Hepatitis C Virus NS5A and Subgenomic Replicon Activate NF-κB via Tyrosine Phosphorylation of IκBα and Its Degradation by Calpain Protease*

Hepatitis C virus nonstructural protein 5A (NS5A) has been implicated in the HCV antiviral resistance, replication, and transactivation of cellular gene expression. We have recently shown that HCV NS5A activates NF-κB via oxidative stress (22). In this study, we investigate the molecular mechanism(s) of NF-κB activation in response to oxidative stress induced by NS5A protein. In contrast to the classic Ser32,36 phosphorylation of IκBα, we report here that tyrosine phosphorylation of IκBα at Tyr42 and Tyr305 residues is induced by the HCV NS5A and the subgenomic replicons in the NF-κB activation process. Use of IκBα-Tyr42,305 double mutant provided the evidence for their key role in the activation of NF-κB. Activation of NF-κB was blocked by a series of tyrosine kinase inhibitors but not by IκB kinase inhibitor BAY 11-7085. More specifically, a ZAP-70 knock-out cell line expressing NS5A and other nonstructural proteins respectively prevented the NF-κB activation, indicating the involvement of ZAP-70 as a probable tyrosine kinase in the activation process. Evidence is also presented for the possible role of calpain proteases in the NS5A-induced IκBα degradation. These studies collectively define an alternate pathway of NF-κB activation by NS5A alone or in the context of the HCV subgenomic replicon. Constitutive activation of NF-κB by HCV has implications in the chronic liver disease including hepatocellular carcinoma associated with HCV infection.

Hepatitis C virus nonstructural protein 5A (NS5A) has been implicated in the HCV antiviral resistance, replication, and transactivation of cellular gene expression. We have recently shown that HCV NS5A activates NF-B via oxidative stress (22). In this study, we investigate the molecular mechanism(s) of NF-B activation in response to oxidative stress induced by NS5A protein. In contrast to the classic Ser 32,36 phosphorylation of IB␣, we report here that tyrosine phosphorylation of IB␣ at Tyr 42 and Tyr 305 residues is induced by the HCV NS5A and the subgenomic replicons in the NF-B activation process. Use of IB␣-Tyr 42,305 double mutant provided the evidence for their key role in the activation of NF-B. Activation of NF-B was blocked by a series of tyrosine kinase inhibitors but not by IB kinase inhibitor BAY 11-7085. More specifically, a ZAP-70 knock-out cell line expressing NS5A and other nonstructural proteins respectively prevented the NF-B activation, indicating the involvement of ZAP-70 as a probable tyrosine kinase in the activation process. Evidence is also presented for the possible role of calpain proteases in the NS5A-induced IB␣ degradation. These studies collectively define an alternate pathway of NF-B activation by NS5A alone or in the context of the HCV subgenomic replicon. Constitutive activation of NF-B by HCV has implications in the chronic liver disease including hepatocellular carcinoma associated with HCV infection.
Human hepatitis C virus (HCV) 1 causes acute and chronic hepatitis (1, 2) with a significant risk of cirrhosis and hepatocellular carcinoma (3)(4)(5). The positive-sense single-stranded enveloped RNA genome is translated in an internal ribosome entry site (IRES)-dependent manner to generate a single precursor polypeptide, which is proteolytically processed into at least three structural proteins (core, E1, and E2) and six nonstructural proteins (NS2-NS5B) (6 -8) (Fig. 1A). The development of subgenomic HCV RNA replicons represents a major break-through in the HCV field (9). The HCV subgenomic replicon is a bi-cistronic RNA, containing a neomycin resistance gene under the translational control of HCV IRES followed by the HCV nonstructural proteins encompassing NS3 through NS5B, and 3Ј-NCR under the translational control of EMCV IRES (Fig. 1B). G418 selection is used to maintain the replication of subgenomic replicon in the Huh7 cells (9). Recently full-length hepatitis C virus RNA genome replicons have been reported (10,11). The functions of non-structural proteins are continually been characterized (12,13).
