Dahlene N. Fusco

IL28B inhibits hepatitis C virus replication through the JAK–STAT pathway

BACKGROUND & AIMS The combination of pegylated interferon (IFN) α and ribavirin (RBV) is the standard therapy for patients with chronic HCV infection. However, it produces a sustained virologic response (SVR) in only half of the treated individuals and is associated with significant side effects. Recently, several single-nucleotide polymorphisms (SNPs) near the IL28B locus, also known as IFNλ3, were identified to be strong predictors of SVR in patients receiving PEG-IFN and RBV. We sought to determine whether IL28B was capable of inhibiting HCV replication and to determine the pathway by which IL28B exhibits anti-HCV activity. METHODS Using the full-length HCV replicon OR6 and the infectious HCV clones JFH1 and Jc1, we assessed the anti-HCV effect of IL28B on HCV and characterized the key steps of the JAK-STAT pathway by real time PCR, luciferase assay, and Western blot. Finally, we evaluated the anti-HCV effect of IL28B in the presence of JAK-STAT pathway inhibitors such as blocking antibodies, a pharmacological inhibitor, and siRNAs. RESULTS We found that IL28B inhibits HCV replication in a dose- and time-dependent manner. Like IFNα, IL28B induces the phosphorylation of STAT1 and STAT2, ISRE-driven transcription, and expression of known ISGs. The anti-HCV effects of IL28A, IL28B, and IL29 were abrogated by an IL10R2 blocking antibody, a pharmacological inhibitor of JAK1/TYK2, and by siRNA against IL28R1, STAT1, STAT2, and IRF9. CONCLUSIONS Our data demonstrate that IL28A, IL28B, and IL29 signal through the JAK-STAT pathway to inhibit HCV. These data suggest possible applications of new approaches in HCV treatment.

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

ABSTRACT Responses to alpha interferon (IFN-α)-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 NS5As 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.

A functional genomic screen reveals novel host genes that mediate interferon-alpha’s effects against hepatitis C virus

BACKGROUND & AIMS The precise mechanisms by which IFN exerts its antiviral effect against HCV have not yet been elucidated. We sought to identify host genes that mediate the antiviral effect of IFN-α by conducting a whole-genome siRNA library screen. METHODS High throughput screening was performed using an HCV genotype 1b replicon, pRep-Feo. Those pools with replicate robust Z scores ≥2.0 entered secondary validation in full-length OR6 replicon cells. Huh7.5.1 cells infected with JFH1 were then used to validate the rescue efficacy of selected genes for HCV replication under IFN-α treatment. RESULTS We identified and confirmed 93 human genes involved in the IFN-α anti-HCV effect using a whole-genome siRNA library. Gene ontology analysis revealed that mRNA processing (23 genes, p=2.756e-22), translation initiation (nine genes, p=2.42e-6), and IFN signaling (five genes, p=1.00e-3) were the most enriched functional groups. Nine genes were components of U4/U6.U5 tri-snRNP. We confirmed that silencing squamous cell carcinoma antigen recognized by T cells (SART1), a specific factor of tri-snRNP, abrogates IFN-αs suppressive effects against HCV in both replicon cells and JFH1 infectious cells. We further found that SART1 was not IFN-α inducible, and its anti-HCV effector in the JFH1 infectious model was through regulation of interferon stimulated genes (ISGs) with or without IFN-α. CONCLUSIONS We identified 93 genes that mediate the anti-HCV effect of IFN-α through genome-wide siRNA screening; 23 and nine genes were involved in mRNA processing and translation initiation, respectively. These findings reveal an unexpected role for mRNA processing in generation of the antiviral state, and suggest a new avenue for therapeutic development in HCV.

Kinetic differences in the induction of interferon stimulated genes by interferon-α and interleukin 28B are altered by infection with hepatitis C virus

