positive effect of the hepatitis c virus nonstructural 5a protein on viral multiplication

Arch Virol (2004) 149: 1353–1371DOI 10.1007/s00705-003-0291-6

Positive effect of the hepatitis C virus nonstructural 5Aprotein on viral multiplication

D. Bonte1, C. Francois1, S. Castelain1, C. Wychowski2, J. Dubuisson2,E. F. Meurs3, and G. Duverlie1

1Laboratoire de Virologie, CHU, Amiens, France2Institut de Biologie de Lille, Institut Pasteur, Lille, France

3Unite Hepacivirus, Institut Pasteur, Paris, France

Received August 25, 2003; accepted December 5, 2003Published online March 17, 2004 c© Springer-Verlag 2004

Summary. Hepatitis C virus infection (HCV), is a major cause of liver diseaseworldwide, and are frequently resistant to the interferon alpha treatment. Thenonstructural (NS) 5A protein of HCV has been proposed to be involved in thisresistance. Additional studies have pointed out a role for NS5A in several othercellular interactions as well as an important role of its adaptative mutations inHCV genome replication. However, no infectious system is available to assess therole of NS5A in the HCV life cycle. Thus, we have constructed a recombinantsystem directly demonstrating for the first time that the expression of NS5Aconfers a multiplicative advantage to Sindbis virus, a virus close to HCV. Thisadvantage seemed to be related to an anti-apoptotic effect of the NS5A protein. Ata later stage, a possible nuclear localization of NS5A was observed, likely due toapoptotic cleavages of this protein. The NS5A protein was also shown to inducethe interleukin-8 (IL-8) mRNA and to activate the NF-κB pathway independentlyof the Sindbis virus. Together, our data suggest that the activation of NF-κB couldlead to the anti-apoptotic activity of NS5A and explain the viral multiplicativeadvantage conferred by the expression of the NS5A protein.

Introduction

Hepatitis C virus infection (HCV) is a major cause of chronic liver diseaseworldwide [27]. Viral clearance can only be achieved in about half of the patients,even after combined pegylated α-interferon (IFN)/ribavirine treatment [30, 50]. Anumber of reports have pointed out to a role for the nonstructural (NS) 5A proteinin the resistance of HCV to IFN [48].A first molecular evidence has been describedby Gale et al. who reported a physical and functional interaction between NS5A

1354 D. Bonte et al.

and the IFN-induced dsRNA-activated serine/threonine protein kinase (PKR),leading to PKR inactivation [13, 14]. PKR is known to be activated in response tothe accumulation of dsRNA in cells, a situation which occurs frequently duringinfections, for instance through viral replicative intermediates [8]. Once activated,PKR inhibits protein synthesis through phosphorylation of serine 51 of the α sub-unit of eukaryotic initiation factor 2 (eIF-2α) [31]. The ability of NS5A to behaveas a PKR inhibitor would therefore explain the resistance of some HCV strains toIFN. However, we and others have shown that the anti-IFN activity of NS5A mayinvolve other pathways [11, 12, 35]. NS5A has since been shown to interact withmany cellular proteins, which suggests a pleiotropic effect for this protein. Forinstance, NS5A was reported to interact with the Grb2-element which might leadto interference with the MAPK/ERK1/2 pathway [43, 44]. Other interactions withcellular proteins have been described, including a cellular transcriptional factorSRCAP [17], p53 [29], and Stat 3 [19]. An interaction with the Karyopherin β3has also been shown and this might interfere with the nuclear transport of RNA[7]. Amphiphysin II has recently been described to interact directly with NS5A[49].

NS5A is a phosphoprotein which is variable both in length and sequencedepending on the genotype [38, 45]. A direct role for NS5A in the replicationcomplex of HCV has not been described, but clinical and recent basic data usingHCV subgenomic RNAs have suggested a correlation between the variabilityof NS5A and the level of HCV replication and/or cellular adaptation [2, 10,34]. Recently, NS5A has been shown to stimulate the IRES-mediated translationin the replicon system [21]. Phosphorylation of NS5A may play an importantrole because it is conserved among viruses in the family Flaviviridae. Putativefunctional domains have been described for NS5A of HCV genotype 1, i.e, a 162–166 stretch involved in the interaction with NS4A [1], a 353–361 region whichpresents a potential nuclear localization signal (NLS) [22], and an endoplasmicreticulum (ER) localization signal in its amino-terminal region 1–31 [4]. Twocleavage sites involving caspase-like proteases at position D154 and D389 havealso been identified [41]. Although NS5A is associated with the ER membrane,cleavage by caspase-like proteases could lead to the transport of an NS5A domaininto the nucleus, and the induction of some transactivation activity by its carboxy-terminal region [23].

