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Cellular Signalling 17

Herpes virus proteins ICP0 and BICP0 can activate NF-nB by catalyzing

InBa ubiquitination

Lirong Diaoa,b, Bianhong Zhanga, Junkai Fana, Xiang Gaoa, Shaogang Suna, Kai Yanga,

Dan Xinb, Naihe Jina, Yunqi Gengb, Chen Wanga,*

aLaboratory of Molecular and Cellular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences,

Chinese Academy of Sciences, Box 49, 320 Yue Yang Road, Shanghai 200031, P.R. ChinabCollege of Life Science, Nan Kai University, Tianjin 300071, P.R. China

Received 4 July 2004; received in revised form 12 July 2004; accepted 12 July 2004

Available online 25 August 2004

Abstract

The immediate early proteins ICP0 and BICP0 from Herpes virus are promiscuous activators of both viral and cellular genes and play a

critical role in virus life cycle. Here we report that ICP0 and BICP0 could induce NF-nB translocation from cytoplasm into nucleus and

strongly activate NF-nB responsive genes specifically. This process was dependent on the RING domain of both proteins. In addition, ICP0

interacted specifically with InBa and its activating effect was attenuated by Ubch5A(C85A) and MG132, but not by InBa(S32A/S36A).Remarkably, InBa was poly-ubiquitinated by both ICP0 and BICP0, in vitro and in vivo. These data indicate that ICP0 and BICP0,

functioning as ubiquitin ligases, are bona fide activators of NF-nB signaling pathway. Our study identifies a new way ICP0 and BICP0

explore to regulate gene expression.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Herpes virus; ICP0; BICP0; Ubiquitin; InBa

1. Introduction

Herpes simplex viruses (HSV) are nuclearly replicating,

icosahedral, enveloped DNA viruses. They are the culprits

of cold sores and other more serious diseases [1]. HSV-1 has

developed a successful strategy to establish a life-long latent

infection in the nervous system after initial entry into the

epithelial cells [2]. In response to stress, it can reactivate

into lytic cycle and express the immediate-early genes such

as ICP0, ICP4, ICP27, which then turn on the early and late

genes [3]. Remarkably, HSV-1 functional counterpart in

bovine has been characterized as bovine herpes virus 1

(BHV-1) that shares a number of biological properties with

HSV-1 in terms of life cycle and protein functions [4].

0898-6568/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.cellsig.2004.07.003

* Corresponding author. Tel.: +86 21 54921185.

E-mail addresses: yqgeng@public.tpt.tj.cn (Y. Geng)8

cwang01@sibs.ac.cn (C. Wang).

ICP0 is a 775-amino-acid multifunctional protein that is

expressed immediately after HSV-1 infection or reactivation

[5]. It is critical for the efficient initiation of lytic infection

and sufficient to induce HSV-1 reactivation from neuronal

latency [6]. The underlying mechanisms have been under

intensive study. Initially, ICP0 was found to promote both

viral and cellular protein production by stimulating mRNA

synthesis [7]. However, it failed to identify signature

promoter sequence recognized by ICP0; nor was it able to

demonstrate any affinity between ICP0 and DNA, which

suggested that the mode of ICP0 action was not a simple

case of direct transcriptional activation [5]. This conjecture

was partly substantiated by the observation that ICP0 co-

localized with the nuclear substructures called ND10 and

mediated the disruption of them [8–10]. Recently, ICP0 was

shown to interact specifically with a diverse collection of

proteins including USP7, cyclin D3, elongation factor EF-

1y, transcription factor BMAL1 and HSV-1 ICP4 [11–15].

Importantly, ICP0 contains a RING domain at its N-

(2005) 217–229

L. Diao et al. / Cellular Signalling 17 (2005) 217–229218

terminus and displays intrinsic ubiquitin ligase activity [16–

19]. In parallel, BICP0 is bovine homologue of ICP0 that

harbors a RING domain at its N-terminus and exhibits

ubiquitin ligase activity too (see data below).

Ubiquitin (Ub) is a 76-amino-acid globular protein that

plays an important role in regulating many aspects of

cellular processes, including NF-nB signaling pathway [20].

Conjugation of Ub onto other proteins requires the

sequential actions of three enzymes: Ub is first activated

in an ATP-dependent way by Ub activating enzyme (E1)

and then transferred to a Cys residue in the Ub-conjugating

enzyme (E2). With the help of Ub ligase (E3), Ub is

attached via its C-terminus to a q-amino group of the Lys

residues in the substrate proteins. Since Ub contains seven

lysines itself, poly-Ub chain can be formed by covalently

attaching one Ub to another Ub in the same way [21]. In

general, proteins with RING domain are members of the E3

family that are responsible for catalyzing ubiquitination of

target proteins [22]. Classically, Ub was regarded as a

bdeath-tagQ to target proteins for degradation by 26S

proteasome. However, new mechanisms of Ub actions have

been emerged and characterized [23,24].

Found in essentially all mammalian cell types, the

transcription factor NF-nB(p65/p50) regulates a wide rangeof genes important in inflammation, immunity, development

and apoptosis. It is normally sequestered in the cytoplasm

by virtue of its association with inhibitor InBa. A myriad of

stimuli can lead to the activation of InB kinase complex

(IKK) and subsequently phosphorylation of the serine

residues 32 and 36 in InBa. The phospho-InBa is then

ubiquitinated by a specific E3 called SCFh-TrCP and

degraded by the 26S proteasome. This releases NF-nBfrom its anchor, which then translocates into nucleus and

activates target gene expressions [25–29]. Interestingly,

ubiquitination was also used to regulate NF-nB signaling

pathway in other distinct ways [30].

Although it was known that manipulating NF-nB signal-

ing pathway could promote viral replication, host cell

survival or evasion of the immune response, different

viruses employed different mechanisms to do so [31].

Previously, it was demonstrated that ICP0 from HSV-1

could trans-activate the LTR promoter of HIV [32,33].

