The mechanisms underlying the persistence of HCV infection are poorly understood. HCV E2 and NS5A have been suggested to inhibit cellular antiviral responses; however, data have been controversial. In collaboration with Dr. Michael Gale's group, we recently demonstrated that the NS3/4A serine protease blocks virus-induced phosphorylation and activation of interferon regulatory factor 3 (IRF-3), a key transcriptional factor in initiating cellular antiviral responses. However, the detailed consequences of this blockade, i.e. the alterations of downstream hepatocellular antiviral defensive gene expression, which may contribute to the persistence of hepatitis C, remain to be completely elucidated. In addition, the mechanisms by which NS3/4A protease blocks IRF-3 phosphorylation are yet not known. The primary goal of this proposal is to explore the answers of these issues using the recently-developed functional genomics approaches including microarrays and proteomics.
Specific Aim 1, to identify and profile IRF-3 dependent and independent antiviral response genes of hepatocytes that are blocked /suppressed by NS3/4A expression or by genome-length HCV RNA replication. Cells that (1) conditionally express the NS3/4A protease, (2) contain replicating full-length HCV RNA, or (3) conditionally express a dominant negative IRF-3 mutant will be challenged with Sendai virus (SenV), and differences in SenV-activated gene expression will be identified using Affymetrix microarrays and compared with those from clonally matched HCV-negative cells. These experiments should identify antiviral genes downstream of the IRF-3 pathway and other possible pathways that are blocked/suppressed by NS3/4A protease. They will also provide information on whether other HCV proteins and IRF-3 independent pathways contribute to the persistence of HCV infection.
Specific Aim 2, to explore the cellular changes at protein level by which NS3/4A protease blocks IRF-3 phosphorylation using a proteomics approach. Cells that conditionally express the NS3/4A protease will be challenged with SenV, and differences in protein expression before and after challenge, in the presence and absence of NS3/4A will be identified by two-dimensional gel eletrophoresis coupled with automated polypeptide sampling and polypeptide sequencing by mass spectrometry. This approach is likely to identify the virus-activated kinase (VAK) or other signaling components indispensable for virus-induced IRF-3 phosphorylation that are inhibited by the NS3/4A serine protease. This proposal should lead to a better understanding of the mechanisms how HCV disrupts the innate immunity and causes persistent infection in hepatocytes.

Agency
National Institute of Health (NIH)
Institute
National Institute on Drug Abuse (NIDA)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21DA018054-02
Application #
6804132
Study Section
Special Emphasis Panel (ZRG1-GMA-2 (51))
Program Officer
Colvis, Christine
Project Start
2003-09-01
Project End
2006-08-31
Budget Start
2004-09-01
Budget End
2006-08-31
Support Year
2
Fiscal Year
2004
Total Cost
$151,000
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Microbiology/Immun/Virology
Type
Schools of Medicine
DUNS #
800771149
City
Galveston
State
TX
Country
United States
Zip Code
77555
Li, Kui; Lemon, Stanley M (2013) Innate immune responses in hepatitis C virus infection. Semin Immunopathol 35:53-72
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
Devaraj, Santhana G; Wang, Nan; Chen, Zhongbin et al. (2007) Regulation of IRF-3-dependent innate immunity by the papain-like protease domain of the severe acute respiratory syndrome coronavirus. J Biol Chem 282:32208-21
Chen, Zihong; Rijnbrand, Rene; Jangra, Rohit K et al. (2007) Ubiquitination and proteasomal degradation of interferon regulatory factor-3 induced by Npro from a cytopathic bovine viral diarrhea virus. Virology 366:277-92
Ishida, Hisashi; Li, Kui; Yi, Minkyung et al. (2007) p21-activated kinase 1 is activated through the mammalian target of rapamycin/p70 S6 kinase pathway and regulates the replication of hepatitis C virus in human hepatoma cells. J Biol Chem 282:11836-48
Chen, Zihong; Benureau, Yann; Rijnbrand, Rene et al. (2007) GB virus B disrupts RIG-I signaling by NS3/4A-mediated cleavage of the adaptor protein MAVS. J Virol 81:964-76
Yang, Yan; Liang, Yuqiong; Qu, Lin et al. (2007) Disruption of innate immunity due to mitochondrial targeting of a picornaviral protease precursor. Proc Natl Acad Sci U S A 104:7253-8
Loo, Yueh-Ming; Owen, David M; Li, Kui et al. (2006) Viral and therapeutic control of IFN-beta promoter stimulator 1 during hepatitis C virus infection. Proc Natl Acad Sci U S A 103:6001-6
Li, Kui; Foy, Eileen; Ferreon, Josephine C et al. (2005) Immune evasion by hepatitis C virus NS3/4A protease-mediated cleavage of the Toll-like receptor 3 adaptor protein TRIF. Proc Natl Acad Sci U S A 102:2992-7
Li, Kui; Chen, Zihong; Kato, Nobuyuki et al. (2005) Distinct poly(I-C) and virus-activated signaling pathways leading to interferon-beta production in hepatocytes. J Biol Chem 280:16739-47

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