A1

LXa

b

c

a

ARRAA

KHHCNV

1

vtrm(mt

whfipIsp

Sf

0d

Virus Research 163 (2012) 74–81

Contents lists available at SciVerse ScienceDirect

Virus Research

journa l homepage: www.e lsev ier .com/ locate /v i rusres

ssociation of Vpu with hepatitis C virus NS3/4A stimulates transcription of typehuman immunodeficiency virus

ei Kanga, Zhen Luoa,b, Youxing Lia,b, Wenjing Zhanga,b, Wei Suna, Wei Lia, Yanni Chena,b, Fang Liua,b,ueshan Xiac, Ying Zhua,b, Jianguo Wua,b,∗

State Key Laboratory of Virology, College of Life Sciences, and Chinese-French Liver Disease Research Institute at Zhongnan Hospital, Wuhan University, Wuhan 430072, ChinaWuhan Institites of Biotechnology, Gaoxin Road, No. 666, Wuhan East Lake High Technology Development Zone, Wuhan 430075, ChinaCollege of Life Sciences, Kunming University of Science and Technology, Kunming 650224, China

r t i c l e i n f o

rticle history:eceived 5 April 2011eceived in revised form 7 August 2011ccepted 19 August 2011vailable online 25 August 2011

eywords:CV

a b s t r a c t

Type 1 human immunodeficiency virus (HIV-1) and hepatitis C virus (HCV) are deadly bloodborne-transmitting pathogens. Due to sharing the routes of transmission, co-infection of HIV-1 and HCV iscommon with a high rate. Co-infection of HCV affects morbidity and mortality of patients with AIDS andimpairs their tolerance to antiretroviral therapy. In this study, the roles of HCV proteins in the regula-tion of HIV-1 replication and the molecular mechanism involved in such regulation were investigated.We demonstrated that HCV NS3 protein stimulated HIV-1 LTR transcription and that HIV-1 Vpu proteinwas required for the activation of HIV-1 transcription regulated by HCV NS3/4A complex. Further study

IV-1o-infectionS3/4Apu

revealed that Vpu mediated ubiquitination-associated degradation of NS4A, detached NS3/4A complexand release NS3 for nuclear translocation. Since both degradation of NS4A and activation of HIV-1 LTRwere closely correlated and mediated by Vpu, we proposed that Vpu impairs the stability of NS4A andreleases NS3 from NS3/4A complex for the stimulation of HIV-1 transcription. This study enriched ourunderstanding on HIV-1/HCV co-infection and provided new insights in molecular mechanism involved

two v

in the co-infection of the

. Introduction

Type 1 human immunodeficiency virus (HIV-1) and hepatitis Cirus (HCV) are life threatening pathogens, causing severe diseaseso human health (Klenerman and Kim, 2007). Due to sharing theoutes of transmission, co-infection of HIV-1 and HCV is very com-on among individuals frequently exposed to contaminated blood

Zhang et al., 2002). In these cohorts, HCV affects the morbidity andortality of AIDS patients (Monga et al., 2001) and impairs their

olerance to antiretroviral therapy (Greub et al., 2000).HIV-1 primarily infects CD4+ lymphocytes and macrophages,

hile HCV attacks hepatocytes. However, increasing evidencesave indicated that the two viruses are not strictly cell type con-ned. HCV replication is found in chronically infected patients’eripheral blood mononuclear cells (PBMCs) (Lerat et al., 1998).

n addition, study of PBMCs isolated from 10 HIV-positive patientshows that monocytes/macrophages and CD4+ lymphocytes sup-ort HCV replication (Laskus et al., 2000). On the other hand,

∗ Corresponding author at: State Key Laboratory of Virology, College of Lifeciences, Wuhan University, Wuhan 430072, China, Tel.: +86 276 875 4979;ax: +86 276 875 4592.

E-mail address: jwu@whu.edu.cn (J. Wu).

168-1702/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.virusres.2011.08.011

iruses.© 2011 Elsevier B.V. All rights reserved.

transcripts of HIV chemokine co-receptors CCR5 and CXCR4 havebeen recently detected in human hepatic stellate cells (Brunoet al., 2009). CD4-independent HIV-1 strain utilizing the above co-receptors for efficient infection of human hepatocytes is isolated(Xiao et al., 2008). These discoveries suggest that the two virusesmight reside, replicate and even mutually interact in the same cells.

HIV-1 is a positive strand RNA virus. Extensive studies haveshown that the long terminal repeat (LTR) region on viral genomeis modulated either by viral regulatory proteins (Marzio et al.,1998; Varin et al., 2003, 2005), host factors (Ballana et al., 2010;Darnell et al., 2006), or by opportunistically infected pathogens(Gomez-Gonzalo et al., 2001; Ranjbar et al., 2009). Previous studyhas reported that HIV-1 LTR is activated by HCV NS3/4A complex(Wu et al., 2008).

HCV NS3/4A complex comprises NS3 and its co-factor NS4A.C-terminus of NS3 exhibits helicase (Tai et al., 1996) and NTPase(Dumont et al., 2006) activities and facilitates viral replication. ItsN-terminus with serine protease domain is responsible for the mat-uration of viral non-structural proteins (Hahm et al., 1995). Thecentral portion of NS4A protein forms � strand and lies into NS3

N-terminal � barrel, while the hydrophobic N-terminus of NS4Aserves as anchor to locate NS3/4A on endoplasmic reticulum (ER)membrane (Kim et al., 1996). It has been reported that the 12–23residues of NS3 are also involved in the membrane association of

esearc

Nei

rva2cpbfn

HtNtHVNH

2

2

r(epst(cponsHalkgIcwnpctpp(Hw

2

baBs

L. Kang et al. / Virus R

S3/4A (Brass et al., 2008). Through binding to NS3, NS4A not onlynhances its helicase activity on RNA (Pang et al., 2002) but alsoncreases its stability and hydrolytic activity (Wolk et al., 2000).

In HCV infected cells, NS3/4A complex plays an importantole in mediating immune evasion. It particularly antagonizesiral RNA-induced activation of both Toll-like receptor 3 (TLR3)nd retinoic acid-inducible gene I (RIG-I) pathways (Foy et al.,005) through cleavage of Toll/IL-1 receptor resistance domain-ontaining adaptor inducing IFN (TRIF) (Li et al., 2005) and IFN-�romoter-stimulator-1 (IPS-1) (Loo et al., 2006), respectively. Sinceoth TLR3 and RIG-I lead to the activation of interferon regulatoryactor-3 (IRF-3), NS3/4A suppresses the translocation of IRF-3 toucleus and inactivate the induction of IFN-� (Foy et al., 2003).

In this study, we investigated the roles of proteins of HIV-1 andCV in the regulation of HIV-1 replication during the co-infection of

he two viruses. We demonstrated that HIV-1 Vpu protein and HCVS3/4A complex were involved in the activation of HIV-1 transcrip-

ion. The mechanism by which Vpu regulated NS3/4A to modulateIV-1 replication was also further elucidated. During this process,pu protein mediated ubiquitination-conjugated degradation ofS4A to release NS3 for nuclear translocation and thus, to activateIV-1 transcription.