Since its suggestive role in IFN-resistance, NS5A has been the subject of intense investigations. NS5A has been shown to interact with several cellular targets. The interferon sensitive determining region (ISDR) of NS5A has been shown to bind double-stranded RNA-dependent protein kinase (PKR) (14), and inhibit its kinase activity (15). Interaction between NS5A and PKR was shown to interfere with the phosphorylation of eIF-2␣, thus leaving protein synthesis unaffected (16). Among other cellular targets of NS5A, interactions with cellular transcription factor SRCAP, and a membrane fusion protein VAP-30, have been described (17,18). NS5A has been shown to bind NS5B (19). A surprising finding was its ability to function as a transcriptional transactivator, being a cytoplasmic protein (20,21). NS5A protein transcriptionally down-regulated the cyclindependent kinase inhibitor p21/waf1 gene and promoted cell growth (17). We have previously shown that association of HCV NS5A with the ER membrane induced a series of intracellular events including alteration of Ca 2ϩ homeostasis, elevation of reactive oxygen species (ROS), and activation of transcription factors, STAT-3 and NF-B (22).
NF-B, a sequence specific transcription factor, regulates expression of numerous cellular and viral genes and plays important roles in inflammation, innate immune responses, tumorigenesis, and cell survival (23,24). In resting cells, NF-B stays latent in the cytoplasm complexed with IB␣, its inhibitory subunit (25)(26)(27). Activation of NF-B occurs via IB kinase (IKK) complex-mediated Ser 32,36 phosphorylation of IB␣ followed by ubiquitin-proteasome-dependent degradation after exposure to a variety of agonists (25, 28 -30). An alternative mechanism for the activation of NF-B involves phosphorylation of IB␣ at Tyr 42 , and PEST (Pro-Glu-Ser-Thr) sequences under oxidative stress (31,32). Livolsi et al. (31) have shown that tyrosine kinases act at several levels to dissociate IB␣-NF-B complexes. Tyrosine phosphorylation of IB␣ has This article has been withdrawn by the authors. Antonia Livolsi, Véronique Imbert, and Jean-François Peyron participated in the study by providing IB mutant plasmids. An investigation by the Journal determined the following. A band was pasted over in the NS5A immunoblot in Fig Fig. 9A were duplicated. The authors who performed the experiments were unable to clarify the Journal's concerns with original data. Gulam Waris does not agree that the actin immunoblot was reused in other publications. Aleem Siddiqui disagrees with the determination of the reuse of actin immunoblots in publications other than the JBC paper being withdrawn. The authors sincerely apologize to the readers. also been observed during ischemia/reprefusion of the liver (33), suggesting its functional role in this pathway.
In this study, we investigated the mechanism(s) of NF-B activation in human hepatoma cell line expressing NS5A protein and the HCV subgenomic replicons respectively. Here, we demonstrate the ability of NS5A to induce activation of NF-B is mediated by IB␣ tyrosine phosphorylation at tyrosine 42 and 305 residues. Evidence is presented for a possible role of calpain protease in the IB␣ degradation following tyrosine phosphorylation. NF-B activation by NS5A also involves Ca 2ϩ signaling and the generation of ROS in the cells. These data collectively suggest a novel pathway of ER-nucleus signaling by the HCV NS5A protein either alone or in the context of the subgenomic replicon. The intracellular events triggered by the HCV translation and replication activities in the cells are relevant to the mechanism(s) of liver disease pathogenesis associated with the HCV infection.