Polyak et al. have shown that NS5A can induce interleukin-8 (IL-8), leading topartial inhibition of the IFN-induced antiviral response [37], as confirmed recentlyby another group [18]. IL-8 is a pro-inflammatory CXC-chemokine [33]. It isinduced by several viruses and can inhibit the antiviral effect of α-IFN in vitro[25]. The NS5A-mediated induction of IL-8 mRNA seemed to be more importantwhen NS5A-truncated forms were located in the nucleus [37].

The lack of a cell culture system supporting an efficient propagation of HCVlimits the studies in vivo. Although the recently described replicon is the besttool to study HCV replication, this system is not suitable for our purpose sinceno viral particles are produced, and since the selection of the cell line based onthe replicon itself leads to adaptative mutations of NS5A. However, since the

NS5A and virus multiplication 1355

replicons described so far are all sensitive to IFN [2, 34], the variations obtainedduring the selection and the presence of heterologous IRES in the bicistronicconstruct (IRES EMCV) could also modify the cellular interplay of NS5A andother HCV proteins.

Here, we have investigated the effects of NS5A in a multiplicative viral systemclose to HCV using an isolate of Sindbis virus, the prototype member of the familyTogaviridæ, genus Alphavirus. It is an enveloped virus with a single-strandedRNA genome of positive polarity with 11,703 bases, a 5′ cap structure and a 3′poly(A). Sindbis virus recombinants expressing the HCV NS5A or other proteinsas reference have been constructed. In these infectious viral models, we studied theeffects of these recombinant proteins on viral multiplication in comparison withthose already described in other systems. The effects of NS5A were analyzed inregard to the PKR activity, apoptosis, localization, and induction of IL-8.

Materials and methods

Generation of Sindbis virus recombinants

Heterologous cDNAs were cloned into full-length SIN vector (clone pTE3′2J) kindly providedby C. M. Rice (Rockefeller University) [20] (Fig. 1). Sequences of the Green FluorescentProtein (GFP) from the pE-GFP plasmid (Clontech), the Vaccinia virus K3L protein (cloneK3L-HR47, hyper-resistant K3L kindly provided by T. Dever) [24] tagged at its N-terminusby a Myc epitope (EQKLISEEDL), and the NS5A protein of genotype 1b from a chronicallyHCV infected patient non responding to IFN therapy were amplified by PCR and insertedinto the XbaI site of plasmid pTE3′2J. The sequences were verified after cloning and beforegenerating virus stocks. ISDR and PKR-BD sequences of NS5A were identical to otherresistant HCV clones already described [10, 13, 14]. The 5′-capped RNA transcripts weresynthesized by using SP6 RNA polymerase and the plasmid DNA template which had beenlinearized with XhoI. Stocks of wild-type Sindbis virus and Sindbis virus recombinants weregenerated by electroporation with in vitro transcribed RNA as described previously [5]. Forty-eight hours post-electroporation, culture supernatants were harvested and aliquots were storedat −80 ◦C until used.

Viral yield assay

HeLa cells were plated at a density of 5×105 per well in six-well plates. Eighteen hourspost-incubation at 37 ◦C, the cells were washed once with serum-free medium, and theninfected with Sindbis virus recombinants at a multiplicity of infection (MOI) of 1 at 37 ◦C for60 min. The virus-containing medium was removed, and the cells were further incubated incomplete medium. At different times post-infection, the cells were scrapped and centrifuged.The supernatant was collected and the virus yield was determined on Vero VC10 cells. Thedifferent dilutions of the virus in serum-free medium were allowed to adsorb to the cells for60 min, and the cells were covered with DMEM containing 5% serum and 0.6% agarose.Forty-eight hours post-incubation at 37 ◦C, cells were fixed in 10% trichloroacetic acid at4 ◦C for 30 min, the agarose-containing medium was removed. The plaques were visualizedby staining with a solution of 0.1% crystal violet in 20% ethanol. Viral yield was expressedas log pfu/ml.


Reagents

The cell lines were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supple-mented with 10% (vol/vol) fetal bovine serum (FBS), 100 U of penicillin per ml, and 100 µgof streptomycin per ml in 5% CO2 at 37 ◦C. For apoptosis study, the caspase inhibitorZ-VAD-fmk (N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) (RnD) was added tocells before and after infection.

The anti-Myc (ATCC CRL-1725) monoclonal antibody (Mab) was produced in vitro byusing a MiniPerm apparatus (Heraeus) as recommended by the manufacturer. Anti-NS5Aand anti-GFP Mabs were purchased from Biodesign and Clontech, respectively. Rabbitpolyclonal antibody directed against a synthetic 13-residue phosphorylated rat eIF-2α peptidewas obtained from Research Genetics (Huntsville, Ala.). Mouse polyclonal antibody directedagainst eIF-2 was a gift of C. Proud (University of Dundee, UK).