Several genome‐wide association studies (GWAS) have identified a genetic polymorphism associated with the gene locus for interleukin 28B (IL28B), a type III interferon (IFN), as a major predictor of clinical outcome in hepatitis C. Antiviral effects of the type III IFN family have previously been shown against several viruses, including hepatitis C virus (HCV), and resemble the function of type I IFN including utilization of the intracellular Janus kinase signal transducer and activator of transcription (JAK‐STAT) pathway. Effects unique to IL28B that would distinguish it from IFN‐α are not well defined. By analyzing the transcriptomes of primary human hepatocytes (PHH) treated with IFN‐α or IL28B, we sought to identify functional differences between IFN‐α and IL28B to better understand the roles of these cytokines in the innate immune response. Although our data did not reveal distinct gene signatures, we detected striking kinetic differences between IFN‐α and IL28B stimulation for interferon stimulated genes (ISGs). While gene induction was rapid and peaked at 8 hours of stimulation with IFN‐α in PHH, IL28B produced a slower, but more sustained increase in gene expression. We confirmed these findings in the human hepatoma cell line Huh7.5.1. Interestingly, in HCV‐infected cells the rapid response after stimulation with IFN‐α was blunted, and the induction pattern resembled that caused by IL28B. Conclusion: The kinetics of gene induction are fundamentally different for stimulations with either IFN‐α or IL28B in hepatocytes, suggesting distinct roles of these cytokines within the immune response. Furthermore, the observed differences are substantially altered by infection with HCV. (Hepatology 2014;59:1250‐1261)

Rapid Progression to Decompensated Cirrhosis, Liver Transplant, and Death in HIV-Infected Men After Primary Hepatitis C Virus Infection

BACKGROUND We and others have shown that primary hepatitis C (HCV) infection in men infected with human immunodeficiency virus (HIV) causes early-onset liver fibrosis; however, little is known about the long-term natural history of the liver disease in these HIV-infected men. METHODS We followed a cohort of HIV-infected men with primary HCV infection in New York City. RESULTS Four men who were not cured after their primary HCV infection developed decompensated cirrhosis within 17 months to 6 years after primary HCV infection. Three died within 8 years of primary HCV infection, and 1 survived after liver transplant done 2 years after primary HCV infection. Three of the 4 men had AIDS at the time of primary HCV infection, and the most rapid progression occurred in the 2 men with the lowest CD4 counts at the time of HCV infection. Liver histopathology was most consistent with HCV-induced damage even though some had exposures to other potential hepatotoxins. CONCLUSIONS Primary HCV infection resulted in decompensated cirrhosis and death within 2-8 years in 4 HIV-infected men. The rapid onset of fibrosis due to primary HCV infection in HIV-infected men cannot therefore be considered benign. The rate of continued progression to liver failure may be proportional to the degree of underlying immunocompromise caused by HIV infection. More research is needed to better define the mechanisms behind accelerated liver damage.

Novel Therapies for Hepatitis C: Insights from the Structure of the Virus

For the first time since the discovery of the hepatitis C virus (HCV), therapeutic options for hepatitis C have expanded. Several agents directly effective against HCV are now in development, including both direct-acting antiviral agents (DAAs) and host cofactor inhibitors. DAAs have been developed to inhibit several HCV proteins, including the NS3/4A serine protease, the NS5B RNA polymerase, NS5A, and NS4B. Host cofactor inhibitors include, but are not limited to, cyclophilin inhibitors, miR122 antagonists, and statins. Development of these agents represents a major advance in HCV therapeutics. This review provides a guide to HCV drugs in various stages of development, including an introduction to their mechanism of action, state of clinical development, efficacy, and side effects.

A Genetic Screen Identifies Interferon-α Effector Genes Required to Suppress Hepatitis C Virus Replication

BACKGROUND & AIMS Hepatitis C virus (HCV) infection is a leading cause of end-stage liver disease. Interferon-α (IFNα) is an important component of anti-HCV therapy; it up-regulates transcription of IFN-stimulated genes, many of which have been investigated for their antiviral effects. However, all of the genes required for the antiviral function of IFNα (IFN effector genes [IEGs]) are not known. IEGs include not only IFN-stimulated genes, but other nontranscriptionally induced genes that are required for the antiviral effect of IFNα. In contrast to candidate approaches based on analyses of messenger RNA (mRNA) expression, identification of IEGs requires a broad functional approach. METHODS We performed an unbiased genome-wide small interfering RNA screen to identify IEGs that inhibit HCV. Huh7.5.1 hepatoma cells were transfected with small interfering RNAs incubated with IFNα and then infected with JFH1 HCV. Cells were stained using HCV core antibody, imaged, and analyzed to determine the percent infection. Candidate IEGs detected in the screen were validated and analyzed further. RESULTS The screen identified 120 previously unreported IEGs. From these, we more fully evaluated the following: asparagine-linked glycosylation 10 homolog (yeast, α-1,2-glucosyltransferase); butyrylcholinesterase; dipeptidyl-peptidase 4 (CD26, adenosine deaminase complexing protein 2); glucokinase (hexokinase 4) regulator; guanylate cyclase 1, soluble, β 3; MYST histone acetyltransferase 1; protein phosphatase 3 (formerly 2B), catalytic subunit, β isoform; peroxisomal proliferator-activated receptor-γ-DBD-interacting protein 1; and solute carrier family 27 (fatty acid transporter), member 2; and demonstrated that they enabled IFNα-mediated suppression of HCV at multiple steps of its life cycle. Expression of these genes had more potent effects against flaviviridae because a subset was required for IFNα to suppress dengue virus but not influenza A virus. In addition, many of the host genes detected in this screen (92%) were not transcriptionally stimulated by IFNα; these genes represent a heretofore unknown class of non-IFN-stimulated gene IEGs. CONCLUSIONS We performed a whole-genome loss-of-function screen to identify genes that mediate the effects of IFNα against human pathogenic viruses. We found that IFNα restricts HCV via actions of general and specific IEGs.

