Viruses use a spectrum of immune evasion strategies that enable infection and replication. The acute phase of hepatitis C virus (HCV) infection is characterized by nonspecific and often mild clinical symptoms, suggesting an immunosuppressive mechanism that, unless symptomatic liver disease presents, allows the virus to remain largely undetected. We previously reported that HCV induced the regulatory protein suppressor of cytokine signaling (SOCS)3, which inhibited TNF‐α‐mediated inflammatory responses. However, the mechanism by which HCV up‐regulates SOCS3 remains unknown. Here we show that the HCV protein, p7, enhances both SOCS3 mRNA and protein expression. A p7 inhibitor reduced SOCS3 induction, indicating that p7′s ion channel activity was required for optimal up‐regulation of SOCS3. Short hairpin RNA and chemical inhibition revealed that both the Janus kinase–signal transducer and activator of transcription (JAK‐STAT) and MAPK pathways were required for p7‐mediated induction of SOCS3. HCV‐p7 expression suppressed TNF‐α‐mediated IκB‐α degradation and subsequent NF‐κB promoter activity, revealing a new and functional, anti‐inflammatory effect of p7. Together, these findings identify a molecular mechanism by which HCV‐p7 induces SOCS3 through STAT3 and ERK activation and demonstrate that p7 suppresses proinflammatory responses to TNF‐α, possibly explaining the lack of inflammatory symptoms observed during early HCV infection.—Convery, O., Gargan, S., Kickham, M., Schroder, M., O'Farrelly, C., Stevenson, N. J. The hepatitis C virus (HCV) protein, p7, suppresses inflammatory responses to tumor necrosis factor (TNF)‐α via signal transducer and activator of transcription (STAT)3 and extracellular signal‐regulated kinase (ERK)–mediated induction of suppressor of cytokine signaling (SOCS)3. FASEB J. 33, 8732–8744 (2019). www.fasebj.org

ABBREVIATIONS

AP‐1 activating protein 1 CIS cytokine‐inducible Src homology 2 domain‐containing protein ER endoplasmic reticulum EV empty vector GAS γ‐activated site GT1a genotype la GT2a genotype 2a HA human influenza hemagglutinin HCV hepatitis C virus HEK human embryonic kidney ISG. IFN‐stimulated gene ISRE IFN‐stimulated response element JAK Janus kinase Jc1Δp7 Jc1 with a partial p7 deletion NS nonstructural protein PMA phorbol myristate acetate qRT‐PCR quantitative RT‐PCR shRNA short hairpin RNA RPS15 40S ribosomal protein S15 SOCS suppressor of cytokine signaling STAT signal transducer and activator of transcription TRAF TNF receptor‐associated factor

Hepatitis C virus (HCV) infection can lead to cirrhosis, liver failure, and hepatocellular carcinoma (1, 2); however, infected individuals often remain outwardly healthy for many years (3, 4). HCV is detected by pathogen recognition receptors of the innate immune system, which stimulate the secretion of type I interferons (IFNs) (5–7). Binding of IFN‐α activates the IFN‐α receptor complex (IFN‐α receptors 1 and 2), resulting in the subsequent phosphorylation and activation of the receptor‐associated protein tyrosine kinases, Janus kinase (JAK)1 and tyrosine kinase 2, which phosphorylate receptor tyrosine residues in the cytoplasmic region. This enables signal transducer and activator of transcription (STAT) proteins to bind the receptor via their Src homology 2 domains (8). Phosphorylated STAT proteins translocate to the nucleus and bind to γ‐activated sites (GASs) and IFN‐stimulated response elements (ISREs), inducing transcription of IFN‐stimulated genes (ISGs), which are essential for