Hepatitis C Virus Nonstructural 5B Protein Regulates Tumor Necrosis Factor Alpha Signaling through Effects on Cellular IκB Kinase

ABSTRACT Hepatitis C virus (HCV) NS5B protein is a membrane-associated phosphoprotein that possesses an RNA-dependent RNA polymerase activity. We recently reported that NS5A protein interacts with TRAF2 and modulates tumor necrosis factor alpha (TNF-α)-induced NF-κB and Jun N-terminal protein kinase (JNK). Since NS5A and NS5B are the essential components of the HCV replication complex, we examined whether NS5B could modulate TNF-α-induced NF-κB and JNK activation. In this study, we have demonstrated that TNF-α-induced NF-κB activation is inhibited by NS5B protein in HEK293 and hepatic cells. Furthermore, NS5B protein inhibited both TRAF2- and IKK-induced NF-κB activation. Using coimmunoprecipitation assays, we show that NS5B interacts with IKKα. Most importantly, NS5B protein in HCV subgenomic replicon cells interacted with endogenous IKKα, and then TNF-α-mediated IKKα kinase activation was significantly decreased by NS5B. Using in vitro kinase assay, we have further found that NS5B protein synergistically activated TNF-α-mediated JNK activity in HEK293 and hepatic cells. These data suggest that NS5B protein modulates TNF-α signaling pathways and may contribute to HCV pathogenesis.

Hepatitis C virus (HCV) is the major cause of non-A, non-B hepatitis, which often leads to liver cirrhosis and hepatocellular carcinoma (1,14). More than 170 million individuals worldwide are infected with HCV. HCV belongs to a member of the Flaviviridae family and contains a single-stranded, positivesense RNA genome of ϳ9,600 nucleotides in length. The HCV genome encodes a single polyprotein precursor of approximately 3,010 amino acids that is cleaved by both cellular signal peptidase and viral protease to generate structural and nonstructural proteins (19,21,33,35). The N-terminally localized core and envelope proteins (E1 and E2) are viral structural proteins, and the remainders of the genome are nonstructural proteins. The nonstructural protein 5B (NS5B) is an RNAdependent RNA polymerase. The NS5B is the key enzyme that catalyzes the replication of HCV. We have previously demonstrated that NS5B is a phosphoprotein that is predominantly localized in the perinuclear region in the cytoplasm (24). Although RNA-dependent RNA polymerase activities have been demonstrated using both bacterially and baculovirus-expressed NS5B proteins (8,16,34,39,40,60), the detailed mechanism of HCV replication is poorly understood by the lack of an efficient cell culture system. The NS5B has been extensively characterized at the biochemical (8,34) and structural levels (10,30) and has been a prime target for inhibitors of HCV replication.
The nuclear transcription factor NF-B plays a critical role in regulating the expression of many cytokines and immunoregula-tory proteins (4)(5)(6). The NF-B complex is composed of homodimers or heterodimers of Rel and NF-B proteins, including NF-B1, NF-B2, p65, Rel B, and c-Rel (4). The activity of NF-B can be elevated by various stimuli, including tumor necrosis factor (TNF), interleukin 1 (IL-1), and phorbol esters (55). In most cells, NF-B proteins are sequestered in the cytoplasm, where they are complexed with one of three IB proteins, IB␣, IB␤, or IBε (26,36,57). Stimulation of the cells with TNF or several other activators leads to the phosphorylation of IBs on the N-terminal serine residues by IB kinase (IKK) complex. The IBs are polyubiquitinated and then rapidly degraded by the proteasome (3). Then NF-B can be translocated to the nucleus and activates target genes by binding with high affinity to B elements in their promoters (4,5).
Both kinases have two related catalytic subunits and contain an N-terminal kinase domain, a leucine zipper motif, and a helixloop-helix motif (25). IKK␣ and IKK␤ can form either a homodimer or a heterodimer via their leucine zipper motif, but the predominant IKK complex forms heterodimer (49). In IKK␣ and IKK␤ knockout cells, NF-B activation is completely inhibited (31). However, IKK␣ and IKK␤ knockout mice show different phenotypes (23,32,54). IKK␥ is the regulatory subunit of the IKK complex, and it binds to IKK␣ and IKK␤ (49). In IKK␥-deficient primary murine embryonic fibroblasts, NF-B cannot be activated by TNF-␣, IL-1, lipopolysaccharide, and other stimuli (50).
