JVI Accepts, published online ahead of print on 4 November 2009

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J. Virol. doi:10.1128/JVI.01504-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Mouse-specific Residues of Claudin-1 limit HCV Genotype 2a Infection in a Human Hepatocyte Cell Line

Sibylle Haid, Marc P. Windisch, Ralf Bartenschlager, and Thomas Pietschmann^*

TWINCORE- Centre for Experimental and Clinical Infection Research, a joint venture between the Medical School Hannover (MHH) and the Helmholtz Centre for Infection Research (HZI), Braunschweig, Feodor-Lynen-Str. 7, 30625 Hannover, Germany; Department of Molecular Virology, University Heidelberg, Im Neuenheimer Feld 345, 69120 Heidelberg, Germany

^* To whom correspondence should be addressed. Email: thomas.pietschmann{at}twincore.de.

Recently, claudin-1 (CLDN1) was identified as host protein essential for HCV infection. To evaluate CLDN1 function during virus entry, we searched for hepatocyte cell lines permissive for HCV RNA-replication but with limiting endogenous CLDN1 expression, thus permitting receptor complementation assays. These criteria were met by the human hepatoblastoma cell line HuH6, which (i) displays low endogenous CLDN1 levels, (ii) efficiently replicates HCV RNA, and (iii) produces HCV particles with properties similar to those generated in Huh-7.5 cells. Importantly, naïve cells are resistant to HCV genotype 2a infection unless CLDN1 is expressed. Interestingly, complementation of HCV entry by human, rat or hamster CLDN1 was highly efficient, while mouse CLDN1 (mCLDN1) supported HCV genotype 2a infection with only moderate efficiency. These differences were observed irrespective of whether cells were infected with HCV pseudoparticles (HCVpp) or cell culture-derived HCV (HCVcc). Comparatively low entry function of mCLDN1 was observed in HuH6 but not 293T cells, suggesting that species-specific usage of CLDN1 is cell type-dependent. Moreover, it was linked to three mouse-specific residues comprised in the second extracellular loop (L152, I155) and the forth transmembrane helix (V180) of the protein, respectively. These determinants could modulate exposure or affinity of a putative viral binding site on CLDN1, or prevent optimal interaction of CLDN1 with other human co-factors thus precluding highly efficient infection. HuH6 cells represent a valuable model for analysis of the complete HCV replication cycle in vitro and in particular for analysis of CLDN1 function in HCV cell entry.