Immunoprecipitation

HeLa cells were plated at a density of 5 × 105 per well in six-well plates and were mock-infected or infected with either SIN/GFP, SIN/K3L, or SIN/NS5A viruses at 5 pfu per celland incubated at 37 ◦C. At five hours post-infection, cells were washed twice in DMEMwithout methionine and cysteine, and incubated in this medium for 30 min. Then, the proteinswere metabolically labeled by incubating cells in methionine- and cysteine-deficient DMEMcontaining 100 µCi/ml 35S protein labeling mix (NEN) for an hour and a half. The cellswere then washed in PBS and were lysed with TBS (Tris Buffered Saline) (20 mM Tris-HCl(pH = 7.5), 137 mM NaCl, 2 mM EDTA, and 5‰ NaN3) supplemented with 0.5% NP40,20 µg/ml TPCK, and 20 µg/ml aprotinin. The anti-Myc and anti-NS5A Mabs were incubatedat 4 ◦C overnight under agitation with Protein A-Sepharose (Pharmacia) in 1 ml of bufferTBS-I supplemented with 0.2% Igepal (6 µl of Mab per 70 µl of beads). The antibody-beadmixture was then washed by 1 ml of TBS-I and 75 µl of cytoplasmic extracts were added andthe mixture was incubated at 4 ◦C for 45 min under agitation. After 3 washes with TBS-I andone with water, the samples were resuspended in 50 µl of Laemmli buffer (75 mM Tris-HCl(pH = 6.8), 4% sodium dodecyl sulfate (SDS), 100 mM 2-mercaptoethanol, 20% glycerol,and bromophenol blue as tracking dye), and denatured at 75 ◦C for 10 min. Then, the proteinswere separated by SDS-PAGE, the gel was fixed, dried and autoradiographed.

Immunoblotting

HeLa cells were scrapped and washed in PBS. Cytoplasmic extracts were prepared in bufferI (20 mM Tris-HCl (pH = 7.2), 50 mM KCl, 400 mM NaCl, 1% Triton X-100, 5 mM 2-mercaptoethanol, 0.05% aprotinin, 0.2 mM phenylmethylsulfonidfluoride, 20% glycerol) andresuspended in an equal volume of Laemmli buffer. The proteins were separated by SDS-PAGE and then transferred to Immobilon PVDF membranes (Millipore). The membraneswere saturated at 37 ◦C for 60 min in PBS containing 5% non fat dry milk and incubated at4 ◦C overnight with the primary antibody in PBS containing 1% fetal bovine serum and 0.1%Tween-20. The membranes were washed twice in PBS, and then incubated with the secondaryantibody coupled to horseradish peroxydase (Amersham) for 1 h at room temperature inthe same buffer. After two washes in PBS, the membranes were processed for enhancedchemiluminescence according to the Amersham protocol.

Confocal microscopy

For laser scanning confocal-microscopy experiments, HeLa cells were cultured in eight-wellchamber slides and infected with Sindbis virus recombinants in the presence or absence of


Z-VAD-fmk. The viruses were allowed to adsorb to the cells for 60 min, then the cells wereincubated with DMEM containing 10% serum. Seven or fifteen hours post-infection, cellswere fixed with paraformaldehyde and permeabilized with 0.5% Triton-X100. After washeswith PBS and PBS-0.05% Tween-20, the primary antibodies were incubated in PBS-0.05%Tween-20 for 1 hour. The cells were stained with anti-mouse antibodies conjugated to FITC(Rockland) and propidium iodide (PI) to stain the nuclear DNA. Green and red fluorescencewere collected simultaneously, and then separated digitally.

Analyses of IL-8 mRNA

IL-8 mRNA was detected by reverse transcription-PCR (RT-PCR) [37]. Total RNAs wereextracted from cells infected with Sindbis virus recombinants in the presence or not of Z-VAD-fmk, using the total RNA isolation kit (Roche). The primers span introns on the IL-8gene and produce a PCR fragment of 289 bp, only if mRNA is amplified.