However, it remains unknown how ICP0 activates the LTR

promoter. Accidentally, we observed that stimulation by

BICP0 of the bovine immunodeficiency virus 1 (BIV-1)

enhancer LTR was severely impaired when a nB-consensusmotif within the LTR was deleted. In light of the facts that

ICP0 and BICP0 are promiscuous activator of gene

transcription, we hypothesized that ICP0 and BICP0 played

a key role in NF-nB activation. In this study, we

demonstrated that ICP0 and BICP0 could specifically

stimulate NF-nB responsive reporter gene expression in

different cell lines and this effect could be attenuated by

Ubch5A(C85A) and MG132. In addition, ICP0 and BICP0

could induce p65-NF-nB to translocate from cytoplasm into

nucleus and promote NF-nB DNA binding affinity.

Remarkably, ICP0 interacted with InBa and stimulated

InBa poly-ubiquitination in vitro and in vivo. However,

this process was independent of InBa phosphorylation. Our

data indicate that ICP0 and BICP0 are bona fide activators

of NF-nB signaling pathway and they function as ubiquitin

ligases to directly catalyze InBa ubiquitination.

2. Experimental procedure

2.1. Plasmids and reagents

cDNA constructs for ICP0, BICP0 and BICP0(13G/51A)

were gifts from Dr. Yange Zhang of University of Nebraska-

Lincoln. These cDNAs were subcloned into pDNA3.1-N-

Flag(a gift from Dr. Hai Wu in UT Southwestern Medical

Center). These constructs were expressed in mammalian

cells for functional analysis and immunoprecipitated with

anti-flag beads for in vitro assays. Truncating mutants of

ICP0 and BICP0 were made either by PCR or directly by

cutting out the corresponding segments and cloned into

pDNA3.1-N-Flag. The mutants covered one segment of the

full-length proteins: 1–234 aa for ICP0(exon1+2), 518–775

aa for ICP0(MC), 555–775 aa for ICP0(AC), 1–357 aa for

BICP0(N), 357–676 aa for BICP0(C). pcDNA3-HA-InBaand the truncating mutants were made by PCR of the

corresponding segments of human InBa and cloning them

into pcDNA3-HA plasmid (kindly provided by Professor

Gang Pei). GST-InBa was prepared by subcloning InBainto pGEX-4T-1 (Promega) and purified from E. coli by

Pharmacia kit. Radioactive InBa and the mutants were in

vitro translated using Promega TNT wheat germ system.

GST-ATF was kindly provided by Dr. Meng Li. All these

constructs were confirmed by automatic DNA sequencing.

The rest of the plasmids and proteins including the ubiquitin

system were prepared as described previously (Deng et.al,

2000) [24]. All antibodies were from Santa Cruz Biotech-

nology if not specified otherwise. From Aldrich-Sigma were

purchased anti-Flag bead, protein-A bead, anti-Ub mono-

clonal antibody and MG132, aprotinin, chymostatin, leu-

peptin. Radioactive isotopes were from Amersham

Biotechnology. The mouse polyclonal antibodies against

ICP0 and BICP0 were made by subcutaneously injecting 6-

week Kunming mice with the purified his-ICP0(C) and his-

BICP0 protein expressed from E. coli BL21. Both anti-

bodies recognized only their immunogens specifically.

2.2. Cell culture, transfection, and reporter gene assays

293T and SH-SY5Y cells were from ATCC, USA. HeLa

cells were kindly provided by professor Lin Li. These cells

were cultured in DMEM media supplemented with 10%

fetal bovine serum (FBS), penicillin (50 U/ml) and

streptomycin (50 Ag/ml). Cells (2.0�105 and 1.5�106)

were seeded in six-well plates for luciferase assay and 10-

cm plates for other experiments, respectively. Transfection

L. Diao et al. / Cellular Signalling 17 (2005) 217–229 219

with the indicated plasmids was carried out by either

calcium phosphate precipitation method [24] or lipofect-

amine 2000 (Invitrogen cat. no. 11668-027) according to the

manufacturer’s instructions. Cells were harvested 24–48 h

after transfection as indicated. For some experiments, cells

were treated with the indicated reagents 16 or 24 h after

transfection. For reporter gene assay, cells were harvested

48 h after transfection and gene activity was measured using

the Luciferase Assay System (Promega). Data were nor-

malized in reference to a GFP internal control. Each datum

was from a representative experiment reproducibly repeated

at least three times. The cell lysates for luciferase assay were

all checked with Western blot to ensure protein expression

of the various constructs. A plasmid expressing LacZ was

included in all transfection experiments as negative control.

2.3. Immunofluorescence and confocal microscopy

293T cells were seeded on chamber slides in 35-mm

plate and transiently transfected with 1 Ag of the

designated plasmids. After 16 h, cells were fixed at room

temperature with 4% (w/v) paraformaldehyde–PBS for 20

min and then permeabilized at room temperature by 0.5%

Triton X-100–PBS for 10 min. The cells were then

incubated at 37 8C with mouse anti-NF-nB p65(F-6)

monoclonal antibody for 1 h and then at 4 8C for 16 h.

After washing slides with 0.05% Triton X-100–PBS, the

cells were incubated with FITC conjugated anti-mouse

immunoglobulin G (IgG) for 1 h at 37 8C. The cells were

then co-stained with DAPI. The pictures were taken by

Bio-Rad confocal microscopy system Radiance 2100 with

Lasersharp 2000 software.

