Hepatitis C Virus NS5A Disrupts STAT1 Phosphorylation and Suppresses Type I Interferon Signaling

Abstract

Responses to alpha interferon (IFN-␣)-based treatment are dependent on both host and viral factors and vary markedly among patients infected with different hepatitis C virus (HCV) genotypes (GTs).Patients infected with GT3 viruses consistently respond better to IFN treatment than do patients infected with GT1 viruses. The mechanisms underlying this difference are not well understood. In this study, we sought to determine the effects of HCV NS5A proteins from different genotypes on IFN signaling. We found that the overexpression of either GT1 or GT3 NS5A proteins significantly inhibited IFN-induced IFN-stimulated response element (ISRE) signaling, phosphorylated STAT1 (P-STAT1) levels, and IFN-stimulated gene (ISG) expression compared to controls. GT1 NS5A protein expression exhibited stronger inhibitory effects on IFN signaling than did GT3 NS5A protein expression. Furthermore, GT1 NS5A bound to STAT1 with a higher affinity than did GT3 NS5A. Domain mapping revealed that the C-terminal region of NS5A conferred these inhibitory effects on IFN signaling. The overexpression of HCV NS5A increased HCV replication levels in JFH1-infected cells through the further reduction of levels of P-STAT1, ISRE signaling, and downstream ISG responses. We demonstrated that the overexpression of GT1 NS5A proteins resulted in less IFN responsiveness than did the expression of GT3 NS5A proteins through stronger binding to STAT1. We confirmed that GT1 NS5A proteins exerted stronger IFN signaling inhibition than did GT3 NS5A proteins in an infectious recombinant JFH1 virus. The potent antiviral NS5A inhibitor BMS-790052 did not block NS5A-mediated IFN signaling suppression in an overexpression model, suggesting that NS5A's contributions to replication are independent of its subversive action on IFN. We propose a model in which the binding of the C-terminal region of NS5A to STAT1 leads to decreased levels of P-STAT1, ISRE signaling, and ISG transcription and, ultimately, to preferential GT1 resistance to IFN treatment. More than 170 million individuals are infected with hepatitis C virus (HCV) worldwide, and the disease often progresses to chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (1, 33). Alpha interferon (IFN-␣) has been the backbone of therapy for hepatitis C virus infection. Unfortunately, a number of patients do not respond well to therapy (9,15,20,40). The response rates to IFN therapy are also markedly different among patients infected with different HCV genotypes (GTs). Patients infected with HCV GT1 demonstrate sustained virological response (SVR) rates of 38 to 52%, whereas those infected with GT3 achieve higher SVR rates of 66 to 88% (5, 15, 34). However, the precise mechanisms for HCV GT1 hyporesponsiveness are still not fully understood (38, 40). Several HCV proteins, including core and NS5A, have been demonstrated to contribute to the lack of a response of patients with HCV infection to IFN treatment (35,38,40,47). We have previously reported that HCV replication and core protein expression decrease...

EFTUD2 Is a Novel Innate Immune Regulator Restricting Hepatitis C Virus Infection through the RIG-I/MDA5 Pathway

“…For example, HCV NS3-NS4A blocks RIG-I signaling and cleaves MAVS protein to attenuate innate immunity (35). Our previous studies have demonstrated that HCV infection, HCV core protein, or NS5A protein expression selectively decrease phospho-STAT1 (P-STAT1) accumulation to block the type I IFN pathway response (7,8,9). In this study, we found that HCV evaded the restriction of EFTUD2 by downregulating its expression, providing another mechanism for HCV immune evasion.…”

“…Pathogen recognition triggers downstream signaling to activate transcription factors, such as interferon (IFN) regulatory factors (IRFs), to induce type I interferon secretion and stimulation of the JAK-STAT signaling pathway, leading to the expression of IFN-stimulated genes (ISGs), which presumptively mediate control of viral replica-tion (6). Our previous studies have demonstrated that signal transducer and activator of transcription 1 (STAT1) is the critical signaling component in innate antiviral immunity to HCV and that HCV core protein and nonstructural protein 5A (NS5A) are associated with decreased STAT1 levels (7)(8)(9). However, the precise mechanisms for HCV persistence are still incompletely understood.…”

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“…But in our study, the population at the 12-week treatment in the region ISDR became more similar to the HCV-JFH1 (Figure 3), an IFN sensitive strain. Chimeric subgenomic JFH1 replicon activity before and after IFN treatment HCV resistance to IFN has been attributed, in part, to the function of the NS5A protein (13)(14)(15)(16). To investigate how these NS5A changes on the influence of the virus replication and response to IFN, we constructed subgenomic JFH1 (wild type) and chimeric replicons (Figure 4).…”

Fluoxetine a novel anti-hepatitis C virus agent via ROS-, JNK-, and PPARβ/γ-dependent pathways

“…Whether these therapies directly alleviate HCV-induced host oxidative stress, chronic inflammation, and altered lipid metabolism is still unintelligible. Activating or blocking molecular pathways [i.e., IFN/Janus kinase/ signal transducer and activator of transcription (STAT) (Datta et al, 2011;Kumthip et al, 2012;Miyaaki et al, 2008), Ras/Raf kinase/mitogen-activated protein kinases (MAPK) (Zhang et al, 2012), and peroxisome proliferator-activated receptor (PPAR)-related signal pathways (Agriesti et al, 2012)] may be additive therapeutic targets to IFN-a to further treat CHC patients who otherwise produce an IFN-a resistance.…”