NF-κB activation

Twenty four hours before transfection, HeLa cells were seeded at a density of 20,000/well in96-well microplates. Cells were co-transfected with the pCona-luc or pIgκCona-luc plasmids(a gift of A. Israel) and a plasmid which contains a synthetic Renillia luciferase gene sequenceby the FuGENE 6 system (Roche). At twenty-four hours post-transfection, the cells wereinfected with the recombinant SIN/GFP or SIN/NS5A viruses at the MOI of 10 in thepresence of Z-VAD-fmk. At eighteen hours post-infection, the culture medium was removedand the cells were washed twice with PBS. The cells were lysed in 20 µl of lysis buffer (DualLuciferase Reporter Assay, Promega). A volume of 5 µl of each sample was analyzed forFirefly luciferase and Renillia luciferase activities in a luminometer (Berthold). The resultswere normalized in comparison with the Renillia luciferase expression. Each transfection wasperformed in four different wells, and the results presented below were the average of thefour obtained values.

Results

Characterization of Sindbis virus recombinants expressingGFP, K3L, or NS5A

To better understand the role of NS5A, we expressed this protein with an infectiousSindbis virus vector. The sequence encoding NS5A of genotype 1b resistantto interferon was inserted in a full-length Sindbis virus expression vector [20]downstream of the Sindbis virus structural proteins and positioned in a subgenomiccassette in order to be conveniently expressed under the control of the Sindbis pro-moter (Fig. 1). In addition, wild-type Sindbis virus and Sindbis virus recombinantsexpressing GFP or the Myc-tagged Vaccinia K3L protein were produced and usedas controls. The K3L protein was used as a positive control of interaction withPKR [9]. K3L shares 30% identity with eIF-2α and is a potent inhibitor of eIF-2α kinases, including PKR, by competing with eIF-2α for binding to the kinasecatalytic domain [24]. In this work, we used the hyper-resistant derivative K3L-HR47 [24]. The expression of the three heterologous proteins was analyzed afterinfection with each Sindbis virus recombinant, followed by metabolic labeling andimmunoprecipitation. Bands of the expected size were observed for the NS5A,Myc-K3L and GFP proteins, and this was confirmed by immunoblotting (data not


Fig. 1. Generation of Sindbis virus recombinants. At the top is shown the full-length SINvector (plasmid pTE3′2J) containing an additional subgenomic mRNA promoter (marked byan arrow) located immediately 3′ to the open reading frame encoding the SIN structuralproteins. After linearization of full-length plasmid templates with XhoI, 5′ capped run-off transcripts were produced. The wild-type Sindbis virus and the different Sindbis virusrecombinants were harvested after RNA electroporation and used for subsequent analyses.Numbers above the protein sequences indicate the position of the amino acid residues.For NS5A 1b, numbers below the sequence refer to the position in the HCV polyprotein,the position of the interferon sensibility determining region (ISDR) is also indicated. Theabbreviations 5′NCR and 3′NCR respectively designate the 5′ and the 3′ non coding region

of the Sindbis genome

shown). In the case of GFP, its expression was also confirmed by direct observationof bright fluorescence in living cells.

Enhanced multiplication of Sindbis virus expressing NS5A or K3L

Cells were infected with either wild-type Sindbis virus, SIN/GFP, SIN/K3L orSIN/NS5A at a MOI of 1, the kinetics of infection were followed during 9 h


Fig. 2. Viral yield. Cells were infected at the MOI of 1 pfu per cell with the wild-typeSindbis virus or the SIN/GFP, SIN/K3L, or SIN/NS5A recombinants. At different times post-infection, the viral yields (expressed as log pfu/ml) were titrated on Vero VC10 cells. Thekinetics of infection were performed with wild-type Sindbis (crosses), SIN/GFP (circles),SIN/K3L (squares), and SIN/NS5A (triangles) at 3, 5, 7 and 9 h post-infection. The resultsare the average of three independent experiments. The comparison of the data was analyzed

statistically by one-way analysis of variance (StatViewTM)

(Fig. 2). Each virus grew well with continuous increase in their yield over time.We followed the viral yields during 9 hours to focus the study to one viral lifecycle. No difference of growth was observed between the wild-type Sindbis virusand the recombinant SIN/GFP virus. On the contrary, we depicted significantdifferences in the growth of the recombinant viruses. The yield of the SIN/K3Lwas 2 logs higher than SIN/GFP after 7 h and still 1.7 log higher 9 h post-infection.The yield of SIN/NS5A was also significantly higher than this of SIN/GFP,although the difference was lower than for SIN/K3L (1 log at 7 and 9 h post-infection). Thus, NS5A and K3L, when expressed in the context of the Sindbisvirus, increased the yield of these recombinant viruses. An immediate explanationfor this growth advantage could be the ability of these proteins to inhibit thenegative control of PKR on protein synthesis, since such a mechanism has beenextensively demonstrated for K3L and also reported for NS5A [24]. Then, weinvestigated the effect of the recombinant SIN/K3L and SIN/NS5A viruses onPKR activity.


