifications,
including acetylation and ubiquitination, it was necessary to
demonstrate that the removal of sumoylation, not other types
of modification, was responsible for the loss of K-bZIP repres-
sion activity. In fact, we found that wild-type K-bZIP does not
appear to be acetylated in living cells (unpublished data). Fur-
thermore, the expression of SENP1 effectively removed the
sumoylation from K-bZIP and significantly compromised the
ability of K-bZIP to repress K-Rta transactivation, whereas the
expression of enzymatically inactive SENP1 did not do so un-
der the same conditions. These data, taken together, strongly
suggest that sumoylation plays a primary role in K-bZIP-me-
How does sumoylation contribute to the repression activity
of K-bZIP? Sumoylation regulates the translocation of target
molecules to different subcellular compartments, thereby af-
fecting their functions. K-bZIP is localized primarily in nucle-
oplasm but also in PODs, a nuclear structure rich in sumoy-
lated molecules such as PML, SP100, p53, and Daxx (12, 32,
84). Indeed, the sumoylation of PML has been shown to be
crucial for its structural formation (84). When the subcellular
locations of transiently transfected K-bZIP wild type and
K158R mutant were compared, no difference was detected.
This result, however, needs to be interpreted with caution,
because sumoylated K-bZIP only represents 10% of the pop-
ulation, so its location may be masked by the unmodified form,
which appeared as a diffusive pattern in the nucleus. Never-
theless, the result tends to suggest that sumoylation was not
required for K-bZIP localization to POD, an observation rem-
iniscent of p53 and EBV Zta (1, 39). In a recent report, Ad-
amson and Kenney (1) showed that overexpression of EBV
Zta wild type, but not a sumoylation mutant, disrupts POD and
suggests that Zta may compete with PML for limiting quanti-
ties of SUMO peptides, preventing the formation of PODs.
Our work does not directly address this issue. At least at the
overexpression level studied here, K-bZIP did not disrupt the
Alternatively, sumoylation has been shown to affect protein
stability (11, 77), which is a potential contribution to the re-
pression activity of K-bZIP. We examined the possibility by
comparing the half-life of K-bZIP to that of the sumoylation
mutant K158R. No significant difference was observed, indi-
cating that protein stability is not a major contributing factor to
the repression activity of K-bZIP.
A third possible mechanism, whereby SUMO mediates re-
pression, is by virtue of its ability to recruit Ubc9, the E2
SUMO-conjugating enzyme. Ubc9 binds avidly to SUMO moi-
eties and thus can be recruited by sumoylated K-bZIP to the
K-Rta transcriptional complex. We showed that Ubc9 binds
K-bZIP and is recruited to the viral promoter sites, where
K-bZIP and K-Rta assemble. The ability of Ubc9 to catalyze
sumoylation of the proximal transcriptional factors or histone
4 is one mechanism of repression function (29, 46, 53, 60, 65).
In this regard, K-bZIP functions like a SUMO ligase or SUMO
adaptor to bring Ubc9 to its potential substrate. In addition, a
recent report showed that Ubc9 also exhibited conjugation-
independent repression function (37). Our Ubc9 dominant-
negative mutant experiment (Fig. 5B) suggested that the Ubc9
conjugation activity is crucial in mediating K-bZIP repression.
We therefore favor the former hypothesis.
Aside from recruitment of Ubc9, the SUMO peptide itself
may exhibit repression activity by the recruitment of transcrip-
tional repressors (67). As a preliminary test of this notion, we
attached SUMO directly to the N terminus of K-Rta, which
induces sharp reduction of K-Rta transcriptional activity (data
not shown). By contrast, introducing a GST peptide of similar
size as SUMO showed little effect. This finding suggests that
the SUMO moiety itself is a repressor. A recent report indi-
cated that EBV Rta is naturally sumoylated (5). We were not
able to demonstrate sumoylation of KSHV Rta (Fig. 1A),
suggesting that the regulation of K-Rta by sumoylation is via
K-bZIP. Recently, a SUMO-binding motif (V/I-X-V/I-V/I) has
been identified, and this motif is present in HDAC2 and -6
(67). Recruitment of HDAC2 and -6 to a transcriptional factor
would decrease its trans-activation potential and increase re-
pression activity. This is indeed the case for Elk-1 and p300
(19, 82). These transcriptional factors become repressors upon
sumoylation, and the sumoylated forms of Elk-1 and p300
recruit, respectively, HDAC2 (82) and HDAC6 (19). It is thus
conceivable that sumoylated K-bZIP serves as a platform to
recruit a corepressor(s) to K-Rta, resulting in the repression of
FIG. 8. Recruitment of Ubc9 to K-bZIP binding sites. ChIP assay
was performed by using anti-Ubc9 antibody or rabbit normal serum
(control serum). PCR showed recruitment of Ubc9 to K-bZIP-associ-
9922IZUMIYA ET AL. J. VIROL.