viral clearance (9–11). Specifically, activated STAT3 dimers bind GAS promoter sequences (12, 13), whereas activated STAT1, STAT2, and IFN regulatory factor 9 trimers recognize the ISRE DNA elements (13), resulting in distinct antiviral and proinflammatory gene induction. Interestingly, even though innate immunity provides an immediate and effective response, the acute phase of HCV infection is often asymptomatic (14), for reasons that remain poorly understood. However, this lack of clinical symptoms indicates that HCV has evolved antiinflammatory mechanisms to effectively counteract immune responses. Indeed, cleavage of adaptor proteins, mitochondrial antiviral signaling protein (MAVS), and Toll/interleukin‐1 receptor (TIR) domain‐containing adaptor protein–inducing IFN‐β (TRIF) disrupt retinoic acid inducible gene‐I (RIG‐I) and Toll‐like receptor (TLR)3 signaling, respectively, revealing viral processes that block HCV detection and subsequent type I IFN induction (15–17). We have previously shown that HCV also degrades essential components of the type IIFN JAK‐STAT pathway via the ubiquitin‐proteasome system, thereby blocking induction of functional antiviral ISGs (18). We also found that HCV induces expression of the intracellular inhibitor, suppressor of cytokine signaling (SOCS)3, a negative regulator of both the JAK‐STAT and NF‐κB pathways (19). Therefore, we propose that HCV‐mediated SOCS3 induction, in turn, suppresses TNF‐α′s proinflammatory signaling (19), perhaps providing some explanation for HCV's clinical silence during the period of acute infection. The SOCS family of proteins consists of 8 members [cytokine‐inducible Src homology 2 domain‐containing protein (CIS) and SOCS1–7] that regulate signal transduction (20). Although SOCS classically inhibit the JAK‐STAT pathway through direct interaction with JAKs, or the receptor, or both (21, 22), they also suppress other pathways, including the NF‐κB signaling cascade. In fact, SOCS3 is thought to suppress NF‐κB signaling via association with TNF receptor‐associated factor (TRAF) family member‐associated NF‐κB activator (TANK)‐binding kinase 1 (TBK1) TRAF2, and TRAF6 (19, 23–26). Because SOCS proteins control many inflammatory responses, it is no surprise that viruses have evolved to harness this suppressive power, essentially controlling the host's innate antiviral activity (27). Bode et al. (28) previously reported that HCV's core protein up‐regulated SOCS3, which is thought to block IFN‐α–induced STAT1 phosphorylation. Additionally, Hsieh et al. (29) found that HCV E2 expression led to a dose‐dependent increase in the SOCS3 gene and protein of Huh7 cells. They also showed that E2 expression resulted in insulin receptor substrate‐1 degradation, which was restored following MG132 treatment. The authors suggested that E2‐mediated SOCS3 may target insulin receptor substrate‐1 for degradation, thereby regulating insulin signaling. Although elevated SOCS3 has been observed in the liver of HCV‐infected humans and chimpanzees (30), its high expression may explain the lack of response to pegylated IFN‐α and ribavirin treatment (31, 32). The correlation between chronic HCV infection and increased SOCS3 indicates an important role for this protein in HCV's immune evasion and modulation strategy. However, whether other HCV proteins (apart from HCV core and E2) induce SOCS3 and the exact mechanism by which HCV enhances SOCS3 expression have not been explored. Diverse stimuli, including cytokines, growth factors, and