In the present study, we asked whether TNF-␣-induced NF-B and Jun N-terminal protein kinase (JNK) activations could be modulated by the HCV NS5B protein. Indeed, NS5B protein inhibited TNF-␣-induced NF-B activation in a dosedependent manner. This inhibition was mediated by NS5B-IKK␣ interaction. Interaction between NS5B and IKK␣ was further confirmed in Huh7 cells harboring the HCV subgenomic replicon. Endogenous IKK␣ kinase activity was also inhibited by the NS5B protein. Moreover, TNF-␣-stimulated JNK activity was synergistically elevated by NS5B protein.
These findings thereby provide a potential mechanism for HCV pathogenesis.
Cell culture and DNA transfection. HEK293 cells, Huh7 cells, HepG2 cells, and Cos7 cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 U/ml streptomycin, and 100 U/ml penicillin (Invitrogen). Huh7 cells harboring HCV subgenomic replicon, which was provided by Christoph Seeger (Fox Chase Cancer Center, Philadelphia, Pa.), were cultured in the presence of G418 (0.5 mg/ml). To establish the interferon (IFN)-cured cells, HCV replicon cells were treated with 100 U/ml of IFN-␣ for 2 weeks. Elimination of HCV replicon RNA was confirmed by reverse transcription-PCR and Western blotting and by the loss of resistance to G418. For the transfection experiment, ϳ5 ϫ 10 5 cells plated on 60-mm dishes were transfected with DNA by using either LipofectAMINE (Invitrogen) or the calcium phosphate method as described previously (44).
Reporter gene assay. HEK293 cells and Huh7 cells were transfected by the calcium phosphate method with 2 g of expression plasmid, 0.1 g of either NF-B luciferase reporter (29), and 0.1 g of pCH110 (Amersham Biosciences) reference plasmid. The total DNA concentration in each transfection mixture was kept constant by adjusting with an empty vector. At 24 h after transfection, the cells were stimulated with TNF-␣ (20 ng/ml; Invitrogen) for 6 h. For the AP-1 luciferase reporter assay, the AP-1 reporter gene (38) was cotransfected into HepG2 cells. Luciferase and ␤-galactosidase assays were performed as described previously (44).
In vitro binding assay and coimmunoprecipitation. cDNA corresponding to full-length NS5B was subcloned into the EcoRI and XhoI site of the pGEX-4T expression vector (Novagen). Glutathione S-transferase (GST)-NS5B fusion protein was expressed in Escherichia coli BL21(DE) (Novagen) and purified with glutathione-Sepharose 4B beads (Amersham Biosciences) as specified by the manufacturer. HEK293 cells were lysed in 400 l of cell lysis buffer A (50 mM HEPES, pH 7.6, 250 mM NaCl, 5 mM EDTA, 0.2% Nonidet P-40, and 1 mM phenylmethylsulfonyl fluoride). The cell lysates were further centrifuged at 14,000 rpm for 10 min at 4°C, and pellets were discarded. The protein concentration was determined using a Bio-Rad protein assay kit.
For in vitro binding assay, each cell lysate of Flag-IKK␣, Flag-IKK␤, and Flag-IB␣ was incubated with either GST or GST-NS5B fusion protein for 2 h at 4°C in cell lysis buffer A. Samples were washed five times with cell lysis buffer A, and the bound proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to a nitrocellulose membrane, and detected by immunoblot analysis using anti-Flag monoclonal antibody (Sigma-Aldrich).