K-Rta activity. This hypothesis and the one discussed in the
last paragraph (recruitment of Ubc9) are not mutually exclu-
sive and differ only in details, as both rely on the sumoylation
of K-bZIP to bring in corepressor, be it HDAC or Ubc9.
K-Rta is a central regulator of KSHV lytic replication and is
a potent activator for a large number of KSHV promoters (9,
41, 47, 68, 73, 74). K-Rta acts on these viral promoters by
either direct binding to the enhancer sequences or via the
association with other transcriptional factors, such as RBJ?,
Oct-1, and SP-1 (9, 42, 62). Presumably, depending on the
sequence motif and promoter context, different transcriptional
complexes are assembled. We previously showed that K-bZIP
only targets a subset of the K-Rta complexes for repression. In
addition, K-bZIP represses K-Rta-mediated activation of both
the K-bZIP and the ORF57 promoter but not the nut-1/PAN
RNA promoter (33, 43). Our ChIP data agree with such se-
lectivity: K-bZIP was recruited to the K-bZIP and ORF57
promoters but not the nut-1/PAN RNA promoter. Perhaps,
noncoincidentally, the K-Rta-responsive elements for these
two promoters are very similar (74). The cosmid-based ChIP
approach allows us to quickly scan the entire viral genome for
high-affinity binding sites. The specificity of this approach was
confirmed by the PCRs of hybridized regions. This approach
was useful for mapping of high-affinity binding sites but may
have missed weak binding sites due to the hybridization back-
ground. With this study, we have now used this approach to
map the chromatin binding sites of two herpesvirus bZIP pro-
teins: the Meq of Marek’s disease virus (40) and K-bZIP of
KSHV. This approach can be readily extended to other her-
pesviruses to study the transcriptional and replication factor
assembly sites, as well as the sites of histone modifications,
during lytic and latent phases.
In addition to K-bZIP and ORF57 promoters, K-bZIP was
also recruited to the lytic replication origin, in agreement with
the results of Lin et al. (44) and AuCoin et al. (4). Curiously,
K-bZIP also binds strongly to the general region covered by K1
and ORF27 promoters, raising the issue of whether these are
also target genes of K-bZIP. Our preliminary data showed that
these promoters indeed are modulated by K-bZIP (unpub-
In summary, we showed that K-bZIP was a sumoylated pro-
tein and sumoylation was important for its transcriptional re-
pression activity. K-bZIP also binds Ubc9 E2, SUMO-conju-
gating enzyme, and as such may serve as a viral “SUMO ligase”
to bring Ubc9 to modify the chromatin structure surrounding
the transcriptional complex where K-bZIP resides. We also
mapped the high-affinity binding sites of K-bZIP in the KSHV
genome; these sites include the lytic replication origin and
several viral promoters modulated by K-bZIP. The informa-
tion presented here provides a framework to understand the
complex role of K-bZIP and its posttranslational modification
by sumoylation in KSHV replication.
We thank Ron Hay for providing the pGEXSAE1/SAE2 expression
plasmid and Ren Sun for KSHV cosmid clones.
Y.I. is supported by the California Universitywide AIDS research
program (UARP [F03-D-206]). This work was supported by grants
from UARP and the National Institutes of Health (CA111185 to
1. Adamson, A. L., and S. Kenney. 2001. Epstein-barr virus immediate-early
protein BZLF1 is SUMO-1 modified and disrupts promyelocytic leukemia
bodies. J. Virol. 75:2388–2399.