2.4. Preparation of soluble cell extracts

Infected or mock infected cells were rinsed in PBS twice,

harvested, pelleted by centrifugation, and solubilized at 4 8Cin mammalian cell lysis buffer [0.5% Nonidet P-40/20 mM

Tris, pH 7.5/20 mM h-glycerol phosphate/10 mM NaF/0.5

mM Na3VO4/150 mM NaCl/1 mM DTT/1 mM PMSF/0.2

mM EGTA (pH 7.0)/5 Ag/ml pepstatin, leupetin and

chymostatin]. Fifteen minutes later, lysates were clarified

by centrifugation at 4 8C, 10,000 rpm for 30 min. The

supernatant was used in immunoblotting, immunoprecipita-

tion and pulldown experiments. For electrophoretic mobility

shift assay (EMSA) experimets, cells were resuspended in

CE buffer [0.1% Triton X-100/10 mM HEPES (pH 7.9)/5

mM MgCl2/10 mM KCl/1 mM DTT/1 mM PMSF],

incubated on ice for 15 min and pelleted by centrifugation

at 4 8C, 3500 rpm for 10 min. The supernatant represented

the cytoplasmic extract (CE). The pellet was further

resuspended in NE buffer [20 mM HEPES (pH 7.9)/25%

glycerol/0.42 M NaCl/1.5 mMMgCl2/0.2 mM EDTA/1 mM

DTT/1 mM PMSF], incubated on ice for 20 min and

centrifuged at 4 8C, 14,000 rpm for 10 min. The supernatant

represented the nuclear extract (NE). Protein expression was

checked by Western blot. Both cytoplasmic and nuclear

extracts were stored at �70 8C.

2.5. Electrophoretic mobility shift assay (EMSA)

NE extracts were assayed for NF-nB binding to its

cognate nB site exactly according to manufacturer’s

instruction (Promega cat. no. E3050). Briefly, NE extracts

containing equal amounts of whole proteins were incu-

bated with 32P-labeled nB oligonucleotide (AGTT-

GAGGGGACTTTCCCAGGC) in buffer containing 4%

glycerol, 1 mM MgCI2, 50 mM NaCl, 0.5 mM EDTA, 0.5

mM DTT, 10 mM Tris–HCl (pH 7.5), and 0.05 mg/ml

poly (dI–dC)d (dI–dC). Sp1 oligonucleotide was used as

control. After incubating at 4 8C for 30 min, aliquots were

fractionated at 4 8C on 0.5� TBE, 4% native polyacry-

lamide gels. Gels were dried and exposed at �70 8C to

XAR film with intensifying screens. For the competitive

assay, 100-fold excessive amount of cold nB oligodeox-

ynucleotide was added into the binding reaction. For super

shift assay, 0.2 Ag of indicated antibodies was incubated

with other components of the binding reaction as was done

in EMSA.

2.6. Coimmunoprecipitation and GST pulldown

Endogenous InBa and IKK were immunoprecipitated

with their corresponding antibody coupled onto protein A

beads. Flag-tagged proteins were immunoprecipitated with

anti-Flag beads (Sigma F-2426) directly. Soluble cell lysates

containing equal amounts of whole proteins were incubated

on a rotor with the indicated beads at 4 8C for 2–4 h. The

beads were washed extensively three to four times with cell

lysis buffer and once with TBS. The beads were added with

SDS-PAGE loading buffer and resolved by Western blot

using indicated antibodies. Or the beads were used in the

subsequent reactions. For GST pulldown, reaction products

were incubated with Glutathione Sepharose 4B (Pharmacia

Biotech code no. 17-0756-01) in TBS and 1% NP-40 for 2 h

and the Sepharose beads were washed thoroughly with TBS

and 1% NP-40 three to four times. The protein was eluted

by adding sample buffer, subsequently run on 9% SDS-

PAGE gel and visualized by immunoblotting with anti-Ub

antibody.

2.7. In vitro InBa polyubiquitination assay

In vitro translated 35S-InBa or GST-InBa or control

proteins were incubated with purified ubiquitin (0.1 mM),

E1 (0.1 mM), Ubch5A (0.2 mM), and E3 (ICP0, BICP0 or

control proteins) in the reaction buffer containing 50 mM

Tris, pH 7.5, 5 mM MgCl2, 2 mM ATP, 0.1 AM Ubal. The

mixtures were thoroughly mixed and allowed to react at 37

8C for 90 min. For 35S-InBa as substrate, the reaction

products were loaded onto 9% SDS-PAGE gel and auto-

radiograph was obtained. For GST-InBa as substrate, the

L. Diao et al. / Cellular Signalling 17 (2005) 217–229220

reaction products were processed with GST pulldown and

subjected to immunoblotting with anti-Ub antibody.

2.8. In vitro IKK kinase assay

To monitor endogenous IKK activity, IKK complex was

immunoprecipitated by anti-IKK protein A beads from cell

extracts transfected for 24 h with LacZ, ICP0, BICP0 or

TRAF6. The beads were then incubated with GST-InBa and

0.5 ACi g-32P-ATP together with a kinase buffer containing

50 mM Tris–HCl, pH 7.5, 5 mM MgCl2, 50 AM ATP. After

incubating at 30 8C for 1 h, the reaction products were

resolved by SDS-PAGE and autoradiograph was obtained.

3. Results

3.1. ICP0 and BICP0 stimulate NF-nB-dependent tran-

scription in vivo

Initially, we set out to characterize BIV-1 LTR enhancer

that is inducible by BICP0. It came into our attention that

this induction was significantly impaired when a short

stretch of the LTR was deleted, in which a potential nBsequence was identified (Fig. 1). This led us to speculate

that HSV-1 ICP0 and BHV BICP0 stimulated gene

transcription via promoting NF-nB activity. To explore

this hypothesis, we transfected a construct encoding ICP0

into human neuroblastoma SH-SY5Y cell together with a

nB-Luc reporter gene that expresses luciferase under the

control of three tandem repeats of NF-nB binding sites

(Fig. 2a). Furthermore, a plasmid was included as a

negative control to express LacZ in parallel experiments. A

strong stimulation of the nB-Luc was observed when ICP0

was expressed. The folds of stimulation were in direct

proportion to the amount of ICP0 expressed (Fig. 2b). In

contrast, no stimulation was detected when LacZ was

expressed (data not shown). Likewise, we transfected

another construct encoding BICP0 and found that BICP0

Fig. 1. Activation of BIV-LTR by BICP0 is dependent on a potential nB site on the

the right side with the deleted regions marked. BICP0 was co-transfected into 29

these constructs by BICP0 is represented at the left side as fold increase of luciferas

nB site. TAR: Tat-associated region.

stimulated NF-nB-dependent transcription in SY5Y cell in

a similar, but more potent, way (Fig. 2b). Since HSV-1

infects other cell lineages besides neuron, we undertook

the same luciferase assay experiments to check whether

ICP0 and BICP0 could still do so in 293T and HeLa cells.