For coimmunoprecipitation assay, Cos7 cells were infected with recombinant vaccinia virus (vTF7-3) expressing T7 RNA polymerase (17). Two hours after infection, cells were transfected with 4 g of corresponding plasmids. Following incubation at 37°C for 12 h, cells were harvested, washed twice in cold-phosphate-buffered saline, and incubated in 400 l of cell lysis buffer A. The cell lysates were triturated by 10 passes through a 25-gauge needle on ice and centrifuged at 14,000 rpm for 10 min. The supernatant was incubated with anti-Flag monoclonal antibody for 2 h. The samples were further incubated with protein A beads (Zymed) for 1 h. Beads were washed five times with cell lysis buffer A, and the bound proteins were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and detected by immunoblot analysis using mouse anti-Myc antibody (Santa Cruz Biotechnology). For coimmunoprecipitation assay using HCV subgenomic replicon cells, cell lysates were incubated with anti-IKK␣ polyclonal antibody (Santa Cruz Biotechnology) for 2 h. Following incubation with protein A-Sepharose beads, bound proteins were immunoblotted with anti-NS5B monoclonal antibody.
For in vivo binding assay between NS5B and endogenous IKK proteins, NS5B-Myc was transfected into HEK293 cells. At 36 h after transfection, cells were immunoprecipitated with either anti-IKK␣, anti-IKK␤, or anti-IB␣ antibodies and then bound protein was immunoblotted with anti-Myc antibody.
IB␣ degradation. HEK293 cells were transfected with either an empty vector or NS5B expression plasmid. At 24 h after transfection, cells were treated with TNF-␣ (20 ng/ml) for various times. The cell extracts were resolved by SDS-PAGE, transferred to a nitrocellulose membrane, and detected by immunoblotting with anti-IB␣ polyclonal antibody (Santa Cruz Biotechnology).
IKK kinase assay. The IKK kinase assay was performed as described previously (44). Briefly, cells were treated with TNF-␣ for the indicated times and lysed in 400 l of cell lysis buffer B (20 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, 2.5 mM sodium pyrophosphate, 1 mM ␤-glycerophosphate, 1 mM Na 3 VO 4 , 1 g/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride). Cell extracts were immunoprecipitated with either anti-IKK␣ or anti-IKK␤ antibodies (Santa Cruz Biotechnology) for 2 h at 4°C, and protein A-Sepharose was added for 1 h at 4°C. Immune complexes were washed twice with lysis buffer B and then washed twice with kinase buffer (20 mM HEPES, pH 7.4, 1 mM MnCl 2 , 5 mM MgCl 2 , 10 mM ␤-glycerophosphate, 0.1 mM Na orthovanadate, 2 mM NaF, and 1 mM dithiothreitol). Samples were incubated for 30 min at 30°C in 20 l of kinase buffer containing 5 Ci of [␥-32 P]ATP (NEN Life Science) and 1 g of GST-IB␣ (amino acids [aa] 1 to 54) as a substrate. The reaction mixtures were analyzed by SDS-PAGE and then detected by autoradiography. Kinase activity was quantified by measuring the radioactivity using a densitometric scanner (Bio-Rad).
JNK assay. The JNK assay was performed as described previously (43). Briefly, HEK293 cells were transfected with either an empty vector or NS5B expression vector. At 24 h after transfection, cells were stimulated with TNF-␣ (20 ng/ml) for 15 min. Total cell lysates were prepared, and JNK activity was determined using a JNK assay kit according to the manufacturer's instructions (New England Biolabs).

HCV NS5B protein inhibits TNF-␣-induced NF-B and IKK activations.
We have previously reported that NS5A protein modulated TNF-␣-induced NF-B and JNK activations in HEK293 cells (44,45). Since NS5A is physically associated with NS5B (52) and both NS5A and NS5B are essential components of the HCV RNA replication complex (46), we examined whether NS5B could modulate TNF-␣-induced NF-B signaling pathways. For this purpose, HEK293 cells and Huh7 VOL. 26,2006 HCV NS5B REGULATES TNF SIGNALING 3049 cells transfected with either empty vectors or NS5B expression plasmids were examined to see whether NS5B protein could affect TNF-␣-stimulated NF-B activation, endogenous IKK activity, and IB␣ degradation. As demonstrated by EMSA in Fig. 1A, NF-B was activated by TNF-␣ as a DNA-protein complex in vector control cells. However, TNF-␣-induced NF-B activation was significantly inhibited by HCV NS5B protein in both HEK293 and Huh7 cells. Furthermore, TNF-␣-induced SP1 activation was not affected by NS5B (Fig. 1A), indicating that NS5B specifically inhibits TNF-␣-induced NF-B activation. To examine IKK activity, endogenous IKK␤ was immunoprecipitated with IKK␤ antibody and immunocomplex kinase activity was determined by using GST-IB␣ as a substrate. Similar to NF-B, endogenous IKK␤ activation was also inhibited by NS5B protein (Fig. 1B). We have also found that NS5B protein inhibited the exogenously expressed IKK␤ kinase activity (data not shown). IB␣ is rapidly phosphorylated on Ser 32 and Ser 36 by TNF-␣ stimulation and subsequently degraded by the 26S proteasome (57). Figure 1C and D showed that IB␣ was immediately degraded by TNF-␣ stimulation in vector-transfected cells. However, TNF-␣-induced IB␣ degradation was also inhibited by NS5B protein.