2. Adamson, A. L., and S. C. Kenney. 1998. Rescue of the Epstein-Barr virus
BZLF1 mutant, Z(S186A), early gene activation defect by the BRLF1 gene
product. Virology 251:187–197.
3. Ahn, J.-H., Y. Xu, W. J. Jang, M. J. Matunis, and G. S. Hayward. 2001.
Evaluation of interactions of human cytomegalovirus immediate-early IE2
regulatory protein with small ubiquitin-like modifiers and their conjugation
enzyme Ubc9. J. Virol. 75:3859–3872.
4. AuCoin, D. P., K. S. Colletti, S. A. Cei, I. Papouskova, M. Tarrant, and G. S.
Pari. 2003. Amplification of the Kaposi’s sarcoma-associated herpesvirus/
human herpesvirus 8 lytic origin of DNA replication is dependent upon a
cis-acting AT-rich region and an ORF50 response element and the trans-
acting factors ORF50 (K-Rta) and K8 (K-bZIP). Virology 318:542–555.
5. Chang, L. K., Y. H. Lee, T. S. Cheng, Y. R. Hong, P. J. Lu, J. J. Wang, W. H.
Wang, C. W. Kuo, S. S. Li, and S. T. Liu. 2004. Post-translational modifica-
tion of Rta of Epstein-Barr virus by SUMO-1. J. Biol. Chem. 279:38803–
6. Chang, P. J., D. Shedd, L. Gradoville, M. S. Cho, L. W. Chen, J. Chang, and
G. Miller. 2002. Open reading frame 50 protein of Kaposi’s sarcoma-asso-
ciated herpesvirus directly activates the viral PAN and K12 genes by binding
to related response elements. J. Virol. 76:3168–3178.
7. Chatterjee, M., J. Osborne, G. Bestetti, Y. Chang, and P. S. Moore. 2002.
Viral IL-6-induced cell proliferation and immune evasion of interferon ac-
tivity. Science 298:1432–1435.
8. Chen, H., R. J. Lin, W. Xie, D. Wilpitz, and R. M. Evans. 1999. Regulation
of hormone-induced histone hyperacetylation and gene activation via acet-
ylation of an acetylase. Cell 98:675–686.
9. Chen, J., K. Ueda, S. Sakakibara, T. Okuno, and K. Yamanishi. 2000.
Transcriptional regulation of the Kaposi’s sarcoma-associated herpesvirus
viral interferon regulatory factor gene. J. Virol. 74:8623–8634.
10. Countryman, J., and G. Miller. 1985. Activation of expression of latent
Epstein-Barr herpesvirus after gene transfer with a small cloned subfragment
of heterogeneous viral DNA. Proc. Natl. Acad. Sci. USA 82:4085–4089.
11. Desterro, J. M., M. S. Rodriguez, and R. T. Hay. 1998. SUMO-1 modification
of IkappaBalpha inhibits NF-kappaB activation. Mol. Cell 2:233–239.
12. Doucas, V., M. Tini, D. A. Egan, and R. M. Evans. 1999. Modulation of
CREB binding protein function by the promyelocytic (PML) oncoprotein
suggests a role for nuclear bodies in hormone signaling. Proc. Natl. Acad.
Sci. USA 96:2627–2632.
13. Dukers, N. H., and G. Rezza. 2003. Human herpesvirus 8 epidemiology: what
we do and do not know. AIDS 17:1717–1730.
14. El-Guindy, A. S., and G. Miller. 2004. Phosphorylation of Epstein-Barr virus
ZEBRA protein at its casein kinase 2 sites mediates its ability to repress
activation of a viral lytic cycle late gene by Rta. J. Virol. 78:7634–7644.
15. Fixman, E. D., G. S. Hayward, and S. D. Hayward. 1992. trans-acting re-
quirements for replication of Epstein-Barr virus ori-Lyt. J. Virol. 66:5030–
16. Flemington, E. K. 2001. Herpesvirus lytic replication and the cell cycle:
arresting new developments. J. Virol. 75:4475–4481.