As was shown in Fig. 2c and d, NF-nB-dependenttranscriptions were indeed promoted by ICP0 and BICP0,

suggesting that this activation induced by ICP0 and BICP0

was independent of cell lines although HSV-1 infection

exhibited cell line dependence.

Previously, ICP0 was proposed to activate gene tran-

scriptions by interfering with ND10 substructure. To rule

out the possibility that NF-nB activation by ICP0 was due to

indiscriminate potentiation of transcription factors in the

nucleus, we transfected LEF-Luc, Gal4-Luc, or nB-Lucreporter genes individually into 293T cell together with

ICP0. As was expected, nB-Luc transcription was markedly

increased by ICP0. However, LEF-Luc and Gal4-Luc gene

transcriptions remained constant in the presence or absence

of ICP0. In contrast, LEF-Luc was activated by its cognate

activator (DN)-h-catenin. The same was true for Gal4-Luc

in response to its cognate activator Gal4-VP16. These

results indicated that ICP0 specifically activated NF-nB-dependent gene transcription (Fig. 3a).

It was reported that the N-terminal part of ICP0

contained a RING domain characteristic of ubiquitin E3.

Likewise, the RING domain is also conserved in BICP0, of

which the cysteine residues at position 13 and 51 were

found to be critical in substrate-independent poly-ubiquiti-

nation reaction (Fig. 3d). To test whether the RING domain

is essential for activating NF-nB, BICP0 or BICP0(13G/

51A) constructs were transfected into 293T cell along with

nB-Luc reporter gene. As was shown in Fig. 3c,

BICP0(13G/51A) achieved little stimulation above back-

ground while BICP0 could induce robust activation of NF-

nB, implicating that the RING domain was absolutely

necessary for NF-nB activation and ubiquitin was possibly

involved in the process. We also tested truncations of ICP0

and BICP0 to explore whether ubiquitin ligase activity alone

enhancer. Diagrams of the BIV-LTR and its truncated mutants are shown at

3T cell with BIV-LTR or its truncated mutants, respectively. Stimulation of

e readout. The solid black bar is used to highlight the existence of a potential

Fig. 2. Both ICP0 and BICP0 can activate NF-nB responsive reporter gene in different cells. (a) Schematic representation of nB-Luc luciferase reporter geneused in this study. Note that the nB sequence of this construct is an authentic one that is slightly different from the potential nB site identified in Fig. 1. ICP0 or

BICP0 was co-transfected into cell along with this reporter gene for 48 h. Shown in (b) was fold inductions of the nB-Luc expression by gradient amount of

ICP0 or BICP0 in Neuroblastoma SH-SY5Y cells. The same luciferase assay experiments were carried out in (c) HeLa cells and (d) 293T cells. Bottom panels:

the gradient expression of ICP0 or BICP0 in 293T cells.

L. Diao et al. / Cellular Signalling 17 (2005) 217–229 221

was sufficient to activate NF-nB. It turned out that either

ICP0(N) or ICP0(C) alone could not activate NF-nB-dependent transcription (Fig. 3b), nor did the BICP0(C).

Although BICP0(N) displayed some stimulation on NF-nB,this was due to the fact that BICP0(N) covered not only the

RING domain, but also a much longer stretch of peptide

located C-terminal to the RING domain as compared to the

ICP0(N), which might mediate protein–protein interactions.

Taken together, these results established that both ICP0 and

BICP0 could specifically activate NF-nB-dependent tran-scription in different cell lines, which required the RING

domain.

Fig. 3. The RING domains of both ICP0 and BICP0 are important for their selective activation of NF-nB. (a) ICP0 activates nB-Luc, but not LEF-Luc or Gal4-Luc reporter genes. ICP0 or control plasmids were co-transfected into 293T cell along with the indicated reporter genes. (DN)-h-catenin and Gal4-VP16 are thecognate activators of LEF-Luc and Gal4-Luc, respectively. (b) nB-Luc activation induced by truncations of ICP0. ICP0(exon1+2): N-terminus of ICP0

harboring the RING domain; ICP0(MC) and ICP0(AC): C-terminal parts of ICP0 that do not contain the RING domain. (c) nB-Luc activation induced by

mutants of BICP0. BICP0(N): N-terminal half of BICP0; BICP0(C): C-terminal half of BICP0; BICP0(13G/51A): C13G and C51A point mutation in the

RING domain. (d) BICP0 can catalyze poly-ubiquitin chain formation. The reaction mixture contained Ubiquitin, E1, Ubch5A and ATP besides BICP0 or

BICP0(13G/51A) as indicated. Poly-Ub chains were detected by anti-Ub antibody.

