This R21 Exploratory/Developmental Research project focuses on developing a better understanding of the interactions of hepatitis C virus (HCV) with host innate antiviral response systems within the liver of infected persons. The NS3/4A protease expressed by HCV disrupts early innate cellular antiviral defenses by mediating proteolysis of two critically important adaptor proteins, MAVS (also known as IPS-1, VISA, or Cardif) and TRIF, that are required for virus induction of type 1 IFN-1/2 synthesis through pathways initiated by the pathogen- associated molecular pattern (PAMP) receptors, Toll-like receptor 3 (TLR3) and retinoic acid-inducible gene I (RIG-I). Strong in vitro evidence supports the ability of HCV infection to disrupt these pathways and ablate virus-induced IFN synthesis. Yet equally strong evidence indicates that the synthesis of multiple IFN-stimulated genes (ISGs) is strongly induced within the HCV-infected liver in vivo liver. These conflicting data raise important questions about the relevance of in vitro observations to the pathogenesis of HCV infection in vivo. We propose the use of fluorescent semiconductor quantum dot (Qdot) probes and multiphoton (2PE) microscopy to accurately identify HCV-infected hepatocytes in sections of liver tissue from patients with chronic hepatitis C, and to ascertain the status of these signaling pathways as well as the sources of ISG expression within the HCV-infected liver.
In Specific Aim 1, we will use sensitive direct Qdot conjugate probes and 2PE microscopy to ascertain the intracellular location (nuclear vs. cytoplasmic) and thus activation status of the transcription factors IRF-3, RelA and NF-kB2 p52 expressed by hepatocytes in infected human liver, and determine whether and how they are modulated in relation to HCV infection and the presence of detectable intracellular dsRNA.
In Aim 2, we will assess the intrahepatic expression of critical signaling adaptor proteins that are targeted by HCV (MAVS and TRIF), as well as host cell proteins that dynamically regulate the synthesis of IFN and proinflammatory cytokines (including the PAMP receptors, RIG-I and TLR3, as well as 2'5'OAS, PKR, TRIM25, RNF125, and NLRX1), and determine if their expression is altered by HCV infection.
In Aim 3, we will quantitatively analyze hepatocellular expression of representative ISGs that control HCV replication (PKR, viperin, ISG20, ISG15 and ISG56), and determine how this relates to HCV infection, IRF-3 and NFkB activation, and infiltrating tissue macrophages and plasmacytoid dendritic cells. Parallel studies of archived tissues from experimentally-infected chimpanzees will examine how these events correlate kinetically with infection outcome and the development of adaptive immunity. These studies will provide important new data concerning the pathogenesis of chronic hepatitis C and help to inform the clinical development of future therapies.
Hepatitis C virus is an increasing cause of liver-specific morbidity and mortality in the United States, and the leading infectious cause of cirrhosis, liver failure and liver cancer. Much of this derives from its ability to cause long-term persistent infections, but how the virus successfully escapes immune responses and accomplishes this is not understood. In this project, we will apply recent advances in molecular imaging techniques to directly examine the interactions of the virus with the innate immune system signaling pathways that induce interferon- mediated antiviral responses within the liver.
|Welsch, Christoph; Schweizer, Sabine; Shimakami, Tetsuro et al. (2012) Ketoamide resistance and hepatitis C virus fitness in val55 variants of the NS3 serine protease. Antimicrob Agents Chemother 56:1907-15|
|Shimakami, Tetsuro; Welsch, Christoph; Yamane, Daisuke et al. (2011) Protease inhibitor-resistant hepatitis C virus mutants with reduced fitness from impaired production of infectious virus. Gastroenterology 140:667-75|
|McGivern, D R; Lemon, S M (2011) Virus-specific mechanisms of carcinogenesis in hepatitis C virus associated liver cancer. Oncogene 30:1969-83|
|Lanford, Robert E; Feng, Zongdi; Chavez, Deborah et al. (2011) Acute hepatitis A virus infection is associated with a limited type I interferon response and persistence of intrahepatic viral RNA. Proc Natl Acad Sci U S A 108:11223-8|
|Qu, Lin; Feng, Zongdi; Yamane, Daisuke et al. (2011) Disruption of TLR3 signaling due to cleavage of TRIF by the hepatitis A virus protease-polymerase processing intermediate, 3CD. PLoS Pathog 7:e1002169|
|Helbig, Karla J; Eyre, Nicholas S; Yip, Evelyn et al. (2011) The antiviral protein viperin inhibits hepatitis C virus replication via interaction with nonstructural protein 5A. Hepatology 54:1506-17|
|Zhou, Zhi; Wang, Nan; Woodson, Sara E et al. (2011) Antiviral activities of ISG20 in positive-strand RNA virus infections. Virology 409:175-88|
|Wang, Nan; Dong, Qingming; Li, Jingjing et al. (2010) Viral induction of the zinc finger antiviral protein is IRF3-dependent but NF-kappaB-independent. J Biol Chem 285:6080-90|
|Lemon, Stanley M (2010) Induction and evasion of innate antiviral responses by hepatitis C virus. J Biol Chem 285:22741-7|
|Wang, Nan; Liang, Yuqiong; Devaraj, Santhana et al. (2009) Toll-like receptor 3 mediates establishment of an antiviral state against hepatitis C virus in hepatoma cells. J Virol 83:9824-34|
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