HCV NS5B protein inhibits IKK-mediated NF-B activation.
To address the mechanism of the above finding, we further examined the effect of NS5B protein on TNF-␣-mediated NF-B activation using a reporter gene assay system. HEK293 cells were cotransfected with an NF-B-luciferase reporter (1 to 4 g). As shown in Fig. 2C, NS5B protein significantly inhibited TNF-␣-induced NF-B activation in a dose-dependent manner (ϳ40 to 80% reduction in reporter gene activity), whereas NS5B itself had little effects on the endogenous NF-B activity. To determine whether the inhibition of NF-B activity by HCV NS5B protein is mediated through TNFR1 signaling cascades, we examined the effects of NS5B protein on NF-B activation induced by TNFR1 signaling transducers, including TNFR1, TRADD, TRAF2, MEKK1, IKK␤, and p65. GFP was used as a control. Overexpression of NS5B significantly inhibited TNFR1-, TRADD-, TRAF2-, MEKK1-, and IKK␤-mediated NF-B activation (ϳ80 to 95% reduction in luciferase activity) (Fig. 2D). However, overexpression of NS5B did not inhibit p65-mediated NF-B activation, indicating that NS5B was acting as an upstream transducer of NF-B. Furthermore, IKK␤-mediated NF-B activation was not inhibited by GFP, suggesting that the inhibition of NF-B activity induced by TNFR1 signaling molecules was NS5B specific. These results suggest that HCV NS5B protein acts as an inhibitor of IKK-mediated NF-B activation in TNFR1 signaling transducers.

HCV NS5B protein interacts with both IKK␣ and IKK␤.
To determine how NS5B protein inhibits IKK-mediated NF-B activation, we asked whether NS5B could interact with IKK proteins. We first performed a GST pull-down assay using either GST or GST-NS5B fusion protein. In this experiment, both vector and I〉␣ were used as controls. HEK293 cells were transiently transfected with empty vector, Flag-IKK␣, Flag-IKK␤, and Flag-IB␣ expression plasmid, individually. At 36 h after transfection, cell extracts were incubated with either GST or GST-NS5B beads, and the bound proteins were detected by anti-Flag antibody. As shown in Fig. 3A, GST-NS5B protein was selectively bound to IKK␣ and IKK␤ but not to vector or IB␣. However, GST protein failed to retain IKK proteins. To further substantiate this finding, we employed a coimmunoprecipitation assay. For this purpose, NS5B-Myc was coexpressed with either Flag-IKK␣, Flag-IKK␤, or Flag-IB␣ in Cos7 cells paired with recombinant vaccinia virus (vTF7-3) system (17). Cell lysates were immunoprecipitated with anti-Flag monoclonal antibody, and the coimmunoprecipitated protein was detected by immunoblotting with anti-Myc monoclonal antibody. In Fig. 3B, it can be seen that not only IKK␣ but also IKK␤ is coprecipitated with NS5B. Nevertheless, NS5B bound IKK␤ very poorly in this system. To further confirm this finding, we investigated whether NS5B could interact with endogenous IKK proteins by using a coimmunoprecipitation assay. NS5B-Myc was transfected into HEK293 cells, and cell lysates were immunoprecipitated with either anti-IKK␣, anti-IKK␤, or IB␣ antibody. Prior to in vivo binding assay, the specificity of each antibody was confirmed by immunoprecipitation as shown in Fig. 3C. The coimmunoprecipitated protein was then subsequently detected by immunoblotting with anti-Myc monoclonal antibody. Indeed, NS5B protein interacts with endogenous IKK␣ (Fig. 3C, lane 1) but not with endogenous IKK␤, although these two proteins are 52% identical in sequence (25). Reciprocally, the same cell lysates were immunoprecipitated with anti-Myc antibody, and then bound protein was detected by immunoblotting with anti-IKK␣, IKK␤, and IB␣, respectively. We confirmed that NS5B protein interacted with endogenous IKK␣ (Fig. 3D). We also found that NS5B protein did not interact with IKK␥ (data not shown). Taken together, these data clearly demonstrate that NS5B directly interacts with IKK␣ both in vitro and in vivo.