L. Diao et al. / Cellular Signalling 17 (2005) 217–229222

3.2. ICP0 and BICP0 induce nuclear translocation of p65-

NF-nB and enhance its binding affinity to nB site

Normally, NF-nB is sequestered within cytoplasm and

translocates into nucleus upon activation [26]. To deter-

mine if ICP0 also exploited this mechanism to activate

NF-nB, we investigated whether it could influence the

subcellular distribution of NF-nB. So ICP0, BICP0,

BICP0(13G/51A) and LacZ were transfected into 293T

cell, respectively. After 16 h, the cells were immunos-

tained in accordance with the manufacturers’ recommen-

dations and imaged by Bio-Rad confocal fluorescent

microscope. The distribution of endogenous p65-NF-nBwas revealed by anti-p65 antibody in combination with

FITC-conjugated secondary antibody. Obviously, p65 was

distributed diffusely at the circumference of the cell when

LacZ was transfected, which reflected the stationary state

of NF-nB. In contrast, ICP0 and BICP0 induced p65 to

concentrate in the center of the cell, which was superposed

neatly with the nucleus as revealed by DAPI staining to

the nucleus. This indicated that both ICP0 and BICP0

could drive translocation of p65-NF-nB from cytoplasm

into nucleus. In addition, BICP0(13G/51A) failed to cause

any change to the subcellular distribution of p65, which

again suggested that the RING domain was indispensable

(Fig. 4a).

Another consequence of NF-nB activation is the specific

targeting of NF-nB to its cognate nB sites. To verify that

ICP0 induced the same effect, nuclear extracts were

prepared from 293T cell transfected with either ICP0 or

LacZ for different lengths of time and then incubated with

radioactive synthetic nB oligodeoxynucleotide. Gel shift

assay indicated that a new activity was elicited in the

nucleus in response to ICP0 that could bind tightly to the nB

Fig. 4. ICP0 and BICP0 induce nuclear translocation of NF-nB-p65 and enhance its binding affinity to nB site. (a) ICP0 and BICP0 induce p65-NF-nB to

translocate from cytoplasm into nucleus. ICP0 or control plasmids were transfected into 293T cell. After 16 h, the cells were immunostained and imaged as

described in Section 2. Green: p65; dark blue: nucleus; white: p65 in nucleus; pink: p65 not inside nucleus. (b) ICP0 induces an activity that can bind to

radioactive nB oligonucleotides as revealed by electrophoresis mobility shift assay (EMSA). (c) The EMSA band can be competed away by 100-fold excess of

cold nB oligonucleotides. (d) Addition of anti-p65 antibody to the EMSA reaction mixture can cause a supershift band relative to the normal shift band.

L. Diao et al. / Cellular Signalling 17 (2005) 217–229 223

L. Diao et al. / Cellular Signalling 17 (2005) 217–229224

probe, while LacZ was unable to do so (Fig. 4b).

Furthermore, this activity did not bind to radioactive

synthetic sp1 oligodeoxynucleotide (Data not shown). More

experiments showed that BICP0 could also induce this

activity, while BICP0(13G/51A) was deprived of this

function (Fig. 4c). In order to confirm that the new activity

targeted only the nB oligodeoxynucleotide, 100-fold exces-

sive amount of cold nB probe was added into the binding

reaction. As a result, the cold nB probe competed away all

the ICP0 or BICP0 responsive proteins and led to the

complete diminishing of gel shift bands representing protein

and DNA interaction, demonstrating that the activity

induced by ICP0 or BICP0 recognized nB sequence

specifically (Fig. 4c). Finally, the identity of the induced

activity was confirmed by supershift EMSA. When antibody

specific to h-actin was incubated with nB probe and nuclear

extracts transfected with ICP0 or BICP0, the resulting gel

shift bands ran at the same position as those of the binding

reaction products without adding any antibody. In contrast,

when antibody specific to p65-NF-nB was introduced, the

resulting gel shift bands apparently ran more slowly than

those of the control (Fig. 4d). These proved that both ICP0

and BICP0 were able to cause NF-nB binding specifically to

its cognate nB promoter.

3.3. ICP0 interacts with InBa and its stimulatory effect is

attenuated by Ubch5A(C85A) and MG132

So far, it was well established that both ICP0 and BICP0

had the ability to stimulate NF-nB. We therefore went on to

investigate how they activated this signaling pathway. At

first, we found that ICP0 could not stimulate IKK activity as

revealed by IKK kinase assay (Fig. 5a). It was known that

when Serine 32 and 36 of InBa were all mutated into

alanine, this mutant InBa(S32/36A) became a super-

repressor that could block NF-nB activation by many NF-

nB specific stimuli. For example, TRAF6, an upstream

activator of IKK, could activate NF-nB and this activation

was severely inhibited by InBa(S32/36A). Consequently,we explored whether InBa(S32/36A) had the same effect on

NF-nB activation mediated by ICP0. When ICP0 and nB-Luc were transfected into 293T cell with or without

InBa(S32/36A), NF-nB activation by ICP0 was not affected

at all even in the presence of a large amount of InBa(S32/36A), which again suggested that InBa phosphorylation

was not essential for ICP0-mediated NF-nB activation and

ICP0 acted downstream of IKK, probably targeting InBadirectly (Fig. 5b).

Like InBa (S32/36A), MG132 was a potent inhibitor of

NF-nB signaling pathway by specifically interfering with

the action of 26S proteasome. To determine whether this

was true for ICP0, 293T cell was transfected with ICP0 and

nB-Luc for 24 h, after which MG132 or control reagents

were added into the culture medium and cells were

harvested after an additional 8 h. Luciferase assay revealed

that while DMSO, aprotinin, chymostatin and leupeptin had

no inhibitory effects on ICP0-mediated NF-nB activation,

MG132 produced significant reduction of NF-nB activation

in a dose-dependent manner. This indicated that protein

degradation was required during NF-nB activation and ICP0

followed the canonical NF-nB pathway at the terminal part

(Fig. 5c).

Ubch5A is a kind of ubiquitin conjugating enzyme E2,

and a point mutation at residue 85 from cysteine to alanine

abolished its proper activity completely. In combination

with it, ICP0 was previously shown to catalyze poly-

ubiquitination of p53 [17]. In addition, Ubch5A was

implicated to be an important E2 during InBa ubiquitination

by its endogenous E3, the SCF complex [29]. Therefore, we

investigated whether Ubch5A was important for NF-nBactivation by ICP0. ICP0 and nB-Luc were transfected into

293T cell with or without the mutant Ubch5A(C85A).