Echinacea purpurea aerial extract alters course of influenza infection in mice

Influenza infection is a major clinical problem and Echinacea purpurea, a widely consumed botanical product, is purported to alter the course of respiratory infections including influenza. Mice infected with WSN influenza A and treated with E. purpurea polysaccharide extract had less weight loss than untreated mice but similar pulmonary viral titers. Echinacea-treated mice had lower systemic and pulmonary KC and IL-10 levels and lower systemic IFN-gamma levels following influenza infection. These suggest that E. purpurea alters the clinical course of influenza infection in mice through modulation of cytokines and not direct antiviral activity.

HCV induces transforming growth factor β1 through activation of endoplasmic reticulum stress and the unfolded protein response.

HCV replication disrupts normal endoplasmic reticulum (ER) function and activates a signaling network called the unfolded protein response (UPR). UPR is directed by three ER transmembrane proteins including ATF6, IRE1, and PERK. HCV increases TGF-β1 and oxidative stress, which play important roles in liver fibrogenesis. HCV has been shown to induce TGF-β1 through the generation of reactive oxygen species (ROS) and p38 MAPK, JNK, ERK1/2, and NFκB-dependent pathways. However, the relationship between HCV-induced ER stress and UPR activation with TGF-β1 production has not been fully characterized. In this study, we found that ROS and JNK inhibitors block HCV up-regulation of ER stress and UPR activation. ROS, JNK and IRE1 inhibitors blocked HCV-activated NFκB and TGF-β1 expression. ROS, ER stress, NFκB, and TGF-β1 signaling were blocked by JNK specific siRNA. Knockdown IRE1 inhibited JFH1-activated NFκB and TGF-β1 activity. Knockdown of JNK and IRE1 blunted JFH1 HCV up-regulation of NFκB and TGF-β1 activation. We conclude that HCV activates NFκB and TGF-β1 through ROS production and induction of JNK and the IRE1 pathway. HCV infection induces ER stress and the UPR in a JNK-dependent manner. ER stress and UPR activation partially contribute to HCV-induced NF-κB activation and enhancement of TGF-β1.

The AMPK-related kinase SNARK regulates hepatitis C virus replication and pathogenesis through enhancement of TGF-β signaling

BACKGROUND & AIMS Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. The biological and therapeutic importance of host cellular cofactors for viral replication has been recently appreciated. Here we examined the roles of SNF1/AMP kinase-related kinase (SNARK) in HCV replication and pathogenesis. METHODS The JFH1 infection system and the full-length HCV replicon OR6 cell line were used. Gene expression was knocked down by siRNAs. SNARK mutants were created by site-directed mutagenesis. Intracellular mRNA levels were measured by qRT-PCR. Endogenous and overexpressed proteins were detected by Western blot analysis and immunofluorescence. Transforming growth factor (TGF)-β signaling was monitored by a luciferase reporter construct. Liver biopsy samples from HCV-infected patients were analyzed for SNARK expression. RESULTS Knockdown of SNARK impaired viral replication, which was rescued by wild type SNARK but not by unphosphorylated or kinase-deficient mutants. Knockdown and overexpression studies demonstrated that SNARK promoted TGF-β signaling in a manner dependent on both its phosphorylation and kinase activity. In turn, chronic HCV replication upregulated the expression of SNARK in patients. Further, the SNARK kinase inhibitor metformin suppressed both HCV replication and SNARK-mediated enhancement of TGF-β signaling. CONCLUSIONS Thus reciprocal regulation between HCV and SNARK promotes TGF-β signaling, a major driver of hepatic fibrogenesis. These findings suggest that SNARK will be an attractive target for the design of novel host-directed antiviral and antifibrotic drugs.