HCV NS5B protein does not interact with TRAF2. In the previous studies, we showed that HCV NS5A protein modulated both NF-B and JNK activities through interaction with TRAF2 protein (44,45). Since NS5B is physically associated with NS5A, we examined the potential interaction between NS5B and TRAF2 by GST pull-down assay using E. coli-expressed GST and GST-NS5B fusion protein. Cell extracts containing either TRAF2 or IKK␣ were incubated with either GST or GST-NS5B beads. The NS5B selectively bound to IKK␣, whereas NS5B failed to retain TRAF2 (Fig. 3E), indicating that NS5B modulated TNF signal transduction through effects on IKK protein.
HCV subgenomic replicon inhibits TNF-␣-induced IKK␣ activation through interaction with NS5B and IKK. We then asked how the inhibitory effect of NS5B on NF-B activation might occur in the context of viral infection or even viral RNA replication. For this purpose, both IFN-cured Huh7 cells and HCV subgenomic replicon cells were treated with human TNF-␣ and cell lysates were immunoprecipitated with anti-IKK␣ polyclonal antibody, and then bound proteins were immunoblotted with anti-NS5B monoclonal antibody. As shown activation is significantly decreased in HCV replicon cells.
Since endogenous IKK␣ expression level is not altered in replicon cells, this result clearly shows that NS5B inhibits TNF-␣-induced IKK kinase activation through effects on IKK␣ protein.

HCV NS5B protein enhances TNF-␣-initiated JNK activation.
Since TNF-␣ induces activation of both transcription factor NF-B (6, 41) and the JNK (9, 28) through TNFR1, we have examined whether NS5B could modulate TNF-␣-induced JNK activation. For this purpose, HEK293 cells were transiently transfected with either an empty vector or NS5B expression plasmid. At 24 h after transfection, cells were either untreated or treated with human TNF-␣ (20 ng/ml) for 15 min, and then JNK activity was determined by using GST-c-Jun (1 to 89) fusion protein as a substrate. NS5B protein itself slightly induced JNK activation (Fig. 5A, lane 2). Moreover, TNF-␣stimulated JNK activation was significantly increased by HCV NS5B protein (Fig. 5A, lane 3 versus lane 4), and this was further confirmed by triplicate experiments (Fig. 5B). Since the protein expression level of NS5B was not affected by TNF (Fig.  5A, lanes 2 and 4), JNK activation was not due to the overexpression of NS5B protein. This result indicated that NS5B protein potentiated TNF-␣-induced JNK activation. To investigate if c-Jun phosphorylation is necessarily leading to activity, AP-1 luciferase reporter gene assay was performed by cotransfecting HepG2 cells with AP-1 reporter gene (38) and MEKK1 with increasing amounts of NS5B expression plasmid. MEKK1, MEK kinase 1, is required for the phosphorylation of c-Jun (13). Overexpression of MEKK1 stimulated the AP-1 transcriptional activity, and this MEKK1-mediated AP-1 activity was synergistically elevated by NS5B protein in a dose-dependent manner (Fig. 5C). In a reporter gene assay, we further confirmed that TNF-␣-induced AP-1 activity was synergistically elevated by NS5B protein (data not shown). TNF-in- VOL. 26,2006 HCV NS5B REGULATES TNF SIGNALING 3055 duced c-Jun phosphorylation is mediated through the MEKK1/ SEK1/JNK1 pathway. To investigate whether the increase of JNK activity by NS5B protein is TNF dependent, the effect of NS5B protein on TNF-induced SEK1 phosphorylation was examined. Huh7 cells were cotransfected with SEK1 and NS5B expression plasmids. At 36 h after transfection, cells were treated with TNF (20 ng/ml) for 15 min, and then the phosphorylation level of SEK1 was determined by Western blotting with p-SEK1 antibody (Cell Signaling Technology). Upon TNF treatment, SEK1 was phosphorylated, and this TNF-induced SEK1 phosphorylation was synergistically elevated by NS5B