Lee F. Peng

A Functional Genomic Screen Identifies Cellular Cofactors of Hepatitis C Virus Replication

Hepatitis C virus (HCV) chronically infects 3% of the worlds population, and complications from HCV are the leading indication for liver transplantation. Given the need for better anti-HCV therapies, one strategy is to identify and target cellular cofactors of the virus lifecycle. Using a genome-wide siRNA library, we identified 96 human genes that support HCV replication, with a significant number of them being involved in vesicle organization and biogenesis. Phosphatidylinositol 4-kinase PI4KA and multiple subunits of the COPI vesicle coat complex were among the genes identified. Consistent with this, pharmacologic inhibitors of COPI and PI4KA blocked HCV replication. Targeting hepcidin, a peptide critical for iron homeostasis, also affected HCV replication, which may explain the known dysregulation of iron homeostasis in HCV infection. The host cofactors for HCV replication identified in this study should serve as a useful resource in delineating new targets for anti-HCV therapies.

A small molecule that binds Hedgehog and blocks its signaling in human cells

Small-molecule inhibition of extracellular proteins that activate membrane receptors has proved to be extremely challenging. Diversity-oriented synthesis and small-molecule microarrays enabled the discovery of robotnikinin, a small molecule that binds the extracellular Sonic Hedgehog (Shh) protein and blocks Shh-signaling in cell lines, human primary keratinocytes and a synthetic model of human skin. Shh pathway activity is rescued by small-molecule agonists of Smoothened, which functions immediately downstream of the Shh receptor Patched.

Hepatitis C Virus Regulates Transforming Growth Factor β1 Production Through the Generation of Reactive Oxygen Species in a Nuclear Factor κB–Dependent Manner

BACKGROUND & AIMS The generation of oxidative stress and transforming growth factor beta1 (TGF-beta1) production play important roles in liver fibrogenesis. We have previously shown that hepatitis C virus (HCV) increases hepatocyte TGF-beta1 expression. However, the mechanisms by which this induction occurs have not been well studied. We explored the possibility that HCV infection regulates TGF-beta1 expression through the generation of reactive oxygen species (ROS), which act through > or =1 of the p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and nuclear factor kappaB (NFkappaB) signaling pathways to induce TGF-beta1 expression. METHODS We used small molecule inhibitors and short interfering RNAs to knock down these pathways to study the mechanism by which HCV regulates TGF-beta1 production in the infectious JFH1 model. RESULTS We demonstrated that HCV induces ROS and TGF-beta1 expression. We further found that JFH1 induces the phosphorylation of p38MAPK, JNK, ERK, and NFkappaB. We also found that HCV-mediated TGF-beta1 enhancement occurs through a ROS-induced and p38 MAPK, JNK, ERK1/2, NFkappaB-dependent pathway. CONCLUSIONS These findings provide further evidence to support the hypothesis that HCV enhances hepatic fibrosis progression through the generation of ROS and induction of TGF-beta1. Strategies to limit the viral induction of oxidative stress appear to be warranted to inhibit fibrogenesis.

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.

HIV Increases HCV Replication in a TGF-β1–Dependent Manner

BACKGROUND & AIMS Human immunodeficiency virus (HIV) coinfection increases hepatitis C virus (HCV)-related progression of hepatic fibrosis, increases HCV persistence, and decreases response rates to interferon-based anti-HCV therapy. It has remained unclear how HIV, a nonhepatotropic virus, accelerates the progression of liver disease by HCV. METHODS We explored the possibility that circulating HIV and/or its proteins contribute to the pathogenesis of HCV through engagement of extracellular coreceptors on hepatocytes. RESULTS In this study, we found that inactivated HIV or gp120 increases HCV replication and enhances HCV-regulated transforming growth factor (TGF)-beta1 expression in both a replicon and an infectious model of HCV. This proviral effect of HIV and gp120 on HCV replication is neutralized by antibodies to CCR5 or CXCR4. However, HIV and gp120 did not alter type I interferon-mediated signaling in these HCV models, indicating that HIV regulates HCV replication through an alternative mechanism. Interestingly, we found that human TGF-beta1 also enhanced HCV replication. The effect of HIV on HCV replication was blocked by a neutralizing antibody to TGF-beta1, indicating that its effects on HCV replication are TGF-beta1 dependent. CONCLUSIONS These results suggest a novel mechanism by which HIV not only enhances HCV replication but also contributes to progression of hepatic fibrosis.

HIV and HCV Cooperatively Promote Hepatic Fibrogenesis via Induction of Reactive Oxygen Species and NFκB

HIV/HCV coinfection leads to accelerated hepatic fibrosis progression, with higher rates of cirrhosis, liver failure, and liver death than does HCV mono-infection. However, the profibrogenic role of HIV on hepatocytes and hepatic stellate cells (HSC) has not been fully clarified. We hypothesized that HIV, HCV induce liver fibrosis through altered regulation of the production of extracellular matrix and matrix metalloproteinases. We examined the fibrogenesis- and fibrolysis-related gene activity in LX2 HSC and Huh7.5.1 cells in the presence of inactivated CXCR4 and CCR5 HIV, as well as HCV JFH1 virus. The role of reactive oxygen species (ROS) upon fibrosis gene expression was assessed using the ROS inhibitor. Fibrosis-related transcripts including procollagen α1(I) (CoL1A), TIMP1, and MMP3 mRNA were measured by qPCR. TIMP1 and MMP3 protein expression were assessed by ELISA. We found that inactivated CXCR4 HIV and CCR5 HIV increased CoL1A, and TIMP1 expression in both HSC and Huh7.5.1 cells; the addition of JFH1 HCV further increased CoL1A and TIMP1 expression. CXCR4 HIV and CCR5 HIV induced ROS production in HSC and Huh7.5.1 cells which was further enhanced by JFH1 HCV. The ROS inhibitor DPI abrogated HIV-and HCV-induced CoL1A and TIMP1 expression. HIV and HCV-induced CoL1A and TIMP1 expression were also blocked by NFκB siRNA. Our data provide further evidence that HIV and HCV independently regulate hepatic fibrosis progression through the generation of ROS; this regulation occurs in an NFκB-dependent fashion. Strategies to limit the viral induction of oxidative stress are warranted to inhibit fibrogenesis.

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.

Syntheses of aminoalcohol-derived macrocycles leading to a small-molecule binder to and inhibitor of Sonic Hedgehog.

We report the synthesis and biological activity of a library of aminoalcohol-derived macrocycles from which robotnikinin, a binder to and inhibitor of Sonic Hedgehog, was derived. Using an asymmetric alkylation to set a key stereocenter and an RCM reaction to close the macrocycle, we were able to synthesize compounds for testing. High-throughput screening via small-molecule microarray (SMM) technology led to the discovery of a compound capable of binding ShhN. Follow-up chemistry led to a library of macrocycles with enhanced biological activity relative to the original hit compounds. Differences in ring size and stereochemistry, leading to alterations in the mode of binding, may account for differences in the degree of biological activity. These compounds are the first ones reported that inhibit Shh signaling at the ShhN level.

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)