Virus-host Interaction, Pathogenesis and Therapeutics of Hepatitis C Virus Infection
National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD and surrounding area
The position will be in the Liver Diseases Branch of the NIDDK Intramural Program under the direction of Dr. T. Jake Liang (http://www2.niddk.nih.gov/NIDDKLabs/IntramuralFaculty/LiangJake.htm ). Hepatitis C virus (HCV) is a leading cause of chronic hepatitis and hepatocellular carcinoma worldwide and infects more than 1% of the world population.
Successful vaccine development is pivotal in controlling this global health problem. We currently have several major projects on HCV. 1. Functional genomic screen for HCV-host interaction. HCV, like other viruses, must exploit host cell functions for productive infection. Classical genetic, virologic and biochemical approaches have identified various host factors crucial for viral infection. To systematically identify and characterize host pathways involved in HCV infection, we developed a high-throughput assay by applying an infectious HCV cell culture system (Wakita et al, Nature Medicine 2005; Heller et al, PNAS 2005; Kato et al, J Virology 2007) and performed a genome-wide RNA interference screen (Li et al, PNAS 2009). An innovative high-throughput protocol was designed to screen for host factors involved in various phases of HCV life cycle. In the screen, we identified a total of 517 siRNA pools that significantly affect HCV infection. Bioinformatics confirmed previous identified host factors and uncovered numerous novel pathways important to HCV infection. These HCV host dependencies uncovered from the genome-wide siRNA screen were systematically interrogated by using various in vitro model systems and molecular techniques to define the various steps of HCV lifecycle where these host factors act. An in-depth network map of cellular pathways and machineries that are associated with the complete life cycle of HCV is reconstructed. Studies are underway to further characterize many of these novel host factors and pathways. We have also begum to explore the roles of microRNAs in the life cycle of HCV by applying similar genome-wide screening technology. Preliminary data identifies novel functions of certain miRNAs in HCV life cycle. 2. Mechanisms of action of interferon and ribavirin in HCV therapy. Current treatment of chronic hepatitis C based on combination of peginterferon and ribavirin is only effective in about half of the patients. The mechanisms of interferon action and resistance in the treatment of hepatitis C remain largely unknown. The aims of the second project are to elucidate the molecular pathways of interferon and ribavirin actions and to study the mechanism of interferon nonresponse in treatment of hepatitis C (Huang et al, Gastroenterology 2007; Feld et al, Hepatology 2007; Feld et al, Gastroenterology 2010; Thomas et al 2011 Hepatology). Certain ISGs have been shown to play a role in the antiviral function of interferon. To systematically identify ISGs involved in this process, we performed a functional genomic siRNA screen by applying the siRNA screening protocol above. Preliminary results of the screen identified novel factors and pathways that are important in the anti-HCV functions of interferon.
Our laboratory also has preliminary data that ribavirin, acting via a novel innate mechanism, potentiates the antiviral effect of interferon in the treatment of hepatitis C. Studies are under way to identify and characterize this novel pathway. Understanding the mechanism of action of ribavirin is valuable in identifying novel antiviral molecules that could be used in combination with interferon. 3. High-throughput screen to identify novel antivirals against HCV. Recent development of direct-acting antivirals against HCV such as protease and polymerase inhibitors is promising but still requires combination with peginterferon and ribavirin for maximal efficacy. In addition, these agents are associated with high rate of resistance and many have significant side effects. Due to the lack of a culture system for infectious HCV, the search for new HCV drugs has been greatly hampered. Cell-based screen for HCV inhibitors in use today is based on the HCV replicon system, which only targets the RNA replication step of the viral lifecycle and does not encompass viral entry, processing, assembly and secretion. High-throughput screening (HTS) with an infectious HCV system would cover the complete spectrum of potentially druggable targets in all stages of HCV lifecycle, and would have more biological relevance than other cell-based assays. In collaboration with the NIH Chemical Genomics Center, we are using a highly sensitive and specific assay platform to identify novel HCV inhibitors. 4. Role and mechanism of host genetic factors in viral clearance of HCV infection. Recent identification of IL28B gene polymorphisms associated with hepatitis C virus (HCV) clearance suggests a role for type III interferons (IFNs) in HCV infection. The function of type III IFNs in intrinsic antiviral immunity is poorly understood. We show that HCV infection of primary human hepatocytes results in a robust induction of type III IFNs, leading to IFN-stimulated gene (ISG) expression. Chimpanzees undergoing experimental HCV infection demonstrated a prompt hepatic induction of IL28, associating with ISG upregulation, but a minimal type I IFN induction. Analysis of liver biopsies from HCV-infected patients supported a close correlation among hepatic expression of IL28 and ISGs, but not with type I IFNs. In addition, HCV infection elicits a much broader range of gene expression alterations in addition to ISG induction. The induction of type III IFNs is mediated by IRF3 and NFB-dependent pathways. Type III IFN, aside from upregulating ISGs with a different kinetic profile, induces a distinct set of genes from type I IFN, potentially explaining the functional difference between the two types of IFNs. We are expanding our study to understand the mechanism for the association between IL28, ISG levels and recovery from HCV infection as well as a potential therapeutic strategy for the treatment of non-responders. 5. iPSC-derived human hepatocytes for study of HCV infection. Generation of embryonic stem cells (ESCs) and the technology to developmentally program these cells to various cell lineages offer great promise for cell therapy of various diseases. Recent advances in inducing pluripotent stem cells (iPSCs) from somatic cells by forced expression of the reprogramming factors provide unique opportunities to generate patient-specific cell types of all lineages. We have successfully generated human iPSC lines from primary human fibroblasts and differentiate them to human hepatocytes. We plan to apply this technology platform to establish culture model systems to study HCV infection and pathogenesis.
TO APPLY:
Application should be submitted directly to Amanda Rogers (rogersam@niddk.nih.gov ).
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