. Materials and methods

.1. Plasmids

HIV-1 clone NL4-3.Luc.R-E- competent for a single round ofeplication, which is vpr- and env-, with nef substituted by luciferaseHe et al., 1995) was obtained through NIH AIDS Research and Ref-rence Reagent Program. pNL4-3/Udel with vpu deletion was kindlyrovided by Dr. Klaus Strebel (NIAID, NIH). pLTR-Luc, in which tran-cription of luciferase gene is under the control of HIV-1 LTR (−454o +67), was described previously (Mu et al., 2011). HIV-1 genesgag, pol, vif, tat, rev and vpu) were amplified from NL4-3.Luc.R-E-DNAs and constructed into pCMV-Tag 2B (Stratagene) to generatelasmids expressing the corresponding HIV-1 proteins. Ser-52/-56n Vpu were replaced by glycines in pCMV-Vpu-Mut by mutage-esis. pEGFP-Vpu-WT and pEGFP-Vpu-Mut were constructed byubcloning the vpu gene and its mutant to pEGFP-N3 (Clontech).CV replicon plasmid FL-J6/JFH-5′C19Rluc2AUbi, which carriesmonocistronic full-length viral genome that expresses Renilla

uciferase (Rluc) was reported previously (Tscherne et al., 2006) andindly provided by Dr. Charles Rice (Rockefeller University). HCVenes used in this study were isolated from HCV1b strain (GenBankD: D45172). pSG5-HA (Chen et al., 1999) was a gift from Dr. Stall-up (University of Southern California, CA). HCV ns3 and ns4a genesere subcloned into pSG5-HA, while other HCV genes (ns2, ns3,

s4b, ns5a and ns5b) were subcloned into pCMV-Tag 2B as describedreviously (Lu et al., 2008). HCV ns3 and ns4a genes were alsoonstructed into mammalian two-hybrid vector plasmids (Clon-ech) to yield pVP16-NS3 and pM-NS4A, and into pEGFP-N3 andDsRed-C1 (Clontech) to generate pEGFP-NS4A, pRed-NS3/4A andRed-NS3, respectively. Coding sequence of NS3 protease domain166 N-terminal residues) (Suzich et al., 1993) was amplified fromCV ns3 gene for the generation of pSG5-HA-CT-NS3. All constructsere verified by automatic DNA sequencing.

.2. Antibodies and inhibitor

Commercial antibodies used in this study were monoclonal anti-

odies against HA (sc-57595; Santa Cruz), Flag (F1804; Sigma)nd polyclonal antibodies against HA (CW0093; Beijing CoWiniotech), GAPDH (CW0266; Beijing CoWin Biotech). Rabbit anti-erum against HIV-1 p24 protein was kindly provided by Dr. Bing

h 163 (2012) 74–81 75

Yan Cruz (Wuhan Institute of Virology, Chinese Academy of Sci-ences, P.R. China). Polyclonal antibodies against GFP were preparedby immunizing rabbit with purified His-tagged GFP protein. Ubiq-uitin E1 inhibitor PYR-41 (sc-358737) was purchased from Santa.

2.3. Cell culture, transient transfection and luciferase assay

Human embryonic kidney (HEK) 293 cells and Huh.7 cells werecultured in Dulbecco’s modified Eagle’s medium (Gibco), supple-mented with 10% fetal bovine serum, 100 U/mL penicillin and100 �g/mL streptomycin sulfate. Cells were co-transfected withfirefly and renilla (pRL-TK) luciferase constructs in 10:1 ratiousing Lipofectamine 2000 (Invitrogen). HCV replicon FL-J6/JFH-5′C19Rluc2AUbi RNA for transfection was transcribed in vitro usingMEGAscript Kit (Applied Biosystems). Lysates were analyzed 48 hpost transfection (p.t.) according to the manufacturer’s instructionson Dual-Luciferase Kit (Promega).

2.4. Co-immunoprecipitation and western blot analysis

HEK 293 cells were seeded in 100 mm dishes at 90% con-fluent and transfected with 6 �g GFP-NS4A, 6 �g pSG5-HA-NS3,in combination with 18 �g pCMV-Vpu or its mutant as indi-cated. Cells were harvested 48 h p.t. and lysed in 1 mL RIPAbuffer (50 mmol/L Tris–HCl pH 7.4, 150 mmol/L NaCl, 1% sodiumdeoxycholate, 0.5 mol/L EDTA, 1 mmol/L NaF, 1% Nonidet P-40,supplemented by 10% proteinase inhibitors cocktail). 1/10 lysate(100 �l) was reserved for direct immunoblot analysis while therest was successively incubated with HA monoclonal antibodies(1 �g) overnight and with protein A/G agarose (Sigma) for another1 h. After 5 rounds of washes, proteins were fractionated by tricineSDS-PAGE (Schagger, 2006) and tranferred to nitrocellulose mem-brane (Amersham). Target bands were then immunoblotted withHA polyclonal antibodies and GFP polyclonal antibodies and visu-alized using Immobilon Western Reagents (Millipore).

2.5. Confocal imaging

HEK 293 cells were seeded on coverslip and transfected with4 �g pDsRed-NS3. Culture medium was removed 24 h p.t. Cellswere washed by PBS and incubated with 1 �g/mL DAPI (Roche)-methanol solution for 15 min at room temperature. After washedby methanol and PBS, the coverslip was mounted with 50% glycerol.Fluorescence was observed and imaged under microscope (TCS-SP,LEICA).

2.6. Statistics

All experiments were reproducible and carried out in triplicate.Each set was repeated at least three times and a representative ofsimilar results was shown. Statistical analysis was accomplishedusing SPSS software package, version 17.0. Parallel samples werefirstly determined for normal distribution by Kolmogorov-Smimovtest. Abnormal values were eliminated according to a follow-upGrubbs test. Levene’s test for equality of variances was performed,which provides information for Student’s t test to distinguishthe equality of means. In this study, means were illustrated inhistogram with error bars representing ±SD. And p < 0.05 was con-sidered of statistical significance.

3. Results

3.1. HCV NS3 protein stimulates HIV-1 replication

In order to investigate the effects of HCV proteins on the reg-ulation of HIV-1 replication, HEK 293 cells were co-transfected

76 L. Kang et al. / Virus Resear

Fig. 1. Determination of the roles of HCV NS3 in the regulation of HIV-1 transcrip-tion and replication. (A) HEK 293 cells were transfected with 400 ng pSG5-HA-NS3and/or 400 ng pSG5-HA-NS4A, 200 ng pNL4-3.Luc.R-E-, and 20 ng pRL-TK. Luciferaseassay were performed 48 h p.t. (upper panel). Immunoblot analysis was carriedout at 48 h p.t. using anti-HA, anti-p24 and anti-GAPDH antibodies, respectively(lower panel); (B) HEK 293 cells were co-transfected with 300 ng pNL4-3.Luc.R-E-, 30 ng pRL-TK, in combination with pSG5-HA-CT-NS3 at different concentrationsas indicated. Luciferase assay were performed 48 h p.t. (upper panel). Immunoblotabi

wN(apteelc

upaEtmC

nalysis was carried out at 48 h p.t. using anti-HA, anti-p24 and anti-GAPDH anti-odies, respectively (lower panel). Results of three independent experiments were

llustrated as mean ± SD. Double asterisks in the graph represents p < 0.01.

ith plasmids (pSG5-HA-NS3 and pSG5-HA-NS4A expressingS3 and NS4A protein, respectively), the reporter plasmid

pNL4-3.Luc.R-E-), and pRL-TK. Results from luciferase activityssays showed that NS3 and NS3/4A stimulated the activity of HIV-1romoter by 4.1 and 3.5 folds, respectively, while NS4A alone failedo activate HIV-1 replication (Fig. 1A, top panel). In addition, west-rn blot analysis indicated that the level of HIV-1 p24 protein wasnhanced in the presence of NS3 or NS3/4A, but not NS4A (Fig. 1A,ower panel). These results demonstrated that HCV NS3 or NS3/4Aould activate HIV-1 replication, but NS4A alone failed to act.

To study the role of the serine protease domain of NS3 in the reg-lation of HIV-1 replication, HEK 293 cells were co-transfected withSG5-HA-CT-NS3 (expressing the serine protease but not helicasend NTPase domain) at different concentrations, pNL4-3.Luc.R-

-, and pRL-TK. Results from luciferase activity assays showedhat CT-NS3 activated HIV-1 transcription in a dose-dependent

anner (Fig. 1B, top panel). Western blot analysis showed thatT-NS3 also stimulated the expression of HIV-1 p24 protein in

ch 163 (2012) 74–81

a concentration-dependent fashion (Fig. 1B, lower panel). Theseresults demonstrated that the serine protease domain of NS3 aloneis enough for the activation of HIV-1 replication.