bacterial and viral pathogen‐associated molecular patterns, stimulate SOCS3 induction (33). Transcriptional activation of SOCS genes is classically mediated by the STAT transcription factors (34). STAT3 has been found to regulate SOCS3 induction (35–37), with both ST ATI and STAT5 also playing a role (38, 39). In addition to the JAK‐STAT cascade, there is a growing body of evidence implicating other signaling pathways in the induction of SOCS3, such as that of MAPK (40). The conventional mammalian MAPKs include ERK, JNK and p38 (41). MAPK signaling can be activated by TLR or receptor tyrosine kinase engagement (42, 43), leading to activation of transcription factors, such as activating protein 1 (AP‐1) and ETS‐like protein‐1 (ELK1) (44–49). All 3 MAPKs (ERK, JNK, and p38) have been shown to regulate SOCS3 expression (50–55). Although induction through these alternative pathways was initially a surprise, this insight into SOCS3's transcriptional regulation gives us a distinct advantage when explaining the mechanism by which HCV stimulates its induction. HCV's ion channel, p7, is vital to producing infectious viral particles during viral egress (56). Although immune evasion mechanisms of several HCV proteins have been described, p7's effect on the immune response has gone largely unstudied, with p7 having only been shown to inhibit the induction of the ISG., interferon‐α inducible protein (IFI)6‐16 (57). In this study, we show that p7 enhances SOCS3 mRNA and protein expression in hepatocytes. Using a p7 inhibitor, we discovered that p7 ion channel activity was required for this SOCS3 induction. We found that p7 expression led to enhanced phosphorylated STAT3, whereas short hairpin RNA (shRNA) knockdown of STAT3 prevented p7‐mediated induction of SOCS3. p7 also stimulated activity of the well‐known STAT3‐driven promoter, GAS, revealing a downstream transcriptional effect of this viral protein. In addition, ERK phosphorylation was enhanced by p7, whereas chemical inhibition of upstream MEK suppressed p7‐mediated induction of SOCS3. Furthermore, activation of TNF‐α‐mediated NF‐κB signaling was reduced in the presence of p7, indicating a functional inhibitory effect of p7 upon inflammation. Together, these findings reveal a novel mechanism by which p7 induces SOCS3 and modulates proinflammatory signaling.

MATERIALS AND METHODS Cell culture Huh7, Huh7.5, and human embryonic kidney (HEK)293T cells were grown in MEM, supplemented with 10% fetal calf serum, 100 U/ml penicillin, and 100 µg/ml streptomycin; and cultured at 37°C in 5% C0 2 . Cells were treated with 1000 IU/ml human IFN‐α2A (Roche, Basel, Switzerland), 10 ng/ml human TNF‐α (PeproTech, Rocky Hill, NJ, USA), 50 ng/ml lipopolysaccharide (LPS), or 50 ng/ml phorbol myristate acetate (PMA). During experiments using N‐(n‐Nonyl)deoxynojirimycin (NNDNJ), cells were transfected with 1 µg of HCV‐p7‐human influenza hemagglutinin (HA) or empty vector (EV) control for 16 h, prior to 8 h treatment with 20 or 100 µM of NNDNJ. Constructs p7 genotype 2a (GT2a) was amplified from pWPI_sp_p7_BLR (a kind gift from Ralf Bartenschlager, University of Heidelberg, Heidelberg, Germany) using the primers 5′‐ATGGAGGCCC‐GAATTGC ACT AG AG A AGCTGGTC ATC‐3′ (forward) and 5′‐AGAGATCTCGGTCGATCAAGCATAAGCCTGTTGGGG‐3′ (reverse) and Velocity DNA polymerase (Bioline, London, United Kingdom). The product was inserted into EcoRI‐ and SalI‐(New England Biolabs, Ipswich, MA, USA) digested pCMV vector, in frame with the N‐terminal HA epitope tag by In‐Fusion Cloning (Clontech Laboratories, Mountain View, CA, USA). p7 genotype la (GT1a) was amplified from pCMV‐p7‐HA using the primers 5′‐ATGG AGGCCCGAATTGATGGCTTTGGAGAACCTCG‐3′ (forward) and 5′‐AGAGATCTCGGTCGACTATGCGTATGCCCGCTG‐3′ (reverse) and Velocity DNA polymerase (Bioline). The product was inserted into EcoRI‐ and SaiI‐ (New England Biolabs) digested pCMV vector out of frame with the N‐terminal HA epitope tag by In‐Fusion Cloning (Clontech Laboratories). Transfection Huh7 cells were transfected with 1 or 4 µg of HCV‐p7‐HA (HCV GT1a), HCV‐p7 GT2a, or the corresponding EV controls for 24 h using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions. HEK293T cells were transfected in 6‐well plates with 1 µg of shRNA STAT3 and scrambled control (InvivoGen, San Diego, CA, USA) or with 1 or 4 µg of HCV‐p7 GT1a or HCV‐p7 GT2a‐HA, using Lipofectamine 2000 for 24 h. HCV infection Huh7.5 cells were transfected with 1 µg of Jc1, Jc1 with a partial p7 deletion (Jc1Δp7, a mutant lacking residues 1–32) (58) (kind gifts from Ralf Bartenschlager, Heidelberg University), or an EV control. Supernatants were collected 24 and 48 h post‐transfection. Huh7.5 cells were then infected with serial dilutions of the virus‐containing supernatants for 72 h, and the median tissue culture infectious dose (TCID50) was calculated by measuring HCV–nonstructural protein (NS)2 mRNA expression by quantitative RT‐PCR (qRT‐PCR) using the following primers: 5′‐AGGGTATGCGCTTTGG‐TGAA‐3′ (forward) and 5′‐CCC‐AGTCCGACATAGGTGTG‐3′ (reverse). qRT‐PCR analysis Total RNA was isolated from cells using the Tri Reagent (MilliporeSigma, Burlington, MA, USA) method. RNA yields were assessed using a NanoDrop ND‐1000 spectrophotometer (Thermo Fisher Scientific). One microgram of total cell RNA was reverse‐transcribed into cDNA using the SensiFast cDNA Synthesis Kit (Bioline) according to the manufacturer's instructions. PCR amplification was performed using primer specific pairs, as detailed in Table 1. Each reaction was carried out in duplicate using the following cycling parameters: 95°C for 15 min followed by 40 cycles of 92°C for 30 s, 65°C for 1 min, and 72°C for 30 s. All gene amplifications were normalized to 40S ribosomal protein S15 (RPS15). Data analysis was carried out using the 2‐DD comparative method. Table 1. Primers for PCR amplification Primer sequence, 5′‐3′ Gene Forward Reverse RPS15 CGGACCAAAGCGATCTCTTC CGCACTGTACAGCTGCATCA CIS GATCTGCTGTGCATAGCCAA ACAAAGGGCTGCACCAGTTT SOCS1 CACTTCCGCACATTCCGTTC AGGGGAAGGAGCTCAGGTA SOCS2 GAGCTCGGTCAGACAGGAT CAGAGATGGTGCTGACGTGT SOCS3 ATCCTGGTGACATGCTCCTC CAAATGTTGCTTCCCCCTTA SOCS4 CTTAGATCATTCCTGTGGGC ATGCCACCTAAAGGCTAAATC SOCS5 TACAGCAAGCAGTCAAAGCC ACAGAGAAGAGGTAGTCCTC SOCS6 TCTCACCATTGCTACCTCCA GAGTCCCTGATTGAATGCTC SOCS7 CTTCTCGGAAGGGCTCCTTC AAGGCTGGCTGCAAAGCTGC p7 GT1a GGGAATTCGGGCTTTGGAGAACCTCGTAA ATTTGTCGACTCATGCGTATGCCCGCTG p7 GT2a TTTCGTGGCTGCTTGGTACA GGGCAATGCTAGGAGCAGTA Immunoblotting Cells were harvested in radioimmunoprecipitation assay (RIPA) lysis buffer and supplemented with protease and phosphatase inhibitors (1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 5 µg/ml leupeptin) and 1 mM dithiothreitol (DTT). Following 1 h incubation at 4°C., extracts were pelleted at 12,000 g at 4°C for 10 min. Sodium dodecyl sulfate (SDS) loading buffer was added and samples were boiled at 100°C for 7 min. Equal quantities of lysate were resolved by SDS‐polyacrylamide gel electrophoresis (PAGE), and proteins were transferred from the gel onto a Polyvinylidene fluoride (PVDF) membrane. Primary antibodies were diluted 1:1000 in 5% (w/v) Marvel in 1× Tris‐buffered saline with Tween or 3% (w/v) bovine serum albumin and incubated overnight at 4°C. Primary antibodies against SOCS3 (Abeam, Cambridge, MA, USA), SOCS4 (GeneTex, Irvine, CA, USA), SOCS5 (Santa Cruz Biotechnology, Dallas, TX, USA), phosphorylated STAT3 (Cell Signaling Technology, Danvers, MA, USA), total STAT3 (Santa Cruz Biotechnology), phosphorylated ERK1/2 (Cell Signaling Technology), total ERK1/2 (Cell Signaling Technology), phosphorylated STAT1 (Cell Signaling Technology), total STAT1 (Cell Signaling Technology), HA (Cell Signaling Technology), IκB‐α (Prof. Ron Hay, University of Dundee, Dundee, United Kingdom), β‐actin (MilliporeSigma), and secondary anti‐mouse or anti‐rabbit antibodies (Thermo Fisher Scientific) were used. Membranes were developed using enhanced chemiluminescent horse radish peroxidase substrate (Bio‐Rad, Hercules, CA, USA) and analyzed using Image Lab software from Bio‐Rad. Luciferase reporter gene assays Huh7 cells were plated onto 6‐well plates at a density of 2.5 × 105 cells per ml. After 24 h, cells were transfected using Lipofectamine 2000 with NF‐κB firefly luciferase reporter construct (1 µg), GAS firefly luciferase reporter construct (1 µg), or positive regulatory domain (PRD)IV (AP‐1) firefly luciferase reporter construct (1 µg) (a kind gift from Dr. Andrew MacDonald, Leeds University, Leeds, United Kingdom) (59), constitutively expressed pGL3 luciferase reporter construct, and HCV‐p7‐HA (1 µg), pCMV‐HA (1 µg), Flag‐TRAF6 (1 µg), or Flag‐TRAF2 (1 µg). The activation of ELK1 was determined using Gal4‐firefly luciferase reporter plasmid pFR‐luciferase (1 µg) with the trans‐activator plasmid pFA‐ELKl (activation domain of ELK is fused with the yeast GAL4 DNA binding domain) (1 µg), pGL3 (1 µg), and either HCV‐p7‐HA (1 µg) or pCMV‐HA control (1 µg). Cells were treated with 10 ng/ml of TNF‐α, 1000 IU/ml of IFN‐α, 50 ng/ml of LPS, or 50 ng/ml of PMA for 24 h. Cell extracts were generated 24 h posttreatment using reporter lysis buffer (Promega, Madison, WI, USA), and extracts were assayed for firefly luciferase and Renilla luciferase activity using the luciferase assay system (Promega) and coelenterazine (Thermo Fisher Scientific), respectively. Firefly luciferase values were normalized to Renilla values, and data shown are the means ± sem of at least 3 independent experiments in triplicate. Confocal Huh7 cells were seeded on poly‐l–lysine coated cover slips for 24 h prior to transfection. Cells were fixed in 4% paraformaldehyde for 20 min, permeabilized in 0.5% Triton X‐100 for 15 min, and blocked with 5% bovine serum albumin in PBS‐Tween (0.05%) for 1 h. The cells were then stained with 1:500 anti‐HA (Covance, Princeton, NJ, USA) or anti‐calnexin (Santa Cruz Biotechnology) overnight at 4°C. The cells were then stained with 1:1000 anti‐mouse–Alexa Fluor 594 and anti‐rabbit–Alexa Fluor 488 (Thermo Fisher Scientific) and 1:500 DAPL The cover slips were then mounted in ProLong Antifade Reagent (Thermo Fisher Scientific) before being visualized using the Olympus Fluoview confocal microscope and analyzed using the Olympus Fluoview FV10‐ASW software (Olympus, Tokyo, Japan). Statistical analysis Statistical analysis was carried out using GraphPad Prism version 6 for Mac (GraphPad Software, La Jolla, CA, USA). Statistical analysis was performed using unpaired Student's t test assuming Gaussian distribution. A value of P <0.05 was deemed statistically significant.