17. Francis, A., T. Ragoczy, L. Gradoville, L. Heston, A. El-Guindy, Y. Endo, and
G. Miller. 1999. Amino acid substitutions reveal distinct functions of serine
186 of the ZEBRA protein in activation of early lytic cycle genes and synergy
with the Epstein-Barr virus R transactivator. J. Virol. 73:4543–4551.
18. Giot, J. F., I. Mikaelian, M. Buisson, E. Manet, I. Joab, J. C. Nicolas, and A.
Sergeant. 1991. Transcriptional interference between the EBV transcription
factors EB1 and R: both DNA-binding and activation domains of EB1 are
required. Nucleic Acids Res. 19:1251–1258.
19. Girdwood, D., D. Bumpass, O. A. Vaughan, A. Thain, L. A. Anderson, A. W.
Snowden, E. Garcia-Wilson, N. D. Perkins, and R. T. Hay. 2003. P300
transcriptional repression is mediated by SUMO modification. Mol. Cell
20. Gong, L., S. Millas, G. G. Maul, and E. T. Yeh. 2000. Differential regulation
of sentrinized proteins by a novel sentrin-specific protease. J. Biol. Chem.
21. Gong, L., T. Kamitani, K. Fujise, L. S. Caskey, and E. T. Yeh. 1997. Pref-
erential interaction of sentrin with a ubiquitin-conjugating enzyme, Ubc9.
J. Biol. Chem. 272:28198–28201.
22. Gradoville, L., J. Gerlach, E. Grogan, D. Shedd, S. Nikiforow, C. Metroka,
and G. Miller. 2000. Kaposi’s sarcoma-associated herpesvirus open reading
frame 50/Rta protein activates the entire viral lytic cycle in the HH-B2
primary effusion lymphoma cell line. J. Virol. 74:6207–6212.
23. Gravel, A., V. Dion, N. Cloutier, J. Gosselin, and L. Flamand. 2004. Char-
acterization of human herpesvirus 6 variant B immediate-early 1 protein
modifications by small ubiquitin-related modifiers. J. Gen. Virol. 85:1319–
24. Gruffat, H., S. Portes-Sentis, A. Sergeant, and E. Manet. 1999. Kaposi’s
sarcoma-associated herpesvirus (human herpesvirus-8) encodes a homo-
logue of the Epstein-Barr virus bZip protein EB1. J. Gen. Virol. 80:557–561.
VOL. 79, 2005 REPRESSION VIA SUMO MODIFICATION9923
25. Gwack, Y., H. Byun, S. Hwang, C. Lim, and J. Choe. 2001. CREB-binding
protein and histone deacetylase regulate the transcriptional activity of Ka-
posi’s sarcoma-associated herpesvirus open reading frame 50. J. Virol. 75:
26. Gwack, Y., H. J. Baek, H. Nakamura, S. H. Lee, M. Meisterernst, R. G.
Roeder, and J. U. Jung. 2003. Principal role of TRAP/mediator and SWI/
SNF complexes in Kaposi’s sarcoma-associated herpesvirus RTA-mediated
lytic reactivation. Mol. Cell. Biol. 23:2055–2067.
27. Gwack, Y., S. Hwang, C. Lim, Y. S. Won, C. H. Lee, and J. Choe. 2002.
Kaposi’s Sarcoma-associated herpesvirus open reading frame 50 stimulates
the transcriptional activity of STAT3. J. Biol. Chem. 277:6438–6442.
28. Hayward, G. S. 2003. Initiation of angiogenic Kaposi’s sarcoma lesions.
Cancer Cell. 3:1–3.
29. Holmstrom, S., M. E. Van Antwerp, and J. A. Iniguez-Lluhi. 2003. Direct and
distinguishable inhibitory roles for SUMO isoforms in the control of tran-
scriptional synergy. Proc. Natl. Acad. Sci. USA 100:15758–15763.