3.2. HIV-1 Vpu protein facilitates HCV NS3/4A complex in theactivation of HIV-1 transcription

Based on the above results, we further analyzed and comparedthe roles of HCV NS3 and NS3/4A in the regulation of HIV-1 LTR tran-scription. HEK 293 cells were co-transfected with pSG5-HA-NS3,pSG5-HA-NS4A, pSG5-HA-NS3 plus pSG5-HA-NS4A, pLTR-Luc, andpRL-TK, respectively. Luciferase activity assays indicated that NS3protein enhanced the activity of HIV-1 LTR by 2.6 folds, whileNS4A protein failed to act again (Fig. 2A, top panel). To our sup-prise, the activity of NS3/4A was reduced in the stimulation ofHIV-1 LTR transcription (Fig. 2A, top panel). This conflicted withthe above result, which showed that NS3/4A complex could acti-vate HIV-1 transcription and replication. We speculated that HIV-1proteins were involved in the regulation of HIV-1 replication medi-ated by NS3/4A, since the major difference between pLTR-Luc andpNL4-3.Luc.R-E- is pLTR-Luc lacks six HIV-1 proteins (Gag, Pol,Vif, Tat, Rev and Vpu) encoded by NL4-3.Luc.R-E-, we investigatedthe roles of these proteins in the activation of HIV-1 LTR tran-scription regulated by NS3/4A. HEK 293 cells were co-transfectedwith pSG5-HA-NS3 plus pSG5-HA-NS4A, pLTR-Luc, pRL-TK, andplasmids (pCMV-Gag, pCMV-Pol, pCMV-Vif, pCMV-Tat, pCMV-Revand pCMV-Vpu) encoding individual HIV-1 proteins. Results fromluciferase activity assays showed that HIV-1 Vpu protein signif-icantly enhanced the function of HCV NS3/4A complex in theactivation of HIV-1 LTR transcription (Fig. 2B). However, the Gag,Pol, Vif and Tat proteins of HIV-1 had slight inhibitory effects onthe function of HCV NS3/4A complex, while Rev had no effect onNS3/4A function (Fig. 2B).

To confirm the involvement of Vpu in the facilitation ofHCV NS3/4A to stimulate HIV-1 LTR transcription, HEK 293 cellswere co-transfected with pSG5-HA-NS3, pSG5-HA-NS4A, pSG5-HA-NS3 plus pSG5-HA-NS4A, and pNL4-3/Udel containing theHIV-1 genomic DNA only missing the vpu gene. Western blot anal-ysis demonstrated that the level of intracellular HIV-1 p24 proteinwas obviously increased in the presence of HCV NS3 protein, butremained relatively unchanged in the presence of NS4A or NS3/4A(Fig. 2C). Since NL4-3/Udel carries the HIV-1 genome with vpu genedeleted, the attenuation of NS3/4A function in the regulation ofHIV-1 transcription was due to the absence of Vpu. These resultsdemonstrated that HIV-1 Vpu protein is involved in HCV NS3/4Afunction in stimulating HIV-1 transcription.

3.3. HIV-1 Vpu mediates HCV NS4A degradation to detachNS3/4A complex

The mechanism involved in the role of HIV-1 Vpu in the regu-lation of HIV-1 replication mediated by HCV NS3/4A was furtherinvestigated. First, we attempted to determine whether there isa direct interaction between Vpu and NS3 or NS4A. However,mammalian two-hybrId assay and co-immunoprecipitation anal-ysis provided negative results indicating Vpu could not bind toNS3/4A (data not shown).

The impacts of HIV-1 Vpu on intracellular levels of HCV NS3and NS4A were then investigated. It has been reported previouslythat Vpu mediated ubiquitination events provided its Ser-52/-56were phosphorylated by casein kinase-II (Margottin et al., 1998). Inthis study, a Vpu mutant (Vpu-Mut) was generated by substituting

Ser-52/-56 with glycines to serve as negative control for Vpu-mediated ubiquitination. HEK 293 cells were then co-transfectedwith pSG5-HA-NS3 or pSG5-HA-NS4A, pCMV-Vpu or pCMV-Vpu-Mut for different times as indicated. Western blot analysis showed

L. Kang et al. / Virus Research 163 (2012) 74–81 77

Fig. 2. Analysis of the roles of HIV-1 Vpu in the activatio of HIV-1 transcription mediated by HCV NS3/4A complex. (A) HEK 293 cells were co-transfected with 400 ng pSG5-HA-NS3 and/or 400 ng pSG5-HA-NS4A, 200 ng pLTR-Luc, in combination with 20 ng pRL-TK. Luciferase assay were performed 48 h p.t. (upper panel). Immunoblot analysiswas carried out at 48 h p.t. using anti-HA, and anti-GAPDH antibodies, respectively (lower panel); (B) HEK 293 cells were co-transfected with 300 ng pSG5-HA-NS3 and 300 ngpSG5-HA-NS4A, 300 ng each of the plasmids (pCMV-Gag, pCMV-Pol, pCMV-Vif, pCMV-Tat, pCMV-Rev and pCMV-Vpu) expressing coresponding HIV-1 proteins (Gag, Pol,Vif, Tat, Rev and Vpu), 150 ng pLTR-Luc, together with 15 ng pRL-TK. Luciferase assay were performed 48 h p.t. (upper panel). Reverse transcription PCR was carried out 48 hp.t. for the detection of individual HIV-1 genes, NS3/4A and �-actin mRNAs; (C) HEK 293 cells were transfected with 400 ng pSG5-HA-NS3 and/or 400 ng pSG5-HA-NS4A,in combination with 200 ng pNL4-3/Udel. Luciferase assay were performed 48 h p.t. (upper panel). Immunoblot analysis was carried out at 48 h p.t. using anti-HA, anti-p24a nt expp

tp2wnprni

itwtdbr(wst

wtVMoot(o

ihw(

nd anti-GAPDH antibodies, respectively (lower panel). Results of three independe< 0.01.

hat the levels of NS3 protein remained relatively unchanged in theresence of wild-type and mutated Vpu (Fig. 3A, upper panel, lanevs 1, 4 vs 3, 6 vs 5 and 8 vs 7). However, the levels of NS4A proteinere significantly reduced in the presence of wild-type Vpu, butot affected by Vpu mutant at the same time points (Fig. 3A, loweranel, lane 2′ vs 1′, 4′ vs 3′, 6′ vs 5′ and 8′ vs 7′). These results clearlyevealed that HIV-1 Vpu protein affected the stability of NS4A butot NS3 and that Ser-52/-56 of Vpu were required for Vpu function

n regulating NS4A stability.Since Vpu protein mediates ubiquitination events, we further

nvestigated whether ubiquitination is involved in the degrada-ion of NS4A regulated by Vpu. HEK 293 cells were co-transfectedith pSG5-HA-NS4A and pCMV-Vpu or pCMV-Vpu-Mut for 8 h and

hen treated with PYR-41 (an effective inhibitor for ubiquitination-ependent protein degradation) or DMSO. Results from westernlot analysis showed that in the absence of PYR-41, NS4A level waseduced by the treatment of wild-type Vpu, but not by Vpu-MutFig. 3B). However, the effect of wild-type Vpu on NS4A reductionas not detected in the presence of PYR-41 (Fig. 3B). These results

upported that Vpu mediated ubiquitination-conjugated degrada-ion of Vpu.