NS5A does not inhibit eIF-2α kinase activity

The eIF-2α kinases, such as PKR or PERK, can be activated during a viralinfection. PKR is activated in the presence of dsRNA, a situation which occursfrequently during a viral infection due to the accumulation of replicative inter-mediate forms or intrinsic dsRNA viral structures. The activity of eIF-2α kinasesin cells can be monitored by immunoblotting with antibodies specific for thephosphorylated form of their substrate, eIF-2α. Consequently, any effect resultingin inhibition of eIF-2α kinase activity can be observed by analyzing the decreasein the phosphorylation state of eIF-2α. The eIF-2α kinase activation was expectedto occur in the case of Sindbis virus infection. Indeed, infection with SIN/GFPled to an increase in the levels of phosphorylated eIF-2α (Fig. 3). The maximalincrease was observed at 7 and 9 hours post-infection, with concomitant increase

Fig. 3. eIF-2α phosphorylation. Cells were infected at the MOI of 1 pfu per cell. At differenttimes post-infection, the cells were scrapped and lysed in buffer I as described in Materialsand methods. The proteins were separated by SDS-PAGE and analyzed by immunoblottingto detect the presence of the GFP protein (A), Myc-K3L-HR47 (B) or NS5A (C) with specificantibodies. In parallel experiments, eIF-2α, as well as its phosphorylated form, were analyzed


in the GFP levels, indicating correct ongoing viral expression (Fig. 3A). In thecase of infection with SIN/K3L, no eIF-2α phosphorylation was observed, evenat 7 and 9 h post-infection, at times of K3L accumulation in the cells (Fig. 3B).This confirmed that expression of K3L prevents the phosphorylation of eIF-2α aspreviously reported [24] and, in this particular case, might correlate with the multi-plicative advantage observed for the viral production (Fig. 2). In contrast, infectionwith SIN/NS5A did not result in any inhibition of the eIF-2α phosphorylation inspite of NS5A expression detected at 7 and 9 h post-infection (Fig. 3C). Thesedata indicate that, unlike K3L, NS5A might confer a multiplicative advantageto Sindbis virus, not through inhibition of eIF-2α kinases, but through anothercellular pathway.

NS5A can mimic the effect of anti-apoptotic agents

Apoptosis is one of the cellular defenses to limit viral production. Infection withSindbis virus has been reported to induce cell apoptosis and this effect can bemonitored by DNA fragmentation (data not shown). Thus, it is possible that theviral yield observed for SIN/NS5A, as compared with SIN/GFP, might be due to aninhibitory effect of NS5A on the cellular apoptosis program. Such a situation hasbeen described for other viral gene products [46]. In order to test whether NS5Acould participate in an anti-apoptosis program, cells were infected for 15 hourswith SIN/GFP or SIN/NS5A either alone or in presence of the caspase inhibitorZ-VAD-fmk. The use of this inhibitor conveniently allows to evaluate the viralyield that can be obtained when cellular caspase-dependent apoptosis is prevented.Accordingly, in the case of SIN/GFP, addition of Z-VAD-fmk resulted in 2.6 foldincrease of viral titer (Fig. 4). If NS5A played no anti-apoptotic role, Z-VAD-fmk would be expected to increase this yield by 2.6 folds similarly to SIN/GFP.However, the caspase inhibitor did not significantly boost the SIN/NS5A yield,suggesting that NS5A could promote Sindbis virus multiplication by interferingwith apoptosis. In agreement with the data presented in Fig. 2, the infection withSIN/NS5A resulted in a higher viral yield (1 log difference) than with SIN/GFP.The same experiments have been realized with the SIN/K3L. The obtained resultswere similar to the SIN/NS5A. It is not surprising since K3L is an inhibitor ofPKR which is implicated in apoptosis (data not shown).

Possible nuclear localization of NS5A in cells undergoing apoptosis

NS5A normally localizes in the cytoplasm, in association with ER or ER-likemembranes [4, 36], but its C-terminal part has been reported to migrate to thenucleus as a result of specific cleavages from the cellular apoptosis machinery [41].Here, we used confocal immunomicroscopy to further characterize the cellularlocalization of NS5A in relation with apoptosis. For this purpose, HeLa cells wereinfected with SIN/NS5A, in the presence or absence of Z-VAD-fmk. Developmentof apoptosis during Sindbis infection was assessed by labeling the nuclear DNAwith propidium iodide. A high disorganization of the nuclear morphology could


