The long term goal of these studies is to define the host-virus Interactions that occur during hepatitis C virus (HCV) infection with the hope that this knowledge will lead to the discovery of improved therapeutic approaches to this important human pathogen. HCV is a noncytopathic, positive-strand RNA virus that causes acute and chronic hepatitis and hepatocellular carcinoma. Approximately 2-4 million persons are chronically infected by HCV in the USA and 170 million people are chronically infected worldwide, many of whom will die from liver failure and hepatocelluar carcinoma. Elegant in vitro experiments have revealed that HCV strongly inhibits type 1 IFN production in infected cells by blocking the retinoid inducible gene-I (RIG-I)- mediated pathway. In contrast, HCV strongly Induces type 1 IFN responses in the liver despite this evasion mechanism. This discrepancy suggests that HCV might trigger type 1 IFN production in the liver by cells other than infected hepatocytes. In experiments that form the basis of this application, we showed that human peripheral blood plasmacytoid dendritic cells (pDCs) produce large amounts of IFNa and IFNp when they are cocultured with HCV-infected human hepatoma-derived Huh-7 cells. We also showed that IFNproduction correlates strongly with the HCV RNA content of the Huh-7 cells, that it requires direct cell-cell contact between the pDCs and the infected cells, that it is not induced by free infectious HCV particles, and that it is mediated by Toll-like receptor 7 (TLR7) in the pDCs. Importantly, active viral RNA replication by the hepatocyte, not virus particle formation, is required to produce the pDC-activatIng signal, implying that a unique cell-cell RNA transfer mechanism could mediate this response. These unexpected observations help resolve the contradiction between the ability of HCV to induce type 1 IFN in the Infected liver and its ability to block type 1 interferon production by cells it infects. The host apparently avoids this viral evasion strategy via pDC sensing of the infected cells. To my knowledge, this is a novel aspect of the host response to viral infection that deserves to be characterized and more fully understood. In this appiication, therefore, we will define the cellular and molecular characteristics of this exciting new mechanism.
In Specific Aim 1. we will identify the molecular nature of the HCV-related signal that activates the pDC, focusing on the viral RNA as the likeliest candidate.
In Specific Aim 2, we will examine the cellular mechanisms whereby that signal is produced and how it is transferred to the pDC.
In Specific Aim 3, we will explore the extent to which this mechanism is operative in the chronically HCV infected liver.

Public Health Relevance

Over 170 million people throughout the world are chronically Infected by HCV, 2-4 million of whom live in the United States. Twenty percent of these patients will develop cirrhosis of the liver and about 5% will die from liver failure and cancer. The work described in this proposal will improve our understanding of the host-virus interactions that regulate this infection with the hope that we will identify vulnerabilities in the HCV life cycle that can be exploited to develop safe and effective antiviral drugs to alleviate the human suffering and socioeconomic burden of this worldwide threat to public health.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Program--Cooperative Agreements (U19)
Project #
5U19AI088778-03
Application #
8377278
Study Section
Special Emphasis Panel (ZAI1-BP-M)
Project Start
Project End
Budget Start
2012-04-01
Budget End
2013-03-31
Support Year
3
Fiscal Year
2012
Total Cost
$387,685
Indirect Cost
Name
University of Washington
Department
Type
DUNS #
605799469
City
Seattle
State
WA
Country
United States
Zip Code
98195
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Kell, Alison; Stoddard, Mark; Li, Hui et al. (2015) Pathogen-Associated Molecular Pattern Recognition of Hepatitis C Virus Transmitted/Founder Variants by RIG-I Is Dependent on U-Core Length. J Virol 89:11056-68
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Kell, Alison M; Gale Jr, Michael (2015) RIG-I in RNA virus recognition. Virology 479-480:110-21
Horner, Stacy M; Wilkins, Courtney; Badil, Samantha et al. (2015) Proteomic analysis of mitochondrial-associated ER membranes (MAM) during RNA virus infection reveals dynamic changes in protein and organelle trafficking. PLoS One 10:e0117963
Errett, John S; Gale, Michael (2015) Emerging complexity and new roles for the RIG-I-like receptors in innate antiviral immunity. Virol Sin 30:163-73
Wieland, Stefan; Makowska, Zuzanna; Campana, Benedetta et al. (2014) Simultaneous detection of hepatitis C virus and interferon stimulated gene expression in infected human liver. Hepatology 59:2121-30
Ireton, Reneé C; Gale Jr, Michael (2014) Pushing to a cure by harnessing innate immunity against hepatitis C virus. Antiviral Res 108:156-64
Brownell, Jessica; Bruckner, Jacob; Wagoner, Jessica et al. (2014) Direct, interferon-independent activation of the CXCL10 promoter by NF-*B and interferon regulatory factor 3 during hepatitis C virus infection. J Virol 88:1582-90

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