Since NS3/4A is a component of HCV replication complex (RC),e next wanted to determine whether other non-structural pro-

eins of HCV could protect NS4A from being degraded by HIV-1pu. HEK 293 cells were transfected with pCMV-Vpu or pCMV-Vpu-ut, pEGFP-NS4A, and plasmids encoding non-structural proteins

f HCV. Western blot analysis demonstrated that in the presence ofther non-structural proteins of HCV, wild-type Vpu was still ableo degrade NS4A and Vpu-Mut had no effect on NS4A degradationFig. 3C). These results suggested that Vpu was able to degrade NS4Aff HCV RC.

Next, we attempted to determine whether Vpu affected the

ntegrity of NS3/4A complex. For this purpose, a mammalian two-ybrid system was used. HCV ns3 and ns4a genes were fusedith VP16 activation domain (AD) and GAL4 DNA binding domain

BD), respectively. Because NS3 and NS4A formed complex, their

eriments were illustrated as mean ± SD. Double asterisks in the graph represents

associations would bring the VP16 AD and GAL4 DNA BD together,which in turn stimulated reporter gene transcription. Results fromluciferase activity assays indicated that in the vector and Vpu-Muttreated groups, reporter gene transcription was activated in thepresence of pM-NS4A and pVP16-NS3 (Fig. 3D). However, in wild-type Vpu treated group, the activation of reporter gene expressionwas abolished (Fig. 3D). These results indicated that the integrityof NS3/4A was impaired by Vpu.

The effect of Vpu protein on the integrity of NS3/4A was fur-ther determined by co-immunoprecipitation assay. HEK 293 cellswere co-transfected with pEGFP-NS4A, pSG5-HA-NS3, pCMV-Vpuor pCMV-Vpu-Mut. Results showed that in the cell lysate, NS4Aprotein levels were lower in cells treated with wild-type Vpu thanthose with Vpu-Mut (Fig. 3E, left panel, lane 1 vs 3, lane 2 vs 4).However, after being precipitated by HA antibody, NS4A was hardlydetected in cells treated with wild-type Vpu (Fig. 3E, right panel,lane 2) but detected in those treated with Vpu-Mut (Fig. 3E, rightpanel, lane 4). These results further confirmed that Vpu affected theintegrity of NS3/4A.

Our results suggested that NS3/4A complex was detached andNS4A was degraded by Vpu. We next sought to determine the sub-cellular localization of NS3 protein. NS3 was previously reportedto locate in both cytoplasm and nucleus (Errington et al., 1999).To investigate the effect of Vpu on the localization of NS3, HEK293 cells were transfected with pDsRed-NS3 or pDsRed-NS3/4A, orco-transfected with pDsRed-NS3/4A and pEGFP-Vpu-WT or pEGFP-Vpu-Mut, respectively. Results showed that in cells transfectedwith pDsRed-NS3, NS3 was distributed in the whole cell in theabsence of NS4A, although there was more NS3 protein in thenucleus than in the cytoplasm (Fig. 3Fa, Fb and Fc). In the pres-ence of NS4A, NS3 was mostly located to the cytoplasm by NS4A(Fig. 3Fd, Fe and Ff). More interestingly, in the presence of both

wild-type Vpu (localized in the cytoplasm, Fig. 3Fg) and NS4A, NS3was apparently localized mainly in the nucleus (Fig. 3Fh, Fi andFj). However, in the presence of both Vpu-Mut (localized in thecytoplasm, Fig. 3Fk) and NS4A, NS3 was also mostly localized to

78 L. Kang et al. / Virus Research 163 (2012) 74–81

Fig. 3. Analysis of the effect of HIV-1 Vpu on the formation of HCV NS3/4A complex. (A) HEK 293 cells were co-transfected with 800 ng pSG5-HA-NS3 (upper panel) or 800 ngpSG5-HA-NS4A (lower panel), in combination with 3200 ng pCMV-Vpu or pCMV-Vpu-Mut. Intracellular proteins were immunoblotted 12 h, 24 h, 36 h and 48 h p.t. usinganti-HA, anti-Flag and anti-GAPDH antibodies; (B) HEK 293 cells were co-transfected with 800 ng pSG5-HA-NS4A and 3200 ng pCMV-Vpu or pCMV-Vpu-Mut for 8 h and thentreated with DMSO or PYR-41 (5 �mol/L). Immunoblot analysis was carried out at 48 h p.t. using anti-HA, anti-Flag and anti-GAPDH antibodies, respectively; (C) HEK 293 cellswere transfected with 1000 ng pCMV-Vpu or pCMV-Vpu-Mut, 500 ng pEGFP-NS4A, together with mixed plasmids (pCMV-NS2, pCMV-NS3, pCMV-NS4B, pCMV-NS5A andpCMV-NS5B, 500 ng each) expressing HCV non-structural proteins. Immunoblot analysis was carried out at 48 h p.t. using anti-HA and anti-GAPDH antibodies, respectively;(D) HEK 293 cells were transfected with 500 ng pCMV-Vpu or pCMV-Vpu-Mut, 250 ng pM or pM-NS4A, 250 ng pVP16 or pVP16-NS3, 50 ng pG5-Luc, along with 5 ng pRL-TKas indicated. Luciferase assay were performed 48 h p.t.; (E) HEK 293 cells were transfected with 6 �g pEGFP-NS4A, 6 �g pSG5-HA-NS3, in combination with 18 �g pCMV-Vpuor pCMV-Vpu-Mut as indicated. Co-immunoprecipitation analysis was carried out 48 h p.t. as described in materials and methods section; (F) HEK 293 cells were transfectedw EGFP-c ents wp

tffioN

3

rstiftwocost

ith 4 �g pDsRed-NS3, or 4 �g pDsRed-NS3/4A, or 1 �g pDsRed-NS3/4A and 3 �g parried out 24 h p.t. as described in Section 2. Results of three independent experim< 0.05 and p < 0.01, respectively.

he cytoplasm by NS4A (Fig. 3Fl, Fm and Fn), indicating Vpu-Mutailed to affect subcellular localization of NS3. These results con-rmed that Vpu mediated ubiquitination-conjugated degradationf NS4A in the cytoplasm, detached NS3/4A complex and directedS3 to the nucleus.

.4. Effects of Vpu on HIV-1 and HCV replication

We further determined whether the effect of NS3/4A on theegulation of HIV-1 transcription enhanced by Vpu was the con-equence of impaired stability of NS4A. HEK 293 cells wereransfected with pSG5-HA-NS3, pSG5-HA-NS4A, pLTR-Luc, pRL-TKn combination with mixed pCMV-Vpu and pCMV-Vpu-Mut in dif-erent ratios as indicated. Luciferase activity assays showed that theranscription levels of HIV-1 LTR were not affected in the absence ofild-type Vpu and presence of Vpu-Mut, enhanced in the presence

f wild-type Vpu and Vpu-Mut at the ratio of 1:1, and signifi-

antly stimulated in the presence of wild-type Vpu and absencef Vpu-Mut (Fig. 4A, top panel). In addition, western blot analy-is illustrated that the level of NS4A protein was not affected inhe absence of wild-type Vpu and presence of Vpu-Mut, reduced

Vpu-WT, or 1 �g pDsRed-NS3/4A and 3 �g pEGFP-Vpu-Mut. Confocal imaging wasere illustrated as mean ± SD. Asterisk and double asterisks in the graph represents

in the presence of wild-type Vpu and Vpu-Mut at the ratio of 1:1,and significantly inhibited in the presence of wild-type Vpu andabsence of Vpu-Mut (Fig. 4A, lower panel). These results confirmedthat Vpu affected the stability of NS4A for the activation of HIV-1LTR transcription.

Finally, the effect of Vpu on HCV replication was also inves-tigated. Huh.7 cells were co-transfected with HCV repliconFL-J6/JFH-5′C19Rluc2AUbi RNA and pCMV-Vpu or pCMV-Vpu-Mut.Luciferase assays showed that HCV replication was stimulated bywild-type Vpu and slightly enhanced by Vpu-Mut (Fig. 4B). Theseresults suggested that Vpu activated HCV replication, which alsopartially depended on Ser-52/-56.