Fig. 4. Z-VAD virus rescue. Cells were treated or not with 100 µM of Z-VAD-fmk, theninfected at the same MOI with SIN/GFP or SIN/NS5A. At 18 hours post-infection, the pro-geny viruses were titrated on Vero VC10 cells as described in Materials and methods. Theresults are the average of three independent experiments. The comparison of the data wasanalyzed statistically by one-way analysis of variance (StatViewTM). The asterisk indicates

that p < 0.05 between the results obtained with SIN/GFP in comparison with SIN/NS5A

be observed at 15 h post-infection in cells not treated with Z-VAD-fmk (Fig. 5Aand B). In contrast, the Z-VAD-fmk mediated inhibition of apoptosis was revealedby a decrease in the number of apoptotic nuclei (Fig. 5C). The NS5A protein wasfound to localize in the cytoplasm at 7 hours post-infection and a large proportionappeared in the nucleus after 15 h, concomitantly with apoptosis. In cells treatedwith Z-VAD-fmk, most of NS5A remained in the cytoplasm. These observationsare in agreement with the data of Satoh et al. showing that NS5A can migrate tothe nucleus under apoptosis conditions [41].

NS5A induces IL-8 mRNA

The NS5A protein has recently been shown to transactivate the expression of theIL-8 chemokine, an effect which was found to be correlated with the presenceof truncated forms of NS5A in the nucleus. Interestingly, IL-8 was shown todown-regulate the synthesis of some IFN-induced proteins and therefore couldinterfere with some mechanisms of IFN-induced apoptosis [37]. Here, we in-vestigated whether NS5A was also able to induce IL-8 mRNA when expressedfrom the recombinant SIN/NS5A virus. HeLa cells were infected with SIN/GFP orSIN/NS5A in the presence or absence of Z-VAD-fmk. At 15 h post-infection, totalRNAs were extracted and the presence of IL-8 mRNA was determined by RT-PCR.


Fig. 5. Subcellular localization of NS5A. Cells were cultured in eight-well chamber slidesand infected with the SIN/NS5A in the absence of Z-VAD-fmk. At 7 and 15 h post-infection,the localization of NS5A (green fluorescence) was analyzed (A) and (B), respectively. Thenuclear DNA was stained with propidium iodide (red fluorescence). C shows the localization

of NS5A at 15 h post-infection in the presence of Z-VAD-fmk

We first tested the capacity of the wild-type Sindbis virus to induce IL-8 transcriptRNAs. As shown in Fig. 6A, IL-8 mRNA was detected in cells infected with wild-type Sindbis virus, indicating that the virus itself is a potent inductor of the IL-8mRNA. In the absence of Z-VAD-fmk, both SIN/GFP and SIN/NS5A inducedIL-8 mRNA in normal growth conditions (Fig. 6B). Interestingly, in the presenceof Z-VAD-fmk, the induction of IL-8 mRNA was inhibited in cells infectedwith SIN/GFP but not in those infected by SIN/NS5A (Fig. 6B). Therefore,these data suggest that NS5A presents an intrinsic ability to induce IL-8 mRNA,independently of that mediated during Sindbis virus infection. The fact that thiseffect occurs in Z-VAD-fmk treated cells, in which localization of NS5A ismainly cytoplasmic, indicates that NS5A can induce IL-8 mRNA, regardless ofits localization.


Fig. 6. Induction of the IL-8 mRNA. A Cells were infected or not with wild-type Sindbisvirus. Total RNAs were extracted and the IL-8 mRNA was detected by RT-PCR. Lane Mindicates the molecular size marker. B Cells were infected with SIN/GFP or SIN/NS5A, andtreated or not with Z-VAD-fmk, IL-8 mRNA was also detected by RT-PCR. The level of

GAPDH mRNA was shown as a control (below)

NS5A activates NF-κB independently of Sindbis virus

The IL-8 induction is dependent on the NF-κB, NF-IL-6 and AP-1 transcriptionfactors [33]. The NS5A protein may stimulate either one of their correspondingsignaling pathways. We chose to analyze the effect of NS5A on NF-κB pathway,based on previous data by Gong et al. which showed that NS5A was able to activateNF-κB after modification of the intracellular calcium levels [19]. Cells were co-transfected with the reporter plasmids expressing luciferase under the control of aminimal promoter (pCona-luc), or a promoter containing three upstream NF-κBresponse elements (pIgκCona-luc), and a plasmid containing a synthetic Renillialuciferase gene sequence. The cells were infected and used to monitor NF-κBstimulation as described previously [3]. Infection was carried out with SIN/GFP orSIN/NS5A in the presence of the caspase inhibitor, and we analyzed the inductionof NF-κB independently of the apoptosis cascade. Infection with either virus wasfound to stimulate the expression of the NF-κB-dependent luciferase reporter(Fig. 7). However, the induction of the luciferase gene was higher when the cellswere infected by SIN/NS5A than by SIN/GFP, thus showing that the expressionof NS5A increases the ability of the virus to induce NF-κB (Fig. 7). WhetherNS5A or the virus stimulates the NF-κB signaling pathway through the samemechanism remains to be determined. However, these data clearly show that theNF-κB signaling pathway can be used by NS5A to induce IL-8 mRNA.