30. Hwang, S., Y. Gwack, H. Byun, C. Lim, and J. Choe. 2001. The Kaposi’s
sarcoma-associated herpesvirus K8 protein interacts with CREB-binding
protein (CBP) and represses CBP-mediated transcription. J. Virol. 75:9509–
31. Hwang, S., D. Lee, Y. Gwack, H. Min, and J. Choe. 2003. Kaposi’s sarcoma-
associated herpesvirus K8 protein interacts with hSNF5. J. Gen. Virol. 84:
32. Ishov, A. M., A. G. Sotnikov, D. Negorev, O. V. Vladimirova, N. Neff, T.
Kamitani, E. T. Yeh, J. F. Strauss III, and G. G. Maul. 1999. PML is critical
for ND10 formation and recruits the PML-interacting protein daxx to this
nuclear structure when modified by SUMO-1. J. Cell Biol. 147:221–234.
33. Izumiya, Y., S.-F. Lin, T. Ellison, L. Y. Chen, C. Izumiya, P. Luciw, and H.-J.
Kung. 2003. Kaposi’s sarcoma-associated herpesvirus K-bZIP is a coregula-
tor of K-Rta: physical association and promoter-dependent transcriptional
repression. J. Virol. 77:1441–1451.
34. Izumiya, Y., S.-F. Lin, T. J. Ellison, A. M. Levy, G. L. Mayeur, C. Izumiya,
and H.-J. Kung. 2003. Cell cycle regulation by Kaposi’s sarcoma-associated
herpesvirus K-bZIP: direct interaction with cyclin-CDK2 and induction of
G1 growth arrest. J. Virol. 77:9652–9661.
35. Jeong, J., J. Papin, and D. Dittmer. 2001. Differential regulation of the
overlapping Kaposi’s sarcoma-associated herpesvirus vGCR (orf74) and
LANA (orf73) promoters. J. Virol. 75:1798–1807.
36. Katano, H., K. Ogawa-Goto, H. Hasegawa, T. Kurata, and T. Sata. 2000.
Human-herpesvirus-8-encoded K8 protein colocalizes with the promyelo-
cytic leukemia protein (PML) bodies and recruits p53 to the PML bodies.
37. Kobayashi, S., H. Shibata, I. Kurihara, K. Yokota, N. Suda, I. Saito, and T.
Saruta. 2004. Ubc9 interacts with chicken ovalbumin upstream promoter-
transcription factor I and represses receptor-dependent transcription. J.
Mol. Endocrinol. 32:69–86.
38. Krishnan, H. H., P. P. Naranatt, M. S. Smith, L. Zeng, C. Bloomer, and B.
Chandran. 2004. Concurrent expression of latent and a limited number of
lytic genes with immune modulation and antiapoptotic function by Kaposi’s
sarcoma-associated herpesvirus early during infection of primary endothelial
and fibroblast cells and subsequent decline of lytic gene expression. J. Virol.
39. Kwek, S. S., J. Derry, A. L. Tyner, Z. Shen, and A. V. Gudkov. 2001.
Functional analysis and intracellular localization of p53 modified by
SUMO-1. Oncogene 20:2587–2599.
40. Levy, A. M., Y. Izumiya, P. Brunovskis, L. Xia, M. S. Parcells, S. M. Reddy,
L. Lee, H. W. Chen, and H.-J. Kung. 2003. Characterization of the chromo-
somal binding sites and dimerization partners of the viral oncoprotein Meq
in Marek’s disease virus-transformed T cells. J. Virol. 77:12841–12851.
41. Liang, Y., and D. Ganem. 2004. RBP-J (CSL) is essential for activation of the
K14/vGPCR promoter of Kaposi’s sarcoma-associated herpesvirus by the
lytic switch protein RTA. J. Virol. 78:6818–6826.
42. Liang, Y., J. Chang, S. J. Lynch, D. M. Lukac, and D. Ganem. 2002. The lytic
switch protein of KSHV activates gene expression via functional interaction
with RBP-Jkappa (CSL), the target of the Notch signaling pathway. Genes
43. Liao, W., Y. Tang, S.-F. Lin, H.-J. Kung, and C. Z. Giam. 2003. K-bZIP of
Kaposi’s sarcoma-associated herpesvirus/human herpesvirus 8 (KSHV/
HHV-8) binds KSHV/HHV-8 Rta and represses Rta-mediated transactiva-
tion. J. Virol. 77:3809–3815.