4. Discussion and conclusions

Previous study has reported that HCV NS3/4A complex, butnot its serine inactive isoform, stimulates HIV-1 replication (Wu

et al., 2008). It is indicated in the results that NS3 serine proteasedomain is indispensible in activating HIV-1 replication. Since NS3N-terminus also interacts with NS4A (Kim et al., 1996), it is interest-ing to see whether NS4A is involved during the activation of HIV-1

L. Kang et al. / Virus Researc

Fig. 4. Determination of the effects of HIV-1 Vpu on the replication of HIV-1 and HCV.(A) HEK 293 cells were transfected with 200 ng pSG5-HA-NS3, 200 ng pSG5-HA-NS4A, 100 ng pLTR-Luc, 10 ng pRL-TK, in combination with 400 ng mixed plasmidsof pCMV-Vpu and pCMV-Vpu-Mut as indicated. Luciferase assay were performed48 h p.t. (upper panel). Immunoblot analysis was carried out at 48 h p.t. using anti-HA, anti-Flag and anti-GAPDH antibodies, respectively (lower panel); (B) Huh.7cells were transfected with 600 ng pCMV-Vpu or pCMV-Vpu-Mut, 300 ng FL-J6/JFH-5′C19Rluc2AUbi RNA, together with 30 ng pRL-TK. Luciferase assay were performed3At

rttaw

NtLmapNmbptt

6 h p.t. Results of three independent experiments were illustrated as mean ± SD.sterisk and double asterisks in the graph represents p < 0.05 and p < 0.01, respec-

ively.

eplication regulated by NS3/4A complex. In this study, we showedhat NS3 alone and along with NS4A were able to stimulate HIV-1ranscription and replication (Fig. 1A). We also demonstrated thatlthough NS3 alone was able to activate HIV-1 LTR, although alongith NS4A, it failed to activate HIV-1 LTR (Fig. 2A).

These two results seemed to be conflicted, one showed thatS3/4A complex could activate HIV-1 transcription and replication,

he other showed that NS3/4A complex failed to activate HIV-1TR transcription. The major difference between the two experi-ents was that pNL4-3.Luc.R-E- was used in the first experiment

nd pLTR-Luc was used in the second. Since pLTR-Luc lacks sixroteins (Gag, Pol, Vif, Tat, Rev and Vpu) of HIV-1 as compared toL4-3.Luc.R-E-, we thus speculated that HIV-1 protein or proteinsight be involved in the regulation of HIV-1 replication mediated

y NS3/4A. In the process of determining the involvement of HIV-1roteins, we demonstrated that HIV-1 Vpu protein was involved inhe function of HCV NS3/4A complex in the activation of HIV-1 LTRranscription and viral replication (Fig. 2B and C).

h 163 (2012) 74–81 79

Vpu is a 16 kDa type I integral membrane protein, with a trans-membrane helix in the N-terminus (Maldarelli et al., 1993). Itscytoplasmic portion comprises two amphipathic �-helices, whichare jointed by a flexible linker peptide. Phosphorylation of Ser-52/-56 on the linker (Schubert et al., 1994) renders Vpu to recruit�-transducin repeat-containing protein (�-TrCP) (Butticaz et al.,2007; Margottin et al., 1998) and link target protein to ubiquitina-tion. It prevents p53 (Verma et al., 2011) and phosphorylated I�B�(Bour et al., 2001) from being degraded and mediates the degrada-tion of CD4 (Schubert et al., 1998) and tetherin (Douglas et al., 2009).In this way, Vpu facilitates HIV-1 in evasion from host immuneresponses, conquest of cell resources and release of progeny viralparticles.

Here, we speculated that NS4A was an opportunistic target forVpu-mediated ubiquitination event. We showed that the stabilityof NS4A was greatly impaired by Vpu, but not by its Ser-52/-56mutant (Fig. 3A). Using ubiquitin E1 inhibitor (PYR-41), which isirrelevant with interactions between Vpu and ubiquitin E3 ligasecomplex, we found that it prevented the degradation of NS4A medi-ated by Vpu (Fig. 3B). Only two molecules, CD4 and tetherin, havebeen reported previously to commit Vpu-mediated degradation.While CD4 is degraded in ER (Schubert et al., 1998), degradationof tetherin appears to occur in trans-Golgi network or early endo-somes (Douglas et al., 2009). Despite the differences in degradationplaces, both CD4 and tetherin are categorized as integral membraneproteins. Interestingly, Vpu is also an integral protein, it seems thatmembrane-association benefits Vpu-mediated degradation event.Same as Vpu, NS4A is known to locate on ER membrane (Kim et al.,1996). Our results confirmed the co-localization of NS3/4A and Vpuin the cytoplasm (Fig. 3Fn). It is possible that the degradation ofNS4A mediated by Vpu was on ER membrane. Since there was nodirect interaction between Vpu and NS4A (data not shown), anotherprotein might act as adapter between them. NS4A does not havelysine residues to form isopeptide bond with ubiquitin, its degra-dation is thus not via classic ubiquitination. Recent studies haverevealed that cysteine, serine and threonine can also be attachedto ubiquitin to trigger protein degradation (Cadwell and Coscoy,2005; Wang et al., 2007). We speculates that the amino acid residueof NS4A responsible for its degradation probably belongs to thiscategory.

In the process of determining the association betweenVpu-mediated NS4A degradation and NS3/4A-regulated HIV-1transcription, we revealed that the activity of HIV-1 LTR wasincreased as the concentration of wild-type Vpu increased, whilethe levels of NS4A protein were decreased (Fig. 4A). We demon-strated that increase of Vpu levels correlated with the reduction ofNS4A and the activation of HIV-1 LTR. Thus, it suggested that Vpufacilitated NS3/4A-mediated activation of HIV-1 transcription bydegrading NS4A. However, we also revealed that increase of wild-type Vpu levels inhibited LTR basal activation in the absence ofNS3/4A (Fig. 4A). Vpu is reported to prevent degradation of phos-phorylated I�B� (Bour et al., 2001) to disturb NF-�B activation.So it would interfere with basal LTR transcription which relieson NF-�B signaling. One recently published paper also confirmsthat Vpu efficiently prevents p53 degradation (Verma et al., 2011).p53 cooperates with Sp1 in binding to its cis-element on HIV-1 LTR to stimulate HIV-1 transcription (Gualberto and Baldwin,1995). Meanwhile, p53 over expression inhibits phosphorylationof carboxyl terminal domain (CTD) on RNA Pol II, resulting in thedelay of both host and viral transcription (Mukerjee et al., 2010).As negative impact overwhelms in the beginning, rescue of p53by Vpu would suppress basal LTR transcription. Since preven-

tions of both phosphorylated I�B� and p53 require recruitmentsof ubiquitin E3 complex, inhibition on HIV-1 LTR basal activationby Ser-52/-56 mutated Vpu in the absence of NS3/4A did not occur(Fig. 4A).

8 esear

(Nibissntbtt(t

dFoNbfmitsanNsmmAdai(NSNm

btpeesmtrtI(iill(toaHov2

0 L. Kang et al. / Virus R

We proved that Vpu reduces bindings between NS3 and NS4AFig. 3D and E) due to the consequences of its ability to degradeS4A. We also showed that Vpu-Mut and NS3 were co-localized

n the cytoplasm (Fig. 3Fn). So there is possibility that Vpu affectsinding affinity of NS3 to NS4A. However, we have found that there

s no direct interaction between Vpu and NS3 or NS4A (data nothown). Moreover, in mammalian two-hybrid assay, NS3 and NS4Ahould be expressed and then shifted to nucleus by GAL4 and SV40uclear localization sequence, respectively. Since Vpu is located inhe cytoplasm (Fig. 3Fg), it would have little if any influence on theinding affinity between NS3 and NS4A in the nucleus. Therefore,he disassociation between NS3 and NS4A observed in mammalianwo-hybrid analysis (Fig. 3D) and in co-immunoprecipitation assayFig. 3E) are most likely due to the reduction in NS4A protein ratherhan in the binding affinity between NS3 and NS4A.