MATERIALS AND METHODS
Plasmids and Oligonucleotides-Plasmid p3x-B-Luc (luciferase reporter driven by the minimal fos promoter with three upstream NF-B binding sites from MHC class I) was a generous gift of J. Martin (University of Colorado, Boulder). The HCV NS5A expression vector (pCNS5A) and its mutant (pCNSM4) were generated from pCMV 729/ 3010 (22). The NS5A deletion mutant plasmid pCNSM4 encodes the amino acids 2135 to 2343 of HCV NS5A, which is cloned between T7 and SP6 promoters. The plasmid pCMV 729/3010 containing coding sequences of all the nonstructural proteins was kindly provided by Dr. K. Shimotohno (Kyoto University, Japan). The plasmids expressing wildtype IB␣, IB␣ Y42F (Tyr 3 Phe), and IB␣ Y305F (Tyr 3 Phe), and IB␣ tyrosine double mutant (Y42F/Y305F) contain an in-frame Myc tag. The plasmid bearing the IB␣ S32A/S36A (Ser 3 Ala) mutated gene under the control of the CMV promoter was a gift of Robert Scheinman, University of Colorado Health Sciences Center, Denver.
Cell Culture-The human hepatoma cell lines, Huh7 and FCA4, were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal calf serum, 100 units/ml penicillin, and 100 g/ml streptomycin sulfate. FCA4 and GS4.3 cells were generous gift of Dr. C. Seeger (Fox Chase Cancer Center, Philadelphia, PA), were grown in 500 g/ml of G418 (Geneticin, Invitrogen). FCA4 cells are a Huh7 cell line stably expressing a HCV subgenomic replicons with a single adaptive mutation, a deletion of serine residue 1176 (34).
Preparation of Nuclear Extracts-Nuclear lysates were prepared from untransfected and Huh7 cells transfected with 2-3 g of indicated plasmid DNA by Lipofectin 1 mM EDTA, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 3 mg/ml aprotinin, 1 mg/ml pepstatin, 20 mM NaF, and 1 mM dithiothreitol with 0.2% Nonidet P-40) on ice for 10 min. After centrifugation at 4 C (13,000 rpm) for 1 min, the nuclear pellet was resuspended in high salt buffer (hypotonic buffer with 20% glycerol and 420 mM NaCl) at 4°C by rocking for 30 min following centrifugation. The supernatant was collected and stored in aliquots at Ϫ80°C.
EMSA-The oligonucleotide NF-B was radiolabeled at 5Ј-end with [␥-32 P]ATP by T 4 polynucleotide kinase. About 20,000 cpm of gel purified probe was incubated with nuclear lysates from FCA4 cells and Huh7 cells untransfected or transfected with pCNS5A and pCMV729/ 3010 expression vectors respectively in EMSA buffer (20 mM Tris-HCl, pH 7.9, 10 mM MgCl 2 , 50 mM KCl, 16.7 g/ml poly dI⅐dC, 1 mM EDTA, 1 mM dithiothreitol, and 1 M leupeptin) for 20 min on ice. Competition analyses were carried out in the presence of a 100-fold excess of an unlabeled competitor oligonucleotide that was preincubated for 20 min on ice prior to the addition of radiolabeled probe. The DNA-protein complexes were resolved by 5% polyacrylamide gel electrophoresis in 0.5ϫ TBE buffer. The gels were dried and subjected to autoradiography.
Immunoprecipitation and Western Blot Analysis-Exponentially growing FCA4 and Huh7 cells transfected with pCNS5A or pCMV729/ 3010 expression vectors were harvested and cell extracts were prepared . B, organization of the HCV subgenomic replicon. The HCV 5Ј-NCR fused to a small portion of the capsid coding region, the neomycin phosphotransferase gene (Neo r ). These sequences are followed by the EMCV IRES, and the HCV NS3 to NS5B coding regions, terminating at the 3Ј-NCR (9). by incubating in radioimmune precipitation assay (RIPA) buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 1 mM sodium formate, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml aprotinin, 10 g/ml leupeptin) for 30 min on ice. Immunoprecipitation was performed with anti-IB␣ serum. After 4 h of incubation, the immune complexes were captured on protein A-Sepharose, washed three times with RIPA buffer, boiled for 5 min in SDS-PAGE sample buffer and subjected to SDS-PAGE. Gels were electroblotted on to nitrocellulose membrane (Amersham Biosciences) in transfer buffer (25 mM Tris, 192 mM glycine, and 20% methanol). Membranes were treated for 1 h in blocking buffer ((20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.3% polyvinyl pyrolidone (PVP), 0.5% Tween-20 (w/v)), probed with monoclonal anti-phosphotyrosine antibody for 1 h and washed twice for 10 min with blocking buffer followed by incubation with the secondary antibody for 45 min. After an additional washing step with blocking buffer, immunoblots were visualized using ECL detection system (Amersham Biosciences). IB␣ protein was detected by Western blot analysis using either anti-Myc monoclonal or anti-IB␣ polyclonal antibodies.