44. Lin, C. L., H. Li, Y. Wang, F. X. Zhu, S. Kudchodkar, and Y. Yuan. 2003.
Kaposi’s sarcoma-associated herpesvirus lytic origin (ori-Lyt)-dependent
DNA replication: identification of the ori-Lyt and association of K8 bZip
protein with the origin. J. Virol. 77:5578–5588.
45. Lin, S.-F., D. R. Robinson, G. Miller, and H.-J. Kung. 1999. Kaposi’s sar-
coma-associated herpesvirus encodes a bZIP protein with homology to
BZLF1 of Epstein-Barr virus. J. Virol. 73:1909–1917.
46. Long, J., G. Wang, D. He, and F. Liu. 2004. Repression of Smad4 transcrip-
tional activity by SUMO modification. Biochem. J. 379:23–29.
47. Lukac, D. M., L. Garibyan, J. R. Kirshner, D. Palmeri, and D. Ganem. 2001.
DNA binding by Kaposi’s sarcoma-associated herpesvirus lytic switch pro-
tein is necessary for transcriptional activation of two viral delayed early
promoters. J. Virol. 75:6786–6799.
48. Lukac, D. M., J. R. Kirshner, and D. Ganem. 1999. Transcriptional activa-
tion by the product of open reading frame 50 of Kaposi’s sarcoma-associated
herpesvirus is required for lytic viral reactivation in B cells. J. Virol. 73:9348–
49. Martin, D. F., B. D. Kuppermann, R. A. Wolitz, A. G. Palestine, H. Li, C. A.
Robinson, and the Roche Ganciclovir Study Group. 1999. Oral ganciclovir
for patients with cytomegalovirus retinitis treated with a ganciclovir implant.
N. Engl. J. Med. 340:1063–1070.
50. Mauser, A., E. Holley-Guthrie, A. Zanation, W. Yarborough, W. Kaufmann,
A. Klingelhutz, W. T. Seaman, and S. Kenney. 2002. The Epstein-Barr virus
immediate-early protein BZLF1 induces expression of E2F-1 and other
proteins involved in cell cycle progression in primary keratinocytes and
gastric carcinoma cells. J. Virol. 76:12543–12552.
51. Morrison, T. E., and S. C. Kenney. 2004. BZLF1, an Epstein-Barr virus
immediate-early protein, induces p65 nuclear translocation while inhibiting
p65 transcriptional function. Virology 328:219–232.
52. Morrison, T. E., A. Mauser, A. Klingelhutz, and S. C. Kenney. 2004. Epstein-
Barr virus immediate-early protein BZLF1 inhibits tumor necrosis factor
alpha-induced signaling and apoptosis by downregulating tumor necrosis
factor receptor 1. J. Virol. 78:544–549.
53. Muller, S., M. Berger, F. Lehembre, J. S. Seeler, Y. Haupt, and A. Dejean.
2000. c-Jun and p53 activity is modulated by SUMO-1 modification. J. Biol.
54. Nakamura, H., M. Lu, Y. Gwack, J. Souvlis, S. L. Zeichner, and J. U. Jung.
2003. Global changes in Kaposi’s sarcoma-associated virus gene expression
patterns following expression of a tetracycline-inducible Rta transactivator.
J. Virol. 77:4205–4220.
55. Park, J., T. Seo, S. Hwang, D. Lee, Y. Gwack, and J. Choe. 2000. The K-bZIP
protein from Kaposi’s sarcoma-associated herpesvirus interacts with p53 and
represses its transcriptional activity. J. Virol. 74:11977–11982.
56. Paulose-Murphy, M., N. K. Ha, C. Xiang, Y. Chen, L. Gillim, R. Yarchoan,
P. Meltzer, M. Bittner, J. Trent, and S. Zeichner. 2001. Transcription pro-
gram of human herpesvirus 8 (kaposi’s sarcoma-associated herpesvirus).
J. Virol. 75:4843–4853.
57. Polson, A. G., L. Huang, D. M. Lukac, J. D. Blethrow, D. O. Morgan, A. L.
Burlingame, and D. Ganem. 2001. Kaposi’s sarcoma-associated herpesvirus
K-bZIP protein is phosphorylated by cyclin-dependent kinases. J. Virol.