In confocal study, we further confirmed that Vpu rendered theissociation of NS3/4A to translocate NS3 into nucleus (Fig. 3Fg,h, Fi and Fj). Based on these results, we propose that activationf HIV-1 transcription and replication (Figs. 1A, 2B and 4A) byS3/4A depends on the translocation of NS3 to nucleus mediatedy HIV-1 Vpu. After NS4A being degraded by Vpu, NS3 is releasedrom NS3/4A complex and might be brought into nucleus by other

olecules. One study has shown that p53 enhances nuclear local-zation of NS3 (Muramatsu et al., 1997). According to their results,he extent of NS3 translocation to nucleus depends on the expres-ion pattern of p53 in host cells. Since HEK 293 cells yield certainmounts of p53 (Hamid and Kakar, 2004), NS3 accumulated in theucleus (Fig. 3Fc and Ff) might be mediated by p53. In addition,S3 may also be dragged into nucleus by Sm-D1, a component of

mall nuclear ribonucleoprotein (snRNP) associated with autoim-une disease (Iwai et al., 2003). Sm-D1 and p53 are the only twoolecules so far known to bind NS3 protease domain in the nucleus.lthough snRNPs are responsible for RNA splicing, there is no evi-ence that Sm-D1 is associated with HIV-1 replication. As describedbove, p53 displays a negative effect on HIV-1 transcription. NS3s reported to suppress p53-mediated transcriptional activationDeng et al., 2006). But it is not mentioned in their study whetherS3 counteracts p53 in the inhibition of RNA Pol II phosphorylation.ince p53 is known to associate with both HIV-1 transcription andS3 in the nucleus, NS3-mediated activation of HIV-1 replicationost likely is p53-dependent.It has been reported that HCV replication is directly stimulated

y the presence of HIV (Beld et al., 1998). Another study also showshat HCV replication is enhanced by the binding of HIV-1 enveloprotein to the coreceptors (CCR5 and CXCR4) on hepatocytes (Lint al., 2008). We discovered that NS4A protein was reduced by Vpuven in the presence of other HCV non-structural proteins (Fig. 3C),uggesting that HCV RC could not protect NS4A from degradationediated by Vpu. Since NS4A is one of the components of HCV RC,

his result indicates that Vpu may have a negative impact on HCVeplication. In sharp contrast to that, we revealed that HCV replica-ion was enhanced by both wild-type Vpu and Vpu-Mut (Fig. 4B).n all of our results, the degradation of NS4A never reached 100%Figs. 1A, 3A–C, E and 4A). NS4A protein could still be detected evenn the presence of Vpu; Although most of NS3 protein was locatedn the nucleus in the presence of Vpu, there was still a small portioneft in the cytoplasm (Fig. 3Fh and Fg); In addition to NS4A, cytoso-ic NS3 is reported to interact with other non-structural proteinsIshido et al., 1998; Paredes and Blight, 2008). We speculate thathere is a portion of HCV RCs being assembled even in the presencef Vpu. Since both Vpu and HCV RC are localized on ER membrane,n association, either direct or indirect, between Vpu protein and

CV RC components, might exist, which enhances the efficiencyf HCV replication. Moreover, the remodeling of lipid microen-ironments is reported to facilitate HCV replication (Hsu et al.,010). It is not clear whether Vpu would facilitate the remodeling

ch 163 (2012) 74–81

process. Further studies are required to determine the mecha-nism for Vpu-mediated activation of HCV replication. Interestingly,since wild-type Vpu displays more effects on the induction of HCVreplication as compared to its mutant, such activation should alsopartially rely on Ser-52/-56 of Vpu.

Acknowledgments

This research was supported by research grants from theNational Mega Project on Major Infectious Diseases Prevention(2008ZX10002-009 and 2009ZX10004-207), the National MegaProject on Major Drug Development (2009ZX09301-014 and2011ZX09401-302), the Major State Basic Research DevelopmentProgram (973 program) (2007CB512803 and 2012CB518900), theNational Science Foundation of China (30730001, 30872491 and81171525), the Program for Changjiang Scholars and InnovativeResearch Team in University (IRT0745), the Key Project of Chi-nese Ministry of Education (204114208), the Department of Scienceand Technology of Hubei Province (2005ABC003), the Fundamen-tal Research Funds for the Central Universities (1102001), and theSpecialized Research Fund for the Doctoral Program of Higher Edu-cation (20090141110033).

We thank Dr. Klaus Strebel of NIAID, NIH for providing plasmidNL4-3/Udel, Dr. Charles Rice of Rockefeller University for plasmidFL-J6/JFH-5′C19Rluc2Aubi, Dr. Stallcup of University of SouthernCalifornia for pSG5-HA, Dr. Bing Yan of Wuhan Institute of Virology,CAS for HIV-1 p24 antibodies, Dr. Yongxin Mu of Wuhan Univer-sity for antiserum against GFP. We also thank Dr. Wei Wei and Dr.Muhammad Mahmood Mukhtar for proof reading the article.

References

Ballana, E., Senserrich, J., Pauls, E., Faner, R., Mercader, J.M., Uyttebroeck, F., Palou,E., Mena, M.P., Grau, E., Clotet, B., Ruiz, L., Telenti, A., Ciuffi, A., Este, J.A., 2010.ZNRD1 (zinc ribbon domain-containing 1) is a host cellular factor that influencesHIV-1 replication and disease progression. Clin. Infect. Dis. 50 (7), 1022–1032.

Beld, M., Penning, M., Lukashov, V., McMorrow, M., Roos, M., Pakker, N., van denHoek, A., Goudsmit, J., 1998. Evidence that both HIV and HIV-induced immun-odeficiency enhance HCV replication among HCV seroconverters. Virology 244(2), 504–512.

Bour, S., Perrin, C., Akari, H., Strebel, K., 2001. The human immunodeficiency virustype 1 Vpu protein inhibits NF-kappa B activation by interfering with beta TrCP-mediated degradation of Ikappa B. J. Biol. Chem. 276 (19), 15920–15928.

Brass, V., Berke, J.M., Montserret, R., Blum, H.E., Penin, F., Moradpour, D., 2008.Structural determinants for membrane association and dynamic organizationof the hepatitis C virus NS3-4A complex. Proc. Natl. Acad. Sci. U.S.A. 105 (38),14545–14550.

Bruno, R., Galastri, S., Sacchi, P., Cima, S., Caligiuri, A., Defranco, R., Milani, S., Gessani,S., Fantuzzi, L., Liotta, F., Frosali, F., Antonucci, G., Pinzani, M., Marra, F., 2009. TheHIV envelope protein GP120 modulates the biology of human hepatic stellatecells: a link between HIV infection and liver fibrogenesis. Gut 59 (4), 428–429.

Butticaz, C., Michielin, O., Wyniger, J., Telenti, A., Rothenberger, S., 2007. Silencing ofboth beta-TrCP1 and HOS (beta-TrCP2) is required to suppress human immun-odeficiency virus type 1 Vpu-mediated CD4 down-modulation. J. Virol. 81 (3),1502–1505.

Cadwell, K., Coscoy, L., 2005. Ubiquitination on nonlysine residues by a viral E3ubiquitin ligase. Science 309 (5731), 127–130.

Chen, D., Ma, H., Hong, H., Koh, S.S., Huang, S.M., Schurter, B.T., Aswad, D.W., Stallcup,M.R., 1999. Regulation of transcription by a protein methyltransferase. Science284 (5423), 2174–2177.