RESULTS
NS5A Induces Tyrosine Phosphorylation of IB␣-To initiate these studies, we sought to determine the ability of NS5A alone or in the context of all the HCV nonstructural proteins (NS) to induce the tyrosine phosphorylation of IB␣. Cellular lysates from cells expressing NS5A, NS proteins (pCMV729/3010) and those stably expressing HCV subgenomic replicons (FCA4 cells) (34) were immunoprecipitated with anti-IB␣ serum, fractionated by SDS-PAGE, and electroblotted onto a nitrocellulose membrane. The membrane was then incubated with anti-phosphotyrosine monoclonal antibody. This analysis shows that the cells expressing NS5A alone or in the context of all nonstructural proteins, and subgenomic replicon contained tyrosine-phosphorylated IB␣ ( Fig. 2A, lanes 2-4). Neither untransfected ( Fig. 2A, lane 1), nor the NS5A deletion mutant, which does not target to the ER (22) (Fig. 2A, lane 5) and nor the NS5B (RNA-dependent RNA polymerase)-expressing cells ( Fig. 2A, lane 6) induced tyrosine phosphorylation of IB␣. The involvement of ROS and Ca 2ϩ signaling in the IB␣ tyrosine phosphorylation induced by HCV NS5A was further evaluated by using antioxidant PDTC and calcium chelator BAPTA-AM. NS5A expressing cells treated with these reagents effectively inhibited tyrosine phosphorylation of IB␣ ( Fig. 2A, lanes 7 and  8). H 2 O 2 has been previously shown to induce tyrosine phosphorylation of IB␣ in immune cells (31). Here we reproduce this effect in Huh7 cells. H 2 O 2 -treated Huh7 lysates were immunoprecipitated with anti-IB␣ serum and immunoblotted with anti-phosphotyrosine antibody. The results described in (Fig. 2B, lanes 1 and 2) show that H 2 O 2 was able to induce tyrosine phosphorylation of IB␣. To confirm the identity of phosphorylated IB␣, lysates from Huh7 cells expressing NS5A were also immunoblotted with anti-IB␣ serum. The NS5A expressing cells contain both the phosphorylated and the unphosphorylated forms of IB␣ (Fig. 2C, lane 2) whereas untransfected Huh7 cells show the presence of only the unphosphorylated band (lane 1). These observations together demon-strate that NS5A and HCV subgenomic replicons are capable of constitutively phosphorylating IB␣ at tyrosine residues in the absence of a cytokine.