58. Quinlivan, E. B., E. A. Holley-Guthrie, M. Norris, D. Gutsch, S. L. Bachen-
heimer, and S. C. Kenney. 1993. Direct BRLF1 binding is required for
cooperative BZLF1/BRLF1 activation of the Epstein-Barr virus early pro-
moter, BMRF1. Nucleic Acids Res. 21:1999–2007.
59. Ragoczy, T., and G. Miller. 1999. Role of the epstein-barr virus RTA protein
in activation of distinct classes of viral lytic cycle genes. J. Virol. 73:9858–
60. Ross, S., J. L. Best, L. I. Zon, and G. Gill. 2002. SUMO-1 modification
represses Sp3 transcriptional activation and modulates its subnuclear local-
ization. Mol. Cell 10:831–842.
61. Russo, J. J., R. A. Bohenzky, M. C. Chien, J. Chen, M. Yan, D. Maddalena,
J. P. Parry, D. Peruzzi, I. S. Edelman, Y. Chang, and P. S. Moore. 1996.
Nucleotide sequence of the Kaposi sarcoma-associated herpesvirus (HHV8).
Proc. Natl. Acad. Sci. USA 93:14862–14867.
62. Sakakibara, S., K. Ueda, J. Chen, T. Okuno, and K. Yamanishi. 2001.
Octamer-binding sequence is a key element for the autoregulation of Kapo-
si’s sarcoma-associated herpesvirus ORF50/Lyta gene expression. J. Virol.
63. Sarisky, R. T., Z. Gao, P. M. Lieberman, E. D. Fixman, G. S. Hayward, and
S. D. Hayward. 1996. A replication function associated with the activation
domain of the Epstein-Barr virus Zta transactivator. J. Virol. 70:8340–8347.
64. Seeler, J. S., and A. Dejean. 2003. Nuclear and unclear functions of SUMO.
Nat. Rev. Mol. Cell Biol. 4:690–699.
65. Shiio, Y., and R. N. Eisenman. 2004. Histone sumoylation is associated with
transcriptional repression. Proc. Natl. Acad. Sci. USA 100:13225–13230.
66. Sista, N. D., C. Barry, K. Sampson, and J. Pagano. 1995. Physical and
functional interaction of the Epstein-Barr virus BZLF1 transactivator with
the retinoic acid receptors RAR alpha and RXR alpha. Nucleic Acids Res.
67. Song, J., L. K. Durrin, T. A. Wilkinson, T. G. Krontiris, and Y. Chen. 2004.
Identification of a SUMO-binding motif that recognizes SUMO-modified
proteins. Proc. Natl. Acad. Sci. USA 101:14373–14378.
68. Song, M. J., H. J. Brown, T.-T. Wu, and R. Sun. 2001. Transcription activa-
tion of polyadenylated nuclear RNA by Rta in human herpesvirus 8/Kaposi’s
sarcoma-associated herpesvirus. J. Virol. 75:3129–3140.
69. Song, M. J., X. Li, H. J. Brown, and R. Sun. 2002. Characterization of
interactions between RTA and the promoter of polyadenylated nuclear
RNA in Kaposi’s sarcoma-associated herpesvirus/human herpesvirus 8.
J. Virol. 76:5000–5013.
70. Sun, R., S.-F. Lin, K. Staskus, L. Gradoville, E. Grogan, A. Haase, and G.
Miller. 1999. Kinetics of Kaposi’s sarcoma-associated herpesvirus gene ex-
pression. J. Virol. 73:2232–2242.
9924IZUMIYA ET AL. J. VIROL.
71. Sun, R., S.-F. Lin, L. Gradoville, Y. Yuan, F. Zhu, and G. Miller. 1998. A Download full-text
viral gene that activates lytic cycle expression of Kaposi’s sarcoma-associated
herpesvirus. Proc. Natl. Acad. Sci. USA 95:10866–10871.
72. Tatham, M. H., E. Jaffray, Q. A. Vaughan, J. M. Desterro, C. H. Botting,
J. H. Naismith, and R. T. Hay. 2001. Polymeric chains of SUMO-2 and
SUMO-3 are conjugated to protein substrates by SAE1/SAE2 and Ubc9.