Darnell, G.A., Schroder, W.A., Gardner, J., Harrich, D., Yu, H., Medcalf, R.L., Warrilow,D., Antalis, T.M., Sonza, S., Suhrbier, A., 2006. SerpinB2 is an inducible host factorinvolved in enhancing HIV-1 transcription and replication. J. Biol. Chem. 281(42), 31348–31358.

Deng, L., Nagano-Fujii, M., Tanaka, M., Nomura-Takigawa, Y., Ikeda, M., Kato, N., Sada,K., Hotta, H., 2006. NS3 protein of Hepatitis C virus associates with the tumoursuppressor p53 and inhibits its function in an NS3 sequence-dependent manner.J. Gen Virol. 87 (Pt 6), 1703–1713.

Douglas, J.L., Viswanathan, K., McCarroll, M.N., Gustin, J.K., Fruh, K., Moses, A.V., 2009.Vpu directs the degradation of the human immunodeficiency virus restriction

factor BST-2/Tetherin via a {beta}TrCP-dependent mechanism. J. Virol. 83 (16),7931–7947.

Dumont, S., Cheng, W., Serebrov, V., Beran, R.K., Tinoco Jr., I., Pyle, A.M., Bustamante,C., 2006. RNA translocation and unwinding mechanism of HCV NS3 helicase andits coordination by ATP. Nature 439 (7072), 105–108.

esearc

E

F

F

G

G

G

H

H

H

H

I

I

K

K

L

L

L

L

L

L

M

M

L. Kang et al. / Virus R

rrington, W., Wardell, A.D., McDonald, S., Goldin, R.D., McGarvey, M.J., 1999. Sub-cellular localisation of NS3 in HCV-infected hepatocytes. J. Med. Virol. 59 (4),456–462.

oy, E., Li, K., Sumpter Jr., R., Loo, Y.M., Johnson, C.L., Wang, C., Fish, P.M., Yoneyama,M., Fujita, T., Lemon, S.M., Gale Jr., M., 2005. Control of antiviral defenses throughhepatitis C virus disruption of retinoic acid-inducible gene-I signaling. Proc. Natl.Acad. Sci. U.S.A. 102 (8), 2986–2991.

oy, E., Li, K., Wang, C., Sumpter Jr., R., Ikeda, M., Lemon, S.M., Gale Jr., M., 2003. Reg-ulation of interferon regulatory factor-3 by the hepatitis C virus serine protease.Science 300 (5622), 1145–1148.

omez-Gonzalo, M., Carretero, M., Rullas, J., Lara-Pezzi, E., Aramburu, J., Berkhout,B., Alcami, J., Lopez-Cabrera, M., 2001. The hepatitis B virus X protein inducesHIV-1 replication and transcription in synergy with T-cell activation signals:functional roles of NF-kappaB/NF-AT and SP1-binding sites in the HIV-1 longterminal repeat promoter. J. Biol. Chem. 276 (38), 35435–35443.

reub, G., Ledergerber, B., Battegay, M., Grob, P., Perrin, L., Furrer, H., Burgisser, P.,Erb, P., Boggian, K., Piffaretti, J.C., Hirschel, B., Janin, P., Francioli, P., Flepp, M.,Telenti, A., 2000. Clinical progression, survival, and immune recovery duringantiretroviral therapy in patients with HIV-1 and hepatitis C virus coinfection:the Swiss HIV Cohort Study. Lancet 356 (9244), 1800–1805.

ualberto, A., Baldwin Jr., A.S., 1995. p53 and Sp1 interact and cooperate in the tumornecrosis factor-induced transcriptional activation of the HIV-1 long terminalrepeat. J. Biol. Chem. 270 (34), 19680–19683.

ahm, B., Han, D.S., Back, S.H., Song, O.K., Cho, M.J., Kim, C.J., Shimotohno, K., Jang,S.K., 1995. NS3-4A of hepatitis C virus is a chymotrypsin-like protease. J. Virol.69 (4), 2534–2539.

amid, T., Kakar, S.S., 2004. PTTG/securin activates expression of p53 and modulatesits function. Mol. Cancer 3, 18.

e, J., Choe, S., Walker, R., Di Marzio, P., Morgan, D.O., Landau, N.R., 1995. Humanimmunodeficiency virus type 1 viral protein R (Vpr) arrests cells in the G2 phaseof the cell cycle by inhibiting p34cdc2 activity. J. Virol. 69 (11), 6705–6711.

su, N.Y., Ilnytska, O., Belov, G., Santiana, M., Chen, Y.H., Takvorian, P.M., Pau, C., vander Schaar, H., Kaushik-Basu, N., Balla, T., Cameron, C.E., Ehrenfeld, E., van Kup-peveld, F.J., Altan-Bonnet, N., 2010. Viral reorganization of the secretory pathwaygenerates distinct organelles for RNA replication. Cell 141 (5), 799–811.

shido, S., Fujita, T., Hotta, H., 1998. Complex formation of NS5B with NS3 and NS4Aproteins of hepatitis C virus. Biochem. Biophys. Res. Commun. 244 (1), 35–40.

wai, A., Hasumura, Y., Nojima, T., Takegami, T., 2003. Hepatitis C virus nonstructuralprotein NS3 binds to Sm-D1, a small nuclear ribonucleoprotein associated withautoimmune disease. Microbiol. Immunol. 47 (8), 601–611.

im, J.L., Morgenstern, K.A., Lin, C., Fox, T., Dwyer, M.D., Landro, J.A., Chambers, S.P.,Markland, W., Lepre, C.A., O’Malley, E.T., Harbeson, S.L., Rice, C.M., Murcko, M.A.,Caron, P.R., Thomson, J.A., 1996. Crystal structure of the hepatitis C virus NS3protease domain complexed with a synthetic NS4A cofactor peptide. Cell 87 (2),343–355.

lenerman, P., Kim, A., 2007. HCV-HIV coinfection: simple messages from a complexdisease. PLoS Med. 4 (10), e240.

askus, T., Radkowski, M., Piasek, A., Nowicki, M., Horban, A., Cianciara, J., Rakela,J., 2000. Hepatitis C virus in lymphoid cells of patients coinfected withhuman immunodeficiency virus type 1: evidence of active replication in mono-cytes/macrophages and lymphocytes. J. Infect. Dis. 181 (2), 442–448.

erat, H., Rumin, S., Habersetzer, F., Berby, F., Trabaud, M.A., Trepo, C., Inchauspe, G.,1998. In vivo tropism of hepatitis C virus genomic sequences in hematopoieticcells: influence of viral load, viral genotype, and cell phenotype. Blood 91 (10),3841–3849.

i, K., Foy, E., Ferreon, J.C., Nakamura, M., Ferreon, A.C., Ikeda, M., Ray, S.C., Gale Jr.,M., Lemon, S.M., 2005. Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc. Natl.Acad. Sci. U.S.A. 102 (8), 2992–2997.

in, W., Weinberg, E.M., Tai, A.W., Peng, L.F., Brockman, M.A., Kim, K.A., Kim, S.S.,Borges, C.B., Shao, R.X., Chung, R.T., 2008. HIV increases HCV replication in aTGF-beta1-dependent manner. Gastroenterology 134 (3), 803–811.

oo, Y.M., Owen, D.M., Li, K., Erickson, A.K., Johnson, C.L., Fish, P.M., Carney, D.S.,Wang, T., Ishida, H., Yoneyama, M., Fujita, T., Saito, T., Lee, W.M., Hagedorn, C.H.,Lau, D.T., Weinman, S.A., Lemon, S.M., Gale Jr., M., 2006. Viral and therapeuticcontrol of IFN-beta promoter stimulator 1 during hepatitis C virus infection.Proc. Natl. Acad. Sci. U.S.A. 103 (15), 6001–6006.

u, L., Wei, L., Peng, G., Mu, Y., Wu, K., Kang, L., Yan, X., Zhu, Y., Wu, J., 2008. NS3 pro-tein of hepatitis C virus regulates cyclooxygenase-2 expression through multiplesignaling pathways. Virology 371 (1), 61–70.

aldarelli, F., Chen, M.Y., Willey, R.L., Strebel, K., 1993. Human immunodeficiencyvirus type 1 Vpu protein is an oligomeric type I integral membrane protein. J.Virol. 67 (8), 5056–5061.

argottin, F., Bour, S.P., Durand, H., Selig, L., Benichou, S., Richard, V., Thomas, D.,Strebel, K., Benarous, R., 1998. A novel human WD protein, h-beta TrCp, that

h 163 (2012) 74–81 81

interacts with HIV-1 Vpu connects CD4 to the ER degradation pathway throughan F-box motif. Mol. Cell 1 (4), 565–574.