Tyrosine Kinase-mediated Activation of NF-B-To determine whether protein-tyrosine kinase (s) (PTK) is involved in the NF-B activation induced by NS5A and HCV replicon, several known inhibitors of PTK were employed in the EMSA. A distinct DNA-protein complex was formed between NF-B and its cognate oligonucleotide in NS5A expressing cells (Fig.  3A, lane 3) which was abrogated in the presence of PTK inhibitors (Fig. 3A, lanes 4 -6). However, BAY11-7085, an inhibitor of IKK did not affect the appearance of the NF-B DNA protein complex (Fig. 3A, lane 7). The specificity of DNA-protein interaction was confirmed by supershift of DNA-protein complex in the presence of an antibody to the NF-B subunit p65 (Fig. 3B,  lane 3) and using unlabeled competitor oligonucleotides representing the NF-B sequences (Fig. 3B, lane 4). We observed similar inhibition of DNA-protein complexes by PTK inhibitors in HCV subgenomic replicon expressing cells that were either stably (FCA4) or transiently transfected with BM4 -5 RNA (Fig. 3C, lanes 3 and 4; D, lanes 4 -6). Huh7 cellular lysates subjected to EMSA produced a modest level of NF-B protein-DNA interaction (Fig. 3A, lane 2 . Treatment of untransfected Huh7 nuclear lysates with PTK inhibitors did not seem to have any dramatic effect on the intensity of the DNA-protein complex (Fig. 3E). Our results with cell-based reporter assays, which confirm the results shown in Fig. 3, are described in Fig. 4. The NF-B controlled reporter gene activity was similarly reduced in the presence of these tyrosine kinase inhibitors (Fig. 4, A-D). In contrast to the effect of inhibitors of PTK and JAK kinase inhibitor (AG490), treatment of cells with protein kinase C inhibitor, H7 and more importantly the IKK inhibitor, BAY 11-7085 had no effect on NF-B activation induced by NS5A (Fig. 4A), pCMV729/3010 vector transfected (Fig. 4B) and HCV subgenomic replicon expressing FCA4 (Fig. 4C) and GS4.3 cells (Fig. 4D). GS4.3 cells represent a different neomycin-resistant clone of Huh7 cells stably expressing replicon RNA. Treatment of untransfected Huh7 cells (control) lysates with PTK inhibitors had no effect on the NF-B-driven reporter gene expression (Fig. 4E). Huh7 cells transfected with NF-B-controlled reporter gene (p3X-B-Luc) and treated with TNF-␣ were used as a positive control for activation of NF-B (Fig. 4F). TNF-␣Ϫmediated stimulation of NF-B has been reported previously (31). These results suggest that NS5A or the HCV replicon-induced IB␣ phosphorylation and subsequent NF-B activation is dependent on tyrosine kinases and independent of IKK activity.
Since piceatannol, a specific ZAP-70/Syk inhibitor, eliminated NF-B activation, we further investigated the activation of NF-B in a ZAP-70 knock-out cell line. Nuclear lysates from Jurkat (JE6) and ZAP-70 knockout (P116) cells (35) expressing NS5A or HCV nonstructural proteins (729/3010) were incubated with NF-B consensus oligonucleotide probe during EMSA. Expression of NS5A or the nonstructural proteins (729/ 3010) activated NF-B in Jurkat (JE6) cells (Fig. 5A, lanes 3  and 4) but no activation was observed in ZAP-70 deficient (P116) cells (Fig. 5A, lanes 6 and 7). The specificity of DNAprotein complex formation was confirmed by a supershift of the complex in the presence of anti-p65 serum (Fig. 5B, lane 3) and by unlabeled NF-B consensus competitor oligonucleotide (Fig.  5B, lane 4). We further show that ZAP-70 tyrosine kinase is expressed in Huh7 cells as evidenced by Western blot assay (Fig. 5C). Together, these results unambiguously rule out the involvement of IKK and suggest the possible functional role of ZAP-70 kinase as one of the kinases responsible in the NS5Ainduced NF-B activation.
Next, we examined the status of IB␣ tyrosine phosphorylation in cells co-expressing NS5A along with the tyrosine (Y42F and Y305F), and serine (S32A/S36A) IB␣ mutants. Cellular lysates from these cells were immunoprecipitated with anti-IB␣ serum and Western blotted with anti-phosphotyrosine monoclonal antibody. The results show that cells expressing wild-type IB␣ and Ser 32,36 mutant contained tyrosine phosphorylated IB␣ (Fig. 6, lanes 2-4 and lane 6). Similarly, individual tyrosine (Y42F and Y305F) mutant IB␣-expressing cells displayed presence of tyrosine phosphorylated IB␣. Cells co-expressing NS5A and IB␣ tyrosine double mutant (Y42F/ Y305F), however failed to show tyrosine-phosphorylated IB␣ (lane 5). These data suggest that HCV NS5A induced phosphorylation of IB␣ requires both Tyr 42 and Tyr 305 residues but not the Ser 32,36 residues. The fact that IB␣ tyrosine mutation at either Y42F or Y305F map positions retained the tyrosine phosphorylation suggests that either residue is sufficient for tyrosine phosphorylation of IB␣ and subsequent activation of NF-B.