protein, although the protein expression level of SEK1 was not affected by TNF or NS5B (Fig. 5D). This result further confirmed that NS5B enhanced the TNF-induced JNK activity. These data indicate that NS5B protein negatively regulates TNF-␣-induced NF-B activation and acts as a positive regulator of TNF-␣induced JNK signaling cascades. NS5B protein inhibits MEKK1-mediated IKK␣ activity but promotes SEK1 activity. MEKK1 is required for the activation of both transcription factor NF-B and JNK. NF-B activation is mediated through IKK protein (27) and JNK activation is mediated through SEK1 protein (42,61). To investigate the molecular mechanism of JNK activation by NS5B, Huh7 cells were cotransfected with IKK and MEKK1 in the presence or absence of NS5B expression plasmid. At 36 h after transfection, cell lysates were immunoprecipitated with anti-Flag antibody, and kinase assay was performed using an immunocomplex. As shown in Fig. 6A, overexpression of MEKK1 activated both IKK␣ and IKK␤ activities. However, NS5B protein in-   (Fig. 6B, lanes 3 and 6).

DISCUSSION
The most prominent feature of HCV is its ability to persist in virus-infected patients. We speculate that if HCV could modulate the cellular responses to immune stimulatory cytokines, it could be advantageous for the virus to maintain persistent infection in the host. To investigate the molecular mechanism underlying HCV pathogenesis, we have explored the potential involvement of HCV NS5B protein in TNF-␣ signaling pathways, especially TNFR1-mediated NF-B and JNK activations. TNF-␣ has a wide range of actions in inflammation, infection, and immunity (7). Nuclear transcription factor NF-B plays a key role in regulating expression of many cytokines and immunoregulatory proteins (4-6). It has been well established that TNFR1 interacts with a number of cellular proteins including TRADD, RIP, TRAF2, NIK, and IKKs that are responsible for TNF-␣-induced NF-B signaling cascade. Recently, we reported that NS5A modulated TNF-␣stimulated NF-B and JNK activations (44,45). Since NS5A and NS5B are the essential components of the HCV replication complex (46), we asked whether HCV NS5B protein could modulate TNF-␣ signaling pathways, especially TNFR1-mediated NF-B and JNK activations. Using both EMSA and reporter gene assays, we have demonstrated that NS5B protein inhibits TNF-␣-induced NF-B activation in HEK293 cells and hepatic cells. Furthermore, NS5B inhibits TNF-␣-induced IKK activation and subsequent IB␣ degradation. We have further shown that NS5B protein inhibits TNFR1-, TRADD-, TRAF2-, MEKK1-, and IKK-mediated NF-B activation in HEK293 cells. Since NF-B activity is immediately controlled by IKK, these results suggest that the inhibition of TNF-␣-induced NF-B activation by HCV NS5B is mediated through effects on IKK proteins.
We utilized a vaccinia virus T7 expression system (vTF7-3) to analyze the physical association between NS5B and IKKs. Indeed, NS5B protein binds to IKK␣ and IKK␤ in both in vitro GST binding and in vivo coimmunoprecipitation assays. The IKK complex consists of two catalytic subunits, IKK␣ and IKK␤ (also called IKK-1 and IKK-2, respectively) (63), and a regulatory subunit termed IKK-␥ (also named NEMO) (59). Both IKK␣ (p85) and IKK␤ (p87) have 52% identity in sequence and contain an amino-terminal catalytic domain in addition to carboxyl-terminal helix-loop-helix and leucine zipper domains. In our system, NS5B protein strongly associates with IKK␣ but binds to IKK␤ only weakly. Moreover, NS5B protein interacts with endogenous IKK␣ but not with endogenous IKK␤, suggesting that HCV may utilize IKK␣ to evade host immune surveillance or to promote RNA replication. Indeed, a recent study shows that IKK␣ but not IKK␤ regulates mitogenic signaling through transcriptional induction of cyclin D1 (2).