Fig. 7. Activation of NF-κB by NS5A. Cells were co-transfected with pCona-luc or pIgκCona-luc and pRenillia-Luc plasmids. At 24 h post-transfection, cells were infected with SIN/GFP(white boxes) or SIN/NS5A (black boxes) in the presence of Z-VAD-fmk. At 18 h post-infection, the cells were lysed and analyzed for luciferase activity. Luc index is the ratiobetween the firefly luciferase activity and the Renillia luciferase activity. The results are theaverage of four experiments. The comparison of the data was analyzed statistically by one-wayanalysis of variance (StatViewTM). The asterisk indicates that p < 0.05 between the results

obtained with SIN/GFP in comparison with SIN/NS5A

Discussion

Because of a lack of suitable cell culture system for infection with HCV, the roleof the NS5A protein has been analyzed in non-virological systems, expressedfrom inducible vectors either alone or in the presence of other HCV proteins.In such conditions, NS5A was found to interfere with IFN signaling pathwaysand to improve cell growth [15, 28]. Here, for the first time, we expressed NS5Afrom a Sindbis virus recombinant to analyze its direct effect on viral productionin an infectious system. Sindbis virus is an enveloped virus with a positive-stranded RNA genome, like HCV. This system allowed to demonstrate that theexpression of NS5A could confer a multiplicative advantage to Sindbis virus.These experiments were not possible in the replicon systems recently developedsince no viral particles are produced. Also, Sindbis produced an acute infectionwith a related cytopathic effect, which has not been clearly described for HCV as itis thought to produce a chronic infection such as BVDV. However, recently Sunget al. have established B-cell lymphoma cell lines persistently infected with HCV[42]. This cell line showed strong anti-apoptotic effect which could counteract


the cytopathic effect of HCV. Our data are in agreement with these observations,and we have shown for the first time that this effect could be related to the NS5Aprotein of HCV.

Since NS5A has been reported to interact with and inhibit PKR in somecellular systems, we first thought that such an interaction could explain the growthadvantage of SIN/NS5A. This was supported by the observation that anotherSindbis virus recombinant, engineered to express the Vaccinia virus K3L protein,a well-known inhibitor of PKR, increased the viral yield. In addition, we confirmedthat the growth advantage of SIN/K3L could be due to the inhibition of eIF-2α

kinases. However, in the case of SIN/NS5A, no inhibition of the phosphorylationof eIF-2α was observed. This result confirms recent data which showed no evidentrole for NS5A as an eIF-2α kinase inhibitor [11, 12, 35]. It remains possiblethat NS5A inhibits only PKR and not the other eIF-2α kinases. Therefore, themechanisms leading to a multiplicative advantage for the SIN/NS5A recombinantappear to be different from that used by the SIN/K3L. For these experiments, weused the SIN/GFP as a control because this virus expressed an heterologous proteinas the others. Moreover, the viral yields obtained with SIN/GFP was similar withwild-type Sindbis virus and other Sindbis virus recombinants expressing non-relevant proteins (data non shown).

Infection with Sindbis virus triggers cell apoptosis through the pro-apoptoticmembers of the Bcl-2 family, such as Bad [32], an effect which can be blocked bycaspase inhibitors, such as Z-VAD or the vFLIP protein of the human herpes virusHHV8 [40]. Accordingly, we found here that Z-VAD-fmk could increase the yieldof SIN/GFP by 2.6 folds. In contrast, no significant increase in virus yield wasobserved in the case of SIN/NS5A. Since the viral yield of SIN/NS5A was higherthan SIN/GFP, this may indicate that NS5A alone can exert an anti-apoptotic effectwhich would give a multiplicative advantage to the virus. Barber et al. reported thatNS5A could not inhibit induction of apoptosis by treatment with IFN, TRAIL oretoposide [11]. However, NS5A has been reported to have an anti-apoptotic effectby inhibiting the activation of the caspase-3 and leading to a protection againstTNF-α-induced apoptosis [16]. If this was the case, Z-VAD-fmk, an irreversiblepan-caspase inhibitor, would not provide any additional multiplicative advantageto the recombinant SIN/NS5A virus. This is indeed what we observed. Sevenhours post-infection, the Sindbis-induced apoptosis was already triggered (datanot shown), a potential block might explain the multiplicative advantage depictedat 7 and 9 h post-infection. We focused some experiments at 15 h post-infection inorder to validate the observations of these previous effects. The same experimentsperformed at 7 and 9 h post-infection have shown the same trends but the resultsbecame significant only after 15 hours. Finally, the cells died suggesting a partialblock of apoptosis.