Tyrosine 42 and 305 Residues of IB␣ Are Involved in HCV NS5A-induced IB␣ Degradation-To determine the mechanism of NF-B activation by HCV NS5A, we analyzed the degradation of IB␣ serine and tyrosine mutants. Cellular lysates from cells co-expressing NS5A and Myc-tagged IB␣ (wild type) or IB␣ Tyr 42 and Tyr 305 mutants were analyzed by Western blot analysis using anti-Myc monoclonal antibody. As shown in Fig. 7A, wild-type IB␣ was degraded in NS5Aexpressing cells (lane 3), whereas individual Tyr 42 and Tyr 305 mutant IBs were not degraded (Fig. 7A, lanes 5 and 7). Similarly, the wild-type IB␣ was also degraded in FCA4 cells (Fig.  7B, lane 2) but IB␣ Tyr 42 and Tyr 305 mutants were not degraded (Fig. 7B, lanes 3 and 4). Cytokine-induced phosphorylation of IB␣ at Ser 32,36 residues is an essential step for subsequent proteasome-mediated degradation (30). To examine whether IB␣ phosphorylation at these sites is required for HCV NS5A-induced IB␣ degradation, cellular extracts from cells co-expressing IB␣ Ser 32,36 double mutant and NS5A were immunoblotted with anti-IB␣ serum. The IB␣ Ser 32,36 was degraded by HCV NS5A (Fig. 7C, lane 3), indicating the lack of involvement of serine phosphorylation of IB␣ in the NS5A-induced NF-B activation. In summary, our results clearly identify Tyr 42 and Tyr 305 residues of IB␣ as key amino acids in the HCV NS5A and replicon-induced IB␣ tyrosine phosphorylation, followed by its degradation and subsequent NF-B activation. Tyr 305 residue has not been previously implicated in the NF-B activation. To examine the expression levels of IB␣, lysates from Huh7 cells expressing wild-type IB␣, and various IB␣ mutants were immunoblotted with anti-IB␣ serum. The results show similar levels of IB␣ expression (Fig. 7D). The differential migration pattern of these IB␣ proteins reflects different conformations of the IB␣ due to Myc tag addition to the IB␣ gene.
HCV NS5A Induces the Mitochondrial Ca 2ϩ Uptake-We have previously shown that NS5A expression induces alteration of Ca 2ϩ homeostasis (22). Ca 2ϩ released from the ER is readily taken up by mitochondria, located near Ca 2ϩ release channel SERCA (36). To determine whether mitochondrial uptake of Ca 2ϩ is involved in the NF-B activation, we employed ruthenium red (RR) and its derivative Ru360, the known inhibitors of mitochondrial Ca 2ϩ uptake, in this study (37,38). Cells treated with RR and Ru360 significantly reduced the replicon and NS5A-induced NF-B activity during EMSA (Fig.  8A, compare lane 3 with lane 6) and in cell-based luciferase assays (Fig. 8B). The chelation of intracellular calcium with BAPTA-AM and TMB-8 reduced the intensity of the DNAprotein complex (Fig. 8A, compare lane 3 with lanes 4 and 5) and affected the NF-B activity in cell-based assays in both NS5A and pCMV729/3010 (expresses all NS proteins)-transfected cells (Fig. 8B). Treatment of untransfected Huh7 cells with BAPTA-AM, RR, and Ru360 (Fig. 8B) did not affect the marginal NF-B driven reporter gene activity. Similar effects were observed in GS4.3 cells treated with calcium chelators (Fig. 4D). These results demonstrate that Ca 2ϩ signaling is an important aspect of NS5A-induced NF-B activation and that the mitochondrial uptake of calcium is an important step in the pathway.