DISCUSSION SOCS proteins are critical negative regulators of cytokine and growth factor signaling, required to switch off signaling cascades, which, if unregulated, could have pathologic consequences (68). Many viruses have hijacked this mechanism to dampen innate antiviral activity (27). Interestingly, clinicians have long reported that HCV infection has mild pathology, resulting in the virus going undetected in many patients until liver disease presents its own clinical symptoms (69). The lack of inflammatory symptoms, associated with normal viral infection, suggests that HCV has developed mechanisms to suppress the host innate antiviral immune response. Indeed, HCV has evolved multiple strategies of evasion, enabling the silent progression of disease (70). Our laboratory previously discovered that PBMCs from patients infected with HCV had significantly enhanced SOCS3 levels compared with healthy controls and that HCV‐mediated SOCS3 induction inhibited proinflammatory TNF‐α signaling in Huh7 cells (19). Therefore, the mechanism of SOCS3 up‐regulation warranted further investigation. Here, we show that HCV‐p7 significantly induced SOCS3 mRNA and protein expression. Moreover, we also report that infection of Huh7.5 cells with Jc1 HCV virus significantly up‐regulated SOCS3 mRNA expression and that this significance was lost following infection with Jc1Δp7. We observed that SOCS3 was partially induced by Jc1Δp7; however, because Jc1Δp7 is a partial deletion, the remaining section of p7 may be inducing some SOCS3; furthermore, because the HCV core (28) and E2 (29) proteins are known to also up‐regulate SOCS3, they may also account for the partial increase in SOCS3. Because p7 can affect several cellular processes, including membrane permeability and ion flux, future studies using p7 mutants should analyze the specific role of these processes in SOCS3 induction. Because SOCS3 induction is associated with STAT3 activity (36), we speculated that STAT3 might be important for the up‐regulation of SOCS3 expression. Because STAT3 phosphorylation is required for propagation of its pathway (71), we first investigated the effect of p7 upon phosphorylated STAT3. We found that STAT3 phosphorylation was enhanced following p7 expression; indeed, downstream GAS luciferase activity was also significantly up‐regulated. Taken together, these results indicate that in the presence of p7, STAT3 activity was increased and that this may have led to functional promoter activity. Interestingly, when STAT3 was suppressed by shRNA, p7 expression no longer enhanced SOCS3 levels, further indicating that STAT3 is essential for p7‐mediated SOCS3 induction. Indeed, our results are in line with several studies showing that STAT3 deletion prevents SOCS3 induction. Baker et al. (65) illustrated that oncostatin M stimulation leads to the robust recruitment of STAT3 to the SOCS3 promoter and that small interfering RNA‐mediated STAT3 inhibition prevented SOCS3 induction. Additionally, overexpression of STAT3 dominant negative mutants inhibited leukemia inhibitory factor‐mediated SOCS3 expression (72). Previous studies have reported that HCV infection modulates STAT3 signaling, including STAT3 activation by the core and NS5A viral proteins (73, 74), in addition to oxidative stress–induced STAT3 activation by HCV replication (75). Interestingly, NS4B causes ER stress, which activates STAT3 signaling, leading to the induction of STAT3‐dependent genes, including vascular endothelial growth factor (VEGF), c‐Myc, and matrix metalloproteinase (MMP)9 (76). Furthermore, McCartney et al (77) showed that STAT3 is actively phosphorylated in the presence of HCV and that STAT3 knockdown significantly decreases HCV RNA levels, implicating STAT3 as a proviral host factor. Together these findings indicate that STAT3 signaling is important for HCV infection, and our discovery, that p7 modulates STAT3 expression and activation, is in keeping with these published reports. STAT1 is also reported to regulate SOCS3 transcription (72); however, in contrast, we did not observe STAT1 phosphorylation upon p7 expression in Huh7 cells, indicating that unlike STAT3, ST ATI may not be required for p7‐induced SOCS3. In addition to STAT3 induction of SOCS3, MAPK signaling is also known to regulate