Luciferase assay revealed that Ubch5A(C85A) significantly

reduced NF-nB activation elicited by ICP0. In contrast,

another E2 mutant Ubch13(C87A) had no inhibitory effect

on NF-nB activation by ICP0, although this mutant had lost

its catalytic activity too (Fig. 5d).

Based on the above functional analysis, it is very likely

that ICP0 may be integrated into the NF-nB signaling

pathway at the InBa site. To explore this possibility, we co-

transfected flag-ICP0 with IKKh or a series of HA-InBamutants, respectively. Then flag-ICP0 immunoprecipitates

were analyzed to check which protein was pulled down by

ICP0. It turned out that full-length InBa interacted

specifically with ICP0, while IKKh did not show any

affinity to ICP0. In addition, ICP0 interacted with InBa(54–317) and it did not interacted with either InBa(1–225) orInBa(1–275), which suggested that ICP0 recognized the C-

terminus of InBa (Fig. 5e). Taken together, these results

suggested that ICP0 might shortcut the NF-nB signaling

pathway and target InBa directly. Importantly, it implicated

that ICP0 might serve as an ubiquitin ligase E3 in the

process.

3.4. ICP0 and BICP0 catalyze InBa ubiquitination in vivo

and in vitro

In light of the facts that ICP0 was an ubiquitin ligase E3

and it interacted with InBa in vivo, we wondered whether

ICP0 could catalyze the poly-ubiquitination reaction with

InBa as the substrate. To address this hypothesis, we set up

an in vitro reaction system that contained in vitro translated35S-InBa and the purified E1 and E2 components of the

ubiquitin system. When incubating them together, no

modification of InBa was observed. Interestingly, when

ICP0 or BICP0 was introduced into the reaction mixture, a

ladder of InBa species was generated with the molecular

weight spanning from about 40 kDa up to more than 200

kDa, which suggested that InBa was poly-ubiquitinated in

the presence of ICP0 or BICP0. In contrast, neither the

mock bead nor various ICP0 truncation mutants could help

produce such high molecular weight species of InBa. In

Fig. 5. ICP0 interacts with InBa and its function is attenuated by Ubch5A(C85A) and MG132. (a) ICP0 does not stimulate IKK activity. After transfecting

293T cell with the indicated constructs for 24 h, endogenous IKK was immunoprecipitated. Upper panel: IKK activity was checked by standard kinase assay

using bacteria expressed InBa as substrate. Lower panel: equal efficiency of IKK immunoprecipitation. (b) Activation of NF-nB by ICP0 is not inhibited by

InBa(S32/36A). Upper panel: nB-Luc activation by ICP0 or TRAF6 in the presence of gradient amounts of InBa(S32/36A); lower panels: expression of the

indicated constructs. (c) Activation of NF-nB by ICP0 is inhibited by MG132. ICP0 was transfected into 293T cells together with nB-Luc reporter gene. After24 h, cells were mock-treated or treated with MG132, aprotinin, chymostatin or leupeptin at indicated final concentrations. Luciferase activity was measured 8

h later. (d) NF-nB activation by ICP0 is attenuated by Ubch5A(C85A). (e) ICP0 physically interacts with InBa. Flag-ICP0 was co-transfected into 293T cells

with the indicated constructs. ICP0 was immunoprecipitated by anti-flag beads. The beads were checked with anti-HA or anti-IKK antibody.

L. Diao et al. / Cellular Signalling 17 (2005) 217–229 225

addition, BICP0(13G/51A) was unable to catalyze the

modification of InBa, which was consistent with the fact

that BICP0(13G/51A) harbored two point mutations at its

RING domain and it did not possess any ubiquitin E3

activity. As a further control, TRAF6 was also a demon-

strated ubiquitin E3 working at the far upstream of the NF-

nB signaling pathway. However, it failed to catalyze the

modification of InBa when it was added into the reaction

mixture (Fig. 6a). These results indicated that both ICP0 and

BICP0 were responsible for the specific modification of

InBa.

In the previous section, we found that activation of NF-

nB by ICP0 was independent of the phosphorylation of

InBa at either Ser32 or Ser36. Therefore, we continued to

investigate whether this phosphorylation was important for

InBa modification in this context. Consistently, ICP0

promoted indiscriminately the modification of InBa(WT),

InBa(S32A), InBa(S36A) and InBa(S32/36A) to the same

extent (Fig. 6b). These results again supported the notion

that ICP0 targeted InBa directly.

Next was studied the identity of the InBa modification

caused by ICP0 and BICP0. For the sake of protein

Fig. 6. ICP0 and BICP0 catalyze InBa ubiquitination in vivo and in vitro, independent of InBa phosphorylation. (a) ICP0 and BICP0 catalyze modification of35S-InBa. ICP0 or control proteins were incubated with 35S-InBa and purified components of ubiquitin system. The product was resolved with SDS-PAGE and

autoradiographed. (b) The modification of InBa induced by ICP0 is independent of Serine 32/36 phosphorylation. WT: wild type; S32A or S36A: serine 32 or

36 was substituted by alanine; S32/36A: both serines 32 and 36 were changed to alanines. (c) ICP0 and BICP0 catalyze poly-ubiquitination of GST-InBa. Invitro ubiquitination reactions were performed with GST-InBa as substrate and GST or GST-ATF as a control. The reaction products were pulled down with

Glutathione Sepharose beads and probed with anti-Ub antibody. (d) ICP0 and BICP0 catalyze poly-ubiquitination of InBa in an E1-, E2- and Ub-dependent

way. Experiment was carried out as in (c) except that a specific component was left out for each lane. (e) ICP0 and BICP0 stimulate poly-ubiquitination of

InBa in vivo. 293T cell was transfected with indicated constructs for 24 h. Endogenous InBa was immunoprecipitated after treating cell with MG132 for an

additional 8 h.