Calpain-mediated IB␣ Degradation-Since activation of NF-B is associated with the degradation of IB␣, we examined the effect of proteasome inhibitor, MG132 on IB␣ degradation. In cells expressing NS5A, the degradation of IB␣ occurred despite the presence of proteasome inhibitor, MG132 (Fig. 9A,  lane 5). Because Ca 2ϩ signaling has been implicated in the present NF-B activation process (22), we investigated whether calpain protease, which is activated by Ca 2ϩ , is involved in the IB␣ degradation. Treatment of cells with inhib-itors of calpain protease, E64D and ALLM (39,40), blocked the IB␣ degradation in NS5A-transfected (Fig. 9A, lane 4) and replicon-expressing FCA4 cells (Fig. 9C, lanes 2 and 3). The IB␣ Ser 32,36 mutant was degraded (Fig. 9B, lanes 2 and 3) in the presence of NS5A just like the wild-type IB␣ (Fig. 9A, lane  3). The observed degradation was blocked in the presence of calpain inhibitors (Fig. 9B, lanes 4 and 5). Luciferase reporter assays were also used to examine the effect of calpain inhibitors on IB␣ degradation and subsequent NF-B activation. The activation of NF-B was blocked when NS5A and FCA4 cells were treated with calpain inhibitors (data not shown). These results together implicate a potential role of calpain in the IB␣ degradation induced by NS5A alone or in the context of subgenomic replicon. Under normal conditions, calpain and calpastatin exist in a heteromeric complex (41). Ca 2ϩ apparently binds calpain, which leads to its dissociation from the complex (42). Once dissociated, calpastatin is degraded by calpain protease. We examined the status of calpain and calpastatin in NS5A expressing cells. Western blot analysis shows stable expression of calpain in untransfected and NS5A expressing cells (Fig. 9D, lanes 1 and 2), whereas calpastatin appears to be rapidly degraded in the NS5A expressing cells (Fig. 9D, lane 2). This pattern of calpain and calpastatin proteins in the NS5A-expressing lysates is consistent with the model of calpain activation reported previously (42). These data support the view that the pathway of IB␣ degradation induced by HCV is regulated by calpain proteases in contrast to proteasome machinery. DISCUSSION Upon HCV infection cells become programmed to produce large amounts of HCV proteins that must be processed through ER. This causes an ER stress response, which unchecked can lead to cell death. Cells adapt for survival by activating prosurvival genes (24). One of the major players in this process is NF-B (23). The results described here invoke an ER-nucleus signal transduction pathway induced by HCV nonstructural proteins, which ultimately leads to the activation of NF-B. NF-B responsive motifs are found in several cellular genes (24), and a high levels of NF-B activity has been observed in chronic hepatitis C livers and HCC (26,43,44). HCV nonstructural proteins are associated with the ER membrane in the reticular network of the perinuclear region and are believed to form a ribonucleoprotein complex along with the viral RNA genome that engages in RNA replication (10,12). The activities of translation and replication associated with the HCV life cycle cause induction of unfolded protein response (UPR) and ER stress (22,45). One of the consequences of ER stress response is the Ca 2ϩ release from the ER, initiating a series of intracellular events induced by Ca 2ϩ signaling. Our results show that the expression of NS5A is sufficient to activate NF-B via Ca 2ϩ signaling and induction of ROS. Furthermore, we have assayed the NS5A function in the context of either all the NS proteins or the subgenomic replicon. The exact signal NS5A triggers in these intracellular events of ER-nucleus signal transduction pathways remains to be characterized. Since, NS5A alone does not seem to induce ER stress response, 2 these events appear to be triggered by a different signaling pathway.