SOCS3 expression (52, 54, 65, 78). Furthermore, HCV infection has been documented to modify MAPK signaling, including HCV core and E2 induction of p38 and ERK phosphorylation (79–82). HCV infection also promotes ERK phosphorylation and downstream AP‐1 activity (83), whereas blocking ERK signaling reduces intracellular and extracellular HCV RNA copy numbers in human hepatoma cells (84). Our data indicated that ERK phosphorylation was enhanced following p7 expression in Huh7 cells. HCV E2 (85) and core (79) proteins have also been shown to enhance ERK phosphorylation, possibly revealing a conserved immunomodulatory mechanism mediated by several HCV proteins. Furthermore, we observed that MEK inhibition prevented p7‐induced SOCS3 expression. These results are consistent with published data showing that inhibition of MEK and ERK with PD98059 prevents SOCS3 induction (86). Taken together, our results indicate that p7 up‐regulates phosphorylated ERK and that ERK signaling is also required for p7's induction of SOCS3. Interestingly, Wetherill et al (87) found that the human papillomavirus oncoprotein E5 (which, like p7, is also believed to be a virally encoded ion channel) induced ERK phosphorylation, which was blocked by the viroporin inhibitors rimantadine and MV006, suggesting that viral ion channel activity is required for this activation of ERK. We similarly saw that the p7 inhibitor, NNDNJ, reduced SOCS3 induction, linking its ion channel activity to the up‐regulation of SOCS3. Several studies have demonstrated that HCV infection interferes with TNF‐α signaling via its viral proteins, including NS3, NS5B, and core (88–91). Here, we found that expression of p7 inhibited TNF‐α‐mediated IκB‐α degradation and TNF‐α‐mediated NF‐κB promoter activity. We also found that overexpression of p7 prevented both TRAF2‐ and TRAF6‐mediated NF‐κB activation, suggesting a possible mechanism of TNF‐α inhibition. In agreement with this, previous work has shown that SOCS3 interacts with TRAF2 (19,26), and Zhou et al (24) showed that SOCS3 could degrade TRAF6 by poly‐ubiquitination. Furthermore, SOCS3 can inhibit IL‐1 signaling by targeting the TRAF6‐transforming growth factor β‐activated kinase 1 (TAK1) complex (25). p7's inhibition of TNF‐α‐mediated NF‐κB activity sheds further light on HCV's strategies that modulate TNF‐α signaling and may represent another mechanism to suppress proinflammatory signaling. Furthermore, published data show that the viral ion channel from HIV, Viral protein U (Vpu), also inhibits TNF‐α‐induced IκB‐α degradation in both T cells and HeLa cells (92, 93). These findings reveal that viral ion channel activity may control proinflammatory signaling pathways and that this may indeed be a novel and conserved immune evasion mechanism. In summary, our findings suggest a mechanism whereby the HCV‐encoded ion channel, p7, induces the negative regulator SOCS3 via STAT3 and ERK activation. Indeed, these discoveries may reveal a molecular mechanism whereby HCV regulates key inflammatory responses to TNF‐α, possibly explaining the mild clinical symptoms often experienced during acute HCV infection.

ACKNOWLEDGMENTS The authors thank Prof. Ralf Bartenschlager (University of Heidelberg, Heidelberg, Germany) for the Jc1 and p7 constructs, Prof. Ron Hay (University of Dundee, Dundee, United Kingdom) for the IκB‐α antibody, and Dr. Andrew MacDonald (Leeds University, Leeds, United Kingdom) for the PRDIV6 (activating protein 1)‐regulated firefly luciferase construct. This study was funded by the Trinity College Dublin College Award, Health Research Board (POR‐20120‐57), and Science Foundation Ireland (12/IA/1667). The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS O. Convery designed and performed the research, analyzed the data, and wrote the manuscript; S. Gargan designed and performed the research, analyzed the data, and wrote the manuscript; M. Kickham performed the research, analyzed the data, and wrote the manuscript; M. Schroder analyzed the data and helped write the description of the results; C. O'Farrelly designed the research, analyzed the data, wrote and edited the manuscript; and N.J. Stevenson designed the research, analyzed the data, wrote and edited the manuscript.

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