J. Biol. Chem. 276:35368–35374.
73. Ueda, K., K. Ishikawa, K. Nishimura, S. Sakakibara, E. Do, and K. Yaman-
ishi. 2002. Kaposi’s sarcoma-associated herpesvirus (human herpesvirus 8)
replication and transcription factor activates the K9 (vIRF) gene through
two distinct cis elements by a non-DNA-binding mechanism. J. Virol. 76:
74. Wang, S., S. Liu, M. Wu, Y. Geng, and C. Wood. 2001. Kaposi’s sarcoma-
associated herpesvirus/human herpesvirus-8 ORF50 gene product contains a
potent C-terminal activation domain which activates gene expression via a
specific target sequence. Arch. Virol. 146:1415–1426.
75. Wang, S., S. Liu, M. H. Wu, Y. Geng, and C. Wood. 2001. Identification of a
cellular protein that interacts and synergizes with the RTA (ORF50) protein
of Kaposi’s sarcoma-associated herpesvirus in transcriptional activation.
J. Virol. 75:11961–11973.
76. Wang, S. E., F. Y. Wu, H. Chen, M. Shamay, Q. Zheng, and G. S. Hayward.
2004. Early activation of the Kaposi’s sarcoma-associated herpesvirus RTA,
RAP, and MTA promoters by the tetradecanoyl phorbol acetate-induced
AP1 pathway. J. Virol. 78:4248–4267.
77. Weger, S., E. Hammer, and R. Heilbronn. 2004. SUMO-1 modification
regulates the protein stability of the large regulatory protein Rep78 of adeno
associated virus type 2 (AAV-2). Virology 330:284–294.
78. Wu, F. Y., H. Chen, S. E. Wang, C. M. ApRhys, G. Liao, M. Fujimuro, C. J.
Farrell, J. Huang, S. D. Hayward, and G. S. Hayward. 2003. CCAAT/
enhancer binding protein alpha interacts with ZTA and mediates ZTA-
induced p21(CIP-1) accumulation and G1cell cycle arrest during the Ep-
stein-Barr virus lytic cycle. J. Virol. 77:1481–1500.
79. Wu, F. Y., J.-H. Ahn, D. J. Alecendor, W.-J. Jang, J. Xiao, S. D. Hayward,
and G. S. Hayward. 2001. Origin-independent assembly of Kaposi’s sarcoma-
associated herpesvirus DNA replication compartments in transient cotrans-
fection assays and association with the ORF-K8 protein and cellular PML.
J. Virol. 75:1487–1506.
80. Wu, F. Y., Q. Q. Tang, H. Chen, C. ApRhys, C. Farrell, J. Chen, M. Fu-
jimuro, M. D. Lane, and G. S. Hayward. 2002. Lytic replication-associated
protein (RAP) encoded by Kaposi sarcoma-associated herpesvirus causes
p21CIP-1-mediated G1 cell cycle arrest through CCAAT/enhancer-binding
protein-alpha. Proc. Natl. Acad. Sci. USA 99:10683–10688.
81. Xu, Y., J.-H. Ahn, M. Cheng, C. M. Rhys, C. J. Chiou, J. Zong, M. J.
Matunis, and G. S. Hayward. 2001. Proteasome-independent disruption of
PML oncogenic domains (PODs), but not covalent modification by
SUMO-1, is required for human cytomegalovirus immediate-early protein
IE1 to inhibit PML-mediated transcriptional repression. J. Virol. 75:10683–
82. Yang, S. H., and A. D. Sharrocks. 2004. SUMO promotes HDAC-mediated
transcriptional repression. Mol. Cell 13:611–617.
83. Zhang, Q., D. Gutsch, and S. Kenney. 1994. Functional and physical inter-
action between p53 and BZLF1: implications for Epstein-Barr virus latency.
Mol. Cell. Biol. 14:1929–1938.
84. Zhong, S., S. Muller, S. Ronchetti, P. S. Freemont, A. Dejean, and P. P.
Pandolfi. 2000. Role of SUMO-1-modified PML in nuclear body formation.
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