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CD81 and Claudin 1 Coreceptor Association: Role in Hepatitis C Virus Entry ^,

Institute for Biomedical Research, Division of Immunity and Infection, University of Birmingham, Birmingham, United Kingdom,¹ Liver Laboratories, Institute for Biomedical Research, University of Birmingham, and University Hospital Birmingham NHS Foundation Trust, Birmingham, United Kingdom,² Department of Cell Biology and Genetics, Life Science College, Peking University, Beijing, China,³ Department of Pathology, University of Birmingham, and University Hospital Birmingham NHS Foundation Trust, Birmingham, United Kingdom,⁴ Novartis Vaccines and Diagnostics, Inc., Emeryville, California⁵

Received 22 October 2007/ Accepted 27 February 2008

Hepatitis C virus (HCV) is an enveloped positive-stranded RNA hepatotropic virus. HCV pseudoparticles infect liver-derived cells, supporting a model in which liver-specific molecules define HCV internalization. Three host cell molecules have been reported to be important entry factors or receptors for HCV internalization: scavenger receptor BI, the tetraspanin CD81, and the tight junction protein claudin-1 (CLDN1). None of the receptors are uniquely expressed within the liver, leading us to hypothesize that their organization within hepatocytes may explain receptor activity. Since CD81 and CLDN1 act as coreceptors during late stages in the entry process, we investigated their association in a variety of cell lines and human liver tissue. Imaging techniques that take advantage of fluorescence resonance energy transfer (FRET) to study protein-protein interactions have been developed. Aequorea coerulescens green fluorescent protein- and Discosoma sp. red-monomer fluorescent protein-tagged forms of CD81 and CLDN1 colocalized, and FRET occurred between the tagged coreceptors at comparable frequencies in permissive and nonpermissive cells, consistent with the formation of coreceptor complexes. FRET occurred between antibodies specific for CD81 and CLDN1 bound to human liver tissue, suggesting the presence of coreceptor complexes in liver tissue. HCV infection and treatment of Huh-7.5 cells with recombinant HCV E1-E2 glycoproteins and anti-CD81 monoclonal antibody modulated homotypic (CD81-CD81) and heterotypic (CD81-CLDN1) coreceptor protein association(s) at specific cellular locations, suggesting distinct roles in the viral entry process.

Published ahead of print on 12 March 2008.

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