L. Diao et al. / Cellular Signalling 17 (2005) 217–229226

precipitation and Western blot, we used as substrate GST-

InBa purified from bacteria instead of in vitro translated35S-InBa in the following experiments. Once the modifica-

tion reactions were over, the GST-InBa and the control

substrates were pulled down and washed extensively. Then

the products were loaded onto SDS denaturing gel and

probed with antibody specifically against ubiquitin. As was

expected, GST-InBa from reactions containing ICP0 or

BICP0 produced characteristic smears of ubiquitin species

representing different numbers of ubiquitins conjugated

with the GST-InBa. However, neither TRAF6 nor

BICP0(13G/51A) was able to cause GST-InBa to generate

such fingerprint on gel that was detectable by ubiquitin

antibody. Furthermore, GST-ATF and GST alone from

reactions containing ICP0 did not show any characteristic

smears that were recognized by ubiquitin antibody (Fig. 6c).

These data conclusively established that the modified InBaby both ICP0 and BICP0 were indeed poly-ubiquitinated

species of InBa.This conclusion could also be substantiated by examin-

ing contribution of the components in ubiquitin system to

the poly-ubiquitination of InBa. If purified ubiquitin,

ubiquitin activating enzyme E1, ubiquitin conjugating

enzyme Ubch5A or ICP0 was left out one item at a time,

the characteristic smears of the modified InBa disappeared

completely when probing it with ubiquitin antibody.

Consistently, the inactive Ubch5A(C85A) could not help

the generation of the InBa poly-ubiquitin either. Only when

all the components were incubated together could the poly-

ubiquitinated forms of InBa be produced (Fig. 6d). These

L. Diao et al. / Cellular Signalling 17 (2005) 217–229 227

data clearly showed that ICP0 and BICP0 were ubiquitin

ligase E3 for InBa.Finally, we investigated whether endogenous InBa was

modified by ubiquitin in response to ICP0 and BICP0.

Therefore, 293T cells were transfected with ICP0, BICP0,

BICP0(13G/51A), LacZ or pcDNA3.1, respectively.

Twenty-four hours later, MG132 was added into culture

medium to prevent the de-ubiquitination of InBa and the

cells were harvested after an additional 8 h. Endogenous

InBa was immunoprecipitated with InBa specific antibody

coupled onto protein A bead and the immunoprecipitates

were probed with antibody specifically against ubiquitin. It

turned out that immunoprecipitates from BICP0(13G/51A),

LacZ or pcDNA3.1 transfected cells did not contain proteins

that could be detected by ubiquitin antibody, while the

endogenous InBa was among the immunoprecipitates.

Strikingly, the immunoprecipitates from either ICP0 or

BICP0 transfected cells did contain InBa smears that were

clearly recognized by ubiquitin antibody (Fig. 6e). These

results indicated that both ICP0 and BICP0 could catalyze

InBa poly-ubiquitination in vivo.

4. Discussion

There are several means by which HSV-1 can affect the

life cycle of human immunodeficiency virus type I (HIV-1)

[32]. One possible way involved stimulation of HIV-1 LTR

enhancer by the immediate-early protein ICP0 encoded in

HSV-1, which consequently enhanced gene expressions and

replication of HIV-1 per se [33]. However, the mechanism

underlying this trans-activation is poorly understood. One

recent study [34] suggested that ICP0 could functionally

cooperate with HIV Tat to activate LTR even in the absence

of HIV TAR sequence. They proposed that ICP0 recruited

Tat to the vicinity of the LTR enhancer to achieve this end.

Contradictorily, they did not show any physical interaction

between ICP0 and Tat either in vivo or in vitro. Signifi-

cantly, ICP0 has never been shown to bind to DNA in vivo

or in vitro. It seems unlikely that ICP0 functions as a

transcription co-factor in this process. In fact, current model

of ICP0 action favors an indirect mechanism in promoting

transcription of both viral and cellular genes [5]. Previously,

it has been demonstrated that activation and binding of NF-

nB at the HIV LTR is required for and can support enhanced

LTR-mediated transcription in response to cytokines such as

TNF-a and IL-1h, lectins, PMA and bacterial LPS, etc.

[35]. In this study, we found that BICP0, the ICP0

homologue from bovine herpes virus, could robustly induce

activation of the LTR from bovine immunodeficiency virus.

This induction was not affected when the TAR sequence of

the BIV was removed. In contrast, this activation was

severely impaired when a short stretch of DNA upstream to

the U3 segment was deleted, in which a nB consensus

segment was identified. This observation implicated an

alternative model of ICP0 and BICP0 action and led us to

hypothesize that both ICP0 and BICP0 could activate NF-

nB signaling pathway.

This hypothesis is supported by several lines of

evidence in the present study. First, both ICP0 and BICP0

were able to significantly stimulate gene expression of a

luciferase reporter construct that contained nothing derived

from the LTR and was introduced instead with three

tandem repeats of authentic nB sequence at the promoter

region. In addition, ICP0 could only activate this nB-Lucreporter construct and had no stimulatory effect on either