We further asked how the inhibitory effect of NS5B on NF-B activation might occur in the context of viral infection or even viral RNA replication. For this purpose, we performed a coimmunoprecipitation assay to determine whether NS5B and IKK␣ could interact in Huh7 cells harboring HCV subgenomic replicons. Indeed, NS5B protein in HCV subgenomic replicon cells interacts with endogenous IKK␣ regardless of TNF-␣ stimulation. Furthermore, TNF-␣-mediated IKK␣ kinase activation was significantly decreased in HCV replicon cells. This result further suggests that HCV may utilize cellular IKK␣ to modulate a specific signaling pathway.
The NF-B pathway provides an attractive target for viruses to escape immune suppression, leading to persistent infection. Activation of NF-B is an early event that occurs within minutes after exposure to TNF. Many viruses have evolved their gene products to modulate the NF-B pathway for viral replication, host cell survival, and evasion of immune responses. For example, latent membrane protein 1 of Epstein-Barr virus, Tat protein of human immunodeficiency virus type 1, and Tax protein of the human T-cell leukemia virus type 1 can activate NF-B through several distinct mechanisms (18,22). Meanwhile, the adenovirus E1A protein inhibits TNF-induced NF-B activation through suppression of IKK activity and IB␣ phosphorylation (51). The IB-like protein encoded by the African swine fever virus inhibits TNF-induced NF-B activation through the prevention of binding between p50 and p65 (48). It has previously been reported that HCV core protein either enhanced TNF-␣-induced NF-B activation in HEK293 and HeLa cells (12,20) or suppressed TNF-␣-induced NF-B activity in stable cells expressing HCV core (53,64). These conflicting results may be due to the difference in the cell types stably or transiently expressing core protein. In HBV, X protein directly interacts with IB␣ to prevent the reassociation of IB␣ with NF-B (56).
We previously reported that NS5A inhibited TNF-␣-induced NF-B activation through interaction with TRAF2 (44). To examine whether both NS5A and NS5B proteins would work synergistically in TNF-␣-induced NFB signaling pathway, we performed a luciferase reporter assay by cotransfecting both NS5A and NS5B in Huh7 cells. Indeed, both NS5A and NS5B protein inhibited TNF-␣-induced NF-B activation. However, coexpression of both NS5A and NS5B showed no synergism of inhibitory effect on NF-B activation (data not shown). Since TRAF2 is the upstream transducer of IKK, most of the TNF-␣ signaling cascade might be already blocked at TRAF2 by NS5A and, hence, the effect of NS5B on the TNF-␣ signaling cascade was insignificant at the downstream signaling molecule. We speculate that the combinative effects may not be VOL. 26,2006 HCV NS5B REGULATES TNF SIGNALING 3057 expected in natural infection because NS5A and NS5B may function at different stages of infection. Therefore, HCV may utilize either NS5A or NS5B protein in a temporally regulated manner to modulate TNF-␣ signal transduction to evade host immune responses.
In this report, we show that NS5B protein participates as a novel regulator in TNF-␣ signaling events. NS5B showed divergent effects on NF-B and JNK activities. By interacting with IKK proteins, NS5B inhibits TNF-␣-initiated IKK kinase activity and subsequently inhibits NF-B activation. NS5B not only inhibited MEKK1-mediated NF-B activation through interaction with IKK but also enhanced JNK activation by promoting SEK1 activation. The biological function of TNF-␣-induced JNK activation is still controversial. Hence, we are not sure why NS5B shows pleiotropic responses to the signaling pathway. HCV may utilize more than one mechanism for survival and inhibition of apoptosis to evade cellular immune responses. It is also possible that the IKK subunit may function as an accessory protein in the HCV replication complex, although there is no evidence showing that IKK is associated with any viral replication complex. Taken together, modulation of TNF-␣-induced NF-B and JNK activations by NS5B protein may play a key role in HCV pathogenesis.