In the present study, we show that NS5A constitutively activates transcription factor NF-B that involves tyrosine phosphorylation and calpain protease-mediated degradation of IB␣. These activities of NS5A were also examined in the context of all the NS proteins in cells transfected with an expression vector (pCMV729/3010) or in subgenomic replicon expressing cells. A striking observation made in this work is 2 K. D. Tardif and A. Siddiqui, unpublished results. the non-requirement of the well characterized IB␣ Ser 32,36 residues and 26 S proteasome-mediated degradation of IB␣ during NF-B activation. However, the integrity of Tyr 42 and Tyr 305 is required for the IB␣ degradation during NS5A or replicon-induced NF-B activation. Involvement of Tyr 42 phosphorylation in the NF-B activation under oxidative stress conditions has been reported previously (31,32), whereas the role of Tyr 305 in this process is being reported for the first time. We also observed that antioxidant PDTC prevented IB␣ phosphorylation ( Fig. 2A), which support the previous observations that tyrosine phosphorylation of IB␣ occurs under conditions of oxidative stress (46). Our results demonstrate that both Tyr 42 and Tyr 305 residue are important for the IB␣ phosphorylation and subsequent NF-B activation.
In the present analysis, we observed that HCV replicon and NS5A induced-NF-B activation is sensitive to piceatannol, herbimycin A, AG490, and genistein. The blocking of JAK2 with AG490 abolished the NF-B activation (Fig. 4). Although JAK2 is typically associated with the STAT pathway, Siebenlist (47) in a recent report suggested a possible cross-talk between STATs and NF-B pathways and identified JAK2 to be an essential player in this process. This cross-talk may involve the activation of IB kinases, as well as the phosphorylation of IB␣ at tyrosine residues. In the present study we were able to make use of an available ZAP-70 knock-out cell line, since piceatannol, a ZAP-70/Syk specific inhibitor was effective in eliminating NF-B activation. In the ZAP-70 knock-out cell line expressing NS5A and other NS protein, a complete failure of HCV nonstructural protein mediated-NF-B activation was observed (Fig. 5). This is consistent with an earlier report that T cell-specific p56 LCK and ZAP-70 kinases are involved in pervanadate-induced IB␣ phosphorylation and NF-B activation (31). Since HCV does not replicate in Jurkat cells, we could not use the HCV subgenomic replicon expression scheme in transient transfections. ZAP-70 kinase is expressed in Huh7 cells (Fig. 5E).
Previously, it was reported that pervanadate induces NF-B activation without degradation of IB␣ (46). However, our results indicate that HCV NS5A protein and subgenomic replicons induce activation of NF-B via IB␣ tyrosine phosphorylation and its degradation. This discrepancy may be attributed to the kind of stimulus for IB␣ phosphorylation and the cell type being used. The ability of HCV replicon and NS5A to activate NF-B without serine phosphorylation of IB␣ correlates with similar observations obtained in cells treated with UV (UV-C) or pervanadate (48,49).
It has been shown that mitochondria accumulate Ca 2ϩ from intracellular microdomains of high Ca 2ϩ such as ER in the diseased or damaged cells (54). However, the physiological role of Ca 2ϩ transport into mitochondria is not clearly understood. In our model, Ca 2ϩ is effluxed from the ER as a result of HCV NS5A expression either alone or in the context of subgenomic replicon (22). The elevated cytosolic Ca 2ϩ levels can potentially activate calpain protease, which can degrade the tyrosine-phosphorylated IB␣. The mechanism(s) of HCV replicon and NS5A-induced degradation of IB␣ by calpain is under further investigation. While studies relating to the mechanism(s) of calpain activation are currently under intense study, Ca 2ϩ release after exposure to hydrogen peroxide together with calpain activation under oxidative stress conditions is well documented (55,56). Several groups have reported that the PEST sequence is essential for oxidative stress mediated degradation of IB␣ (57)(58)(59). However, we found that IB␣ containing PEST sequence mutation was degraded when expressed in the presence of HCV NS5A (data not shown).
In summary, we show that NF-B activation by HCV NS5A protein alone or in the context of subgenomic replicon involves tyrosine phosphorylation of IB␣ and its subsequent degradation by calpain proteases. Ca 2ϩ signaling via elevation of ROS in mitochondria provides the pivotal link in this pathway of ER-nucleus signal transduction (22). These studies provide important clues to the understanding of the mechanisms of chronic liver disease induced by ER stress and other intracellular events associated with HCV gene expression.