The NS5A sequence presents two interesting features: some putative cleavagesites by a caspase-like protease [41] and a nuclear localization signal (NLS).This later is not functional because of the presence of an ER-retention signallocated in the amino-terminus of the protein [4]. However, under the action ofcaspases, the NLS-containing part of NS5A can separate from its N-terminus,


migrate to the nucleus and function as transactivator. Infection with Sindbisvirus triggers apoptosis and therefore may induce a change in the localizationof NS5A. By confocal immunomicroscopy, we observed that, at the beginning ofinfection, expression of NS5A from SIN/NS5A recombinant was mainly locatedin the cytoplasm, in the perinuclear area as already described [36]. However,later on, changes were evident, and a possible nuclear localization of the NS5Aprotein was progressively observed and it correlated with the ongoing of apoptosis.Interestingly, these results show that Sindbis virus-induced apoptosis might allowthe nuclear translocation of NS5A in agreement with Satoh et al. [41]. In thepresence of the caspase inhibitor, NS5A remained mainly in the cytoplasm. Forthe SIN/GFP, we observed no modification of the GFP localization during theapoptosis (data not shown).

It has been suggested that the translocation of NS5A into the nucleus mighttransactivate the IL-8 promoter and lead to the production of this CXC-chemokine[18]. It has been shown that IL-8 enhances the replication of several viruses,including EMCV, poliovirus, CMV, and HIV [26]. Moreover, IL-8 can inhibit theantiviral activity of α-IFN in vitro and therefore, the NS5A-mediated inductionof IL-8 has been suggested to be involved in HCV resistance to IFN [25, 37].However, IL-8 is known to be induced during infection by a number of viruses[39] and was also induced during infection with our control Sindbis virus. Suchan induction can be inhibited in the presence of the caspase inhibitor Z-VAD-fmk, indicating that during viral infection, at least in the case of Sindbis virus,the IL-8 mRNA induction depends on an apoptosis state according to previousreports [6]. However, in the case of SIN/NS5A infection, IL-8 mRNA inductionpersisted, even in presence of the caspase inhibitor. These data suggest that NS5Acan transactivate the IL-8 gene, independently of the Sindbis-induced apoptosisprogram, either directly by activating the promoter after nuclear translocation, orthrough the activation of signaling pathways for transcription factors responsibleof the transactivation. The IL-8 gene contains three major binding sites for theNF-κB, AP-1 and NF-IL-6 transcription factors [39]. Since it has been shown thatNF-κB can be activated by NS5A [19], we have analyzed whether this would bethe case in our model. We found that NF-κB was activated when cells were infectedwith SIN/GFP or SIN/NS5A, but the induction was higher with SIN/NS5A.Altogether, our data indicate that the expression of NS5A from Sindbis virusallows the induction of IL-8 mRNA, in a pathway different from that used bythe virus alone, and that the NF-κB pathway, at least in part, is involved in thisprocess. Recently, specific activation of the NF-κB pathway by NS5A alone or inthe context of the HCV subgenomic replicon was demonstrated [47].

In conclusion, our results indicate that the NS5A protein has the ability toprovide a multiplicative advantage possibly by an anti-apoptotic effect. Thisadvantage could not be explained by an escape from a control on protein synthesis,since eIF-2α phosphorylation was not affected. In contrast, NS5A was found tofavor IL-8 mRNA induction potentially through activation of NF-κB signalingpathway, independently of the own ability of Sindbis virus to induce IL-8. Inthe case of HCV infection, the situation may be similar, and the NS5A-sustained


production of IL-8 will also likely provide the virus with the possibility to escapesome of the actions of IFN. Together, our data suggest that the activation ofNF-κB could lead to the anti-apoptotic activity of NS5A and explain the viralmultiplicative advantage conferred by the expression of the NS5A protein.

Acknowledgements

The first two authors contributed equally to this work. We are grateful to Emmanuelle Perret(Institut Pasteur, Paris) for assistance in laser confocal microscopy. This work was supportedby a PRFMMIP grant from the French Ministry of Research and by a contract from theEuropean Community HEPAC-RESIST N◦QLK-CT-2002-00954, by the “Centre Nationalde la Recherche Scientifique” or CNRS and the Institut Pasteur, Lille.

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Author’s address: Dr. Gilles Duverlie, Laboratoire de Virologie, CHU d’Amiens, 80054Amiens, Cedex 1, France; e-mail: [email protected]

positive effect of the hepatitis c virus nonstructural 5a protein on viral multiplication

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