Marzio, G., Tyagi, M., Gutierrez, M.I., Giacca, M., 1998. HIV-1 tat transactivatorrecruits p300 and CREB-binding protein histone acetyltransferases to the viralpromoter. Proc. Natl. Acad. Sci. U.S.A. 95 (23), 13519–13524.

Monga, H.K., Rodriguez-Barradas, M.C., Breaux, K., Khattak, K., Troisi, C.L., Velez,M., Yoffe, B., 2001. Hepatitis C virus infection-related morbidity and mortalityamong patients with human immunodeficiency virus infection. Clin. Infect. Dis.33 (2), 240–247.

Mu, Y., Yu, Y., Yue, X., Musarat, I., Gong, R., Zhu, C., Liu, Y., Liu, F., Zhu, Y., Wu, J.,2011. The X protein of HBV induces HIV-1 long terminal repeat transcriptionby enhancing the binding of C/EBPbeta and CREB1/2 regulatory proteins to thelong terminal repeat of HIV-1. Virus Res. 156 (1-2), 81–90.

Mukerjee, R., Claudio, P.P., Chang, J.R., Del Valle, L., Sawaya, B.E., 2010. Transcriptionalregulation of HIV-1 gene expression by p53. Cell Cycle 9 (22), 4569–4578.

Muramatsu, S., Ishido, S., Fujita, T., Itoh, M., Hotta, H., 1997. Nuclear localization ofthe NS3 protein of hepatitis C virus and factors affecting the localization. J. Virol.71 (7), 4954–4961.

Pang, P.S., Jankowsky, E., Planet, P.J., Pyle, A.M., 2002. The hepatitis C viral NS3 proteinis a processive DNA helicase with cofactor enhanced RNA unwinding. EMBO J21 (5), 1168–1176.

Paredes, A.M., Blight, K.J., 2008. A genetic interaction between hepatitis C virus NS4Band NS3 is important for RNA replication. J. Virol. 82 (21), 10671–10683.

Ranjbar, S., Boshoff, H.I., Mulder, A., Siddiqi, N., Rubin, E.J., Goldfeld, A.E., 2009. HIV-1replication is differentially regulated by distinct clinical strains of Mycobac-terium tuberculosis. PLoS One 4 (7), e6116.

Schagger, H., 2006. Tricine-SDS-PAGE. Nat. Protoc. 1 (1), 16–22.Schubert, U., Anton, L.C., Bacik, I., Cox, J.H., Bour, S., Bennink, J.R., Orlowski, M.,

Strebel, K., Yewdell, J.W., 1998. CD4 glycoprotein degradation induced by humanimmunodeficiency virus type 1 Vpu protein requires the function of protea-somes and the ubiquitin-conjugating pathway. J. Virol. 72 (3), 2280–2288.

Schubert, U., Henklein, P., Boldyreff, B., Wingender, E., Strebel, K., Porstmann,T., 1994. The human immunodeficiency virus type 1 encoded Vpu proteinis phosphorylated by casein kinase-2 (CK-2) at positions Ser52 and Ser56within a predicted alpha-helix-turn-alpha-helix-motif. J. Mol. Biol. 236 (1),16–25.

Suzich, J.A., Tamura, J.K., Palmer-Hill, F., Warrener, P., Grakoui, A., Rice, C.M.,Feinstone, S.M., Collett, M.S., 1993. Hepatitis C virus NS3 protein polynucleotide-stimulated nucleoside triphosphatase and comparison with the relatedpestivirus and flavivirus enzymes. J. Virol. 67 (10), 6152–6158.

Tai, C.L., Chi, W.K., Chen, D.S., Hwang, L.H., 1996. The helicase activity associ-ated with hepatitis C virus nonstructural protein 3 (NS3). J. Virol. 70 (12),8477–8484.

Tscherne, D.M., Jones, C.T., Evans, M.J., Lindenbach, B.D., McKeating, J.A., Rice, C.M.,2006. Time- and temperature-dependent activation of hepatitis C virus for low-pH-triggered entry. J. Virol. 80 (4), 1734–1741.

Varin, A., Decrion, A.Z., Sabbah, E., Quivy, V., Sire, J., Van Lint, C., Roques, B.P.,Aggarwal, B.B., Herbein, G., 2005. Synthetic Vpr protein activates activatorprotein-1, c-Jun N-terminal kinase, and NF-kappaB and stimulates HIV-1 tran-scription in promonocytic cells and primary macrophages. J. Biol. Chem. 280(52), 42557–42567.

Varin, A., Manna, S.K., Quivy, V., Decrion, A.Z., Van Lint, C., Herbein, G., Aggarwal,B.B., 2003. Exogenous Nef protein activates NF-kappa B, AP-1, and c-Jun N-terminal kinase and stimulates HIV transcription in promonocytic cells. Rolein AIDS pathogenesis. J. Biol. Chem. 278 (4), 2219–2227.

Verma, S., Ali, A., Arora, S., Banerjea, A.C., 2011. Inhibition of {beta}-TrcP-dependentubiquitination of p53 by HIV-1 Vpu promotes p53-mediated apoptosis in humanT cells. Blood 117 (24), 6600–6607.

Wang, X., Herr, R.A., Chua, W.J., Lybarger, L., Wiertz, E.J., Hansen, T.H., 2007. Ubiquiti-nation of serine, threonine, or lysine residues on the cytoplasmic tail can induceERAD of MHC-I by viral E3 ligase mK3. J. Cell. Biol. 177 (4), 613–624.

Wolk, B., Sansonno, D., Krausslich, H.G., Dammacco, F., Rice, C.M., Blum, H.E., Morad-pour, D., 2000. Subcellular localization, stability, and trans-cleavage competenceof the hepatitis C virus NS3-NS4A complex expressed in tetracycline-regulatedcell lines. J. Virol. 74 (5), 2293–2304.

Wu, X., Ishaq, M., Hu, J., Guo, D., 2008. HCV NS3/4A protein activates HIV-1 tran-scription from its long terminal repeat. Virus Res. 135 (1), 155–160.

Xiao, P., Usami, O., Suzuki, Y., Ling, H., Shimizu, N., Hoshino, H., Zhuang, M., Ashino,Y., Gu, H., Hattori, T., 2008. Characterization of a CD4-independent clinical HIV-1that can efficiently infect human hepatocytes through chemokine (C-X-C motif)

receptor 4. AIDS 22 (14), 1749–1757.

Zhang, C., Yang, R., Xia, X., Qin, S., Dai, J., Zhang, Z., Peng, Z., Wei, T., Liu, H., Pu, D.,Luo, J., Takebe, Y., Ben, K., 2002. High prevalence of HIV-1 and hepatitis C viruscoinfection among injection drug users in the southeastern region of Yunnan,China. J. Acquir. Immune Defic. Syndr. (1999) 29 (2), 191–196.