LEF-Luc or Gal4-Luc reporter construct. Notably, the LEF

binding site was previously identified inside the LTR and

it was functionally implicated in the potentiation of the

LTR by the Wnt signaling pathway [36]. Therefore, a

functional connection could be drawn between ICP0 and

the NF-nB signaling pathway. Second, both ICP0 and

BICP0 could induce translocation of p65-NF-nB from

cytoplasm into nucleus. In addition, NF-nB from ICP0- or

BICP0-treated nuclear extract was able to bind specifically

to its cognate nB site as revealed by EMSA assay, which

clearly indicated that NF-nB was activated, due to the

release of NF-nB from its inhibitor InBa, in response to

ICP0 and BICP0. This possibility was further substantiated

by the finding that activation of NF-nB by ICP0 was

specifically inhibited by proteasome inhibitor MG132,

suggesting that degradation of InBa was essential for the

process and NF-nB activation followed the classical

mechanism in this context. Third, ICP0 was found to

interact with the C-terminus of InBa and it did not

interact with IKKh, the kinase specific for phosphorylat-

ing InBa, which suggested that ICP0 might shortcut the

NF-nB signaling pathway and integrate itself into this

pathway at the InBa point. This conjecture was supported

by the experimental result that the super-repressor

InBa(S32/36A) exhibited no inhibitory effect on NF-nBactivation by ICP0, although this super-repressor was

proved to be a strong inhibitor of NF-nB activation by

many stimuli (e.g., TRAF6) in which phosphorylation of

InBa was an essential step. Consistently, ICP0 did not

influence the kinase activity of IKK complex when ICP0

was expressed inside the cell. Fourth, the RING domains

of both ICP0 and BICP0 were necessary for NF-nBactivation and the base-point mutant of BICP0(13G/51A)

was deprived of the ability to stimulate NF-nB with

respect to the criteria explored in this study. In addition,

the ubiquitin E2 mutant Ubch5A(C85A) was found to

attenuate NF-nB activation in response to ICP0. Given

that ICP0 is an ubiquitin ligase E3 and InBa is poly-

ubiquitinated and degraded before NF-nB activation, we

demonstrated in this investigation that ICP0 and BICP0

could catalyze conjugation of ubiquitin onto InBa,independent of its phosphorylation status. Furthermore,

this process was absolutely dependent on each component

of the ubiquitin system and could be confirmed in vivo by

monitoring endogenous InBa poly-ubiquitination in

response to ICP0 and BICP0.

L. Diao et al. / Cellular Signalling 17 (2005) 217–229228

Although activation of NF-nB in response to microbial

stimuli is normally associated with the initiation of humoral

and cellular immunity, some pathogens have taken advant-

age of this system to enhance their own replication, survival

and dissemination within the host [37]. For example, HTLV-

1 virus is a causative microbial capable of transforming T

cell and is responsible for adult T-cell leukemia. It has been

shown that the oncoprotein Tax from HTLV-1 could interact

with both IKK and another NF-nB inhibitor p100, thus

promoting phosphorylation and degradation of this inhib-

itor. However, Tax itself did not possess any enzymatic

activity. It activated NF-nB by bringing the IKK close to its

substrate and leaving the cellular components to finish the

job [38]. In contrast, ICP0 and BICP0 are ubiquitin ligase

E3 and it could directly catalyze InBa poly-ubiquitination.

Therefore, phosphorylation of InBa became a redundant

process for ICP0 and BICP0 function and Herpes virus

chose a more economical way to activate NF-nB. As an

ubiquitin ligase, ICP0 was previously connected to the

degradation of promyelocytic leukemia antigen (PML) and

Sp100 in ND10 [9,39]. Recently, ICP0 was also shown to

interact with tumor suppressor p53 and catalyze ubiquitina-

tion of the latter [17]. Although the multifunctionality of

ICP0 could be understood in reference to its ubiquitin ligase

activity, it is still a great challenge to integrate the seemingly

disparate targets of ICP0 action in terms of its overall

contribution in HSV-1 life cycle.

In summary, our study identified a new signaling

pathway that ICP0 hijacked to activate a myriad of viral

and cellular genes: when ICP0 is expressed immediately

after HSV-1 initial infection or reactivation, it specifically

recognizes and interacts with the C-terminus of InBa. Incombination with ubiquitin E1 and Ubch5A, ICP0 catalyzes

conjugation of one ubiquitin after another onto InBadirectly. In turn, the poly-ubiquitinated InBa is earmarked

and degraded by the 26S proteasome. This releases NF-nBfrom its cytoplasm anchor and it is free to enter the nucleus

and turns on its target genes, including those regulated by

the LTR enhancer. Interestingly, a nB consensus site was

previously identified in the promoter region of ICP0 gene in

HSV-1. In addition, NF-nB was shown to recognize and

bind this sequence [40]. In light of the new mechanism of

ICP0 action presented here, it is possible that there exists a

positive feedback regulation in ICP0 gene expression in

which more ICP0 can be produced from a tiny amount of

ICP0 via NF-nB signaling pathway. It is known that cellular

stress can induce HSV-1 to exit from the latent phase of its

life cycle and the same signal can activate NF-nB signaling

pathway too. During HSV-1 phase transition, ICP0 plays an

important role in regulating the virus gene expression. But

how, in the first place, is the ICP0 induced in response to

stress? It is likely that stress alone primes NF-nB signaling

pathway, which in turn induces ICP0 expression from HSV-

1. Then the induced ICP0 can augment this effect by

producing much more ICP0 via shortcutting NF-nBactivation directly. As a result, HSV-1 gene program is

turned on and more virus progenies are produced. This

regulation may be important during HSV-1 reactivation

from latency. More experiments are needed to explore this

wonderful model of HSV-1 reactivation.

Acknowledgement

We thank Professors Tom Gilmore(Boston University,

USA), Allan Weissman (National Institute of Health, USA),

Mike Ellison(University of Alberta, Canada), James Chen

(University of Texas Southwestern Medical Center, USA)

for providing plasmids in this study. We are grateful to

professor Lin Li and Youxin Jin for technical help. Naihe Jin

was supported by the National Key Basic Research and

Development Program (2002CB713802). Yunqi Geng was

supported by National Natural Science Foundation

(30170038). Chen Wang was a scholar of bThe Rising Star

ProgramQ from Shanghai Municipal Government and of

bThe Hundred Talents ProgramQ from Chinese Academy of

Sciences. This work was supported in part by bTheDistinguished Young Scholars ProgramQ from National

Natural Science Foundation of China (30225013), CAS

renovation program and 973 Project (2002CB513003).

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