Abbreviations: BB, blocking buffer; DMSO, dimethylsulfoxide; GFP, green ?uorescing protein; HCV, hepatitis C virus; HSP, heat shock protein; IRES, internal ribosome entry site; MSCV, murine stem cell virus; NS5A, nonstructural protein 5A; PCR, polymerase chain reaction; siRNA, small interfering RNA.

From the ¹Department of Pathology and Laboratory Medicine, David Geffen School of Medicine at University of California, Los Angeles, CA; the ²Department of Microbiology, Immunology and Molecular Genetics, University of California, Los Angeles, CA; the ³Department of Pediatrics, David Geffen School of Medicine at University of California, Los Angeles, CA; the ⁴Department of Biological Chemistry, David Geffen School of Medicine at University of California, Los Angeles, CA; the ⁵Department of Chemistry and Biochemistry, University of California, Los Angeles, CA; the ⁶UCLA Molecular Biology Institute, Los Angeles, CA; the ⁷Department of Molecular and Medical Pharmacology, University of California, Los Angeles, CA; ⁸Jonsson Comprehensive Cancer Center, Los Angeles, CA; and the ⁹UCLA AIDS Institute, Los Angeles, CA.

Received April 2, 2009; accepted August 1, 2009.

Supported by the Cure Digestive Diseases Research Center (S.W.F.), Stein Oppenheimer Endowment Award (S.W.F.), National Institutes of Health (NIH) grant 1K22CA120147-01A1 (S.W.F.), and NIH grant AI 072180 (A.D.).

Address reprint requests to: Samuel W. French, M.D., Ph.D., Assistant Professor, Liver and Gastrointestinal Pathology, Department of Pathology and Laboratory Medicine, UCLA Center for Health Sciences, 10833 Le Conte Avenue, Los Angeles, CA 90095-1732. E-mail: sfrench@mednet.ucla.edu; fax: 310-267-2058.

Copyright © 2009 by the American Association for the Study of Liver Diseases.

Published online in Wiley InterScience (www.interscience.wiley.com).

DOI 10.1002/hep.23232

Potential con?ict of interest: Nothing to report.

epatitis C virus (HCV) infection has a worldwide prevalence of 3% and is the main entity responsible for liver transplantation for treatment of cirrhosis in developed countries.¹ In the United States, HCV is the most common chronic blood-borne infection, affecting 1.8% of the population, and it appears to be the major causative factor responsible for the recent doubling of hepatocellular carcinoma in the United States.² Current therapy consists of pegylated interferon-alpha and ribavirin, which results in 76% to 82% sustained virological response in patients infected with genotypes 2 and 3.³ Unfortunately, 70% of patients in the United States are infected with genotype 1, for which sustained virological response is signi?cantly lower at 42% to 46%. Generally, therapy of all genotypes can be accompanied by adverse effects, and contraindications to therapy are not infrequent.⁴ For these reasons, there is a need to develop additional therapies that are less toxic and result in higher sustained virological response either as adjuncts or replacement therapies.

Heat shock proteins (HSPs), including HSP40 and HSP70, are required for cell survival during stress and have classically been designated as protein chaperones required for proper protein folding, protein degradation, and reactivation of damaged proteins.^5,6 More recently HSPs have been found to be highly expressed in malignancies, including hepatocellular carcinoma, which has elevated levels of HSP70 compared with benign liver and benign liver proliferations.⁷ Infection by viruses, including human immunode?ciency virus 1, polyomaviridae, molluscum contagiosum, in?uenza, hepatitis B, human T-lymphotropic virus, papilloma-virus, and adenovirus, has been shown to induce HSPs and facilitate viral production.⁸ Two viruses actually encode their own HSP homologs. Closteroviridae encode an HSP70 homolog, and polyomaviridae encode a DNAJ homolog containing protein (T antigen) that activates HSP70 in a similar fashion to HSP40 (a DNAJ motif containing protein).

The HCV nonstructural protein 5A (NS5A) protein is a 56-kDa to 59-kDa phosphoprotein that has been found to be associated with the viral replicase complex. It has been implicated in the regulation of HCV genome replication, internal ribosome entry site (IRES)-mediated translation of the viral polypeptide, and viral assembly.^9-12 NS5A also appears to regulate cell signaling through interaction with several key proteins, including GRB2, PI3K, SRC proteins, P53, BAX, and CDK1.¹¹ Additionally, NS5A interacts with double-stranded RNA-dependent protein kinase blocking the antiviral response and apoptosis.¹³

Here, we report identi?cation of HSP70 and HSP40 in complex with NS5A and implicate HSPs in HCV IRES–mediated translation. Furthermore, treatment with the HSP expression inhibitor Quercetin^14,15 reduced IRES translation. Treatment of HCV infection with Quercetin in tissue culture lowered intracellular viral accumulation and infectious particle production.