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Journal of Virology, June 2009, p. 6211-6221, Vol. 83, No. 12
0022-538X/09/$08.00+0     doi:10.1128/JVI.00246-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Polarization Restricts Hepatitis C Virus Entry into HepG2 Hepatoma Cells{triangledown}

Christopher J. Mee,1 Helen J. Harris,1 Michelle J. Farquhar,1 Garrick Wilson,1 Gary Reynolds,2 Christopher Davis,1 Sven C. D. van IJzendoorn,3 Peter Balfe,1* and Jane A. McKeating1

Hepatitis C Research Group, 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/Membrane Cell Biology, University Medical Centre Groningen, University of Groningen, Groningen, The Netherlands3

Received 4 February 2009/ Accepted 31 March 2009

The primary reservoir for hepatitis C virus (HCV) replication is believed to be hepatocytes, which are highly polarized with tight junctions (TJ) separating their basolateral and apical domains. HepG2 cells develop polarity over time, resulting in the formation and remodeling of bile canalicular (BC) structures. HepG2 cells expressing CD81 provide a model system to study the effects of hepatic polarity on HCV infection. We found an inverse association between HepG2-CD81 polarization and HCV pseudoparticle entry. As HepG2 cells polarize, discrete pools of claudin-1 (CLDN1) at the TJ and basal/lateral membranes develop, consistent with the pattern of receptor staining observed in liver tissue. The TJ and nonjunctional pools of CLDN1 show an altered association with CD81 and localization in response to the PKA antagonist Rp-8-Br-cyclic AMPs (cAMPs). Rp-8-Br-cAMPs reduced CLDN1 expression at the basal membrane and inhibited HCV infection, supporting a model where the nonjunctional pools of CLDN1 have a role in HCV entry. Treatment of HepG2 cells with proinflammatory cytokines, tumor necrosis factor alpha and gamma interferon, perturbed TJ integrity but had minimal effect(s) on cellular polarity and HCV infection, suggesting that TJ integrity does not limit HCV entry into polarized HepG2 cells. In contrast, activation of PKC with phorbol ester reduced TJ integrity, ablated HepG2 polarity, and stimulated HCV entry. Overall, these data show that complex hepatocyte-like polarity alters CLDN1 localization and limits HCV entry, suggesting that agents which disrupt hepatocyte polarity may promote HCV infection and transmission within the liver.

* Corresponding author. Mailing address: University of Birmingham, Division of Immunity and Infection, IBR, Vincent Drive, Birmingham B15 2TT, United Kingdom. Phone: (44) 121 414 8174. Fax: (44) 121 414 3599. E-mail: p.balfe{at}bham.ac.uk

{triangledown} Published ahead of print on 8 April 2009.

Journal of Virology, June 2009, p. 6211-6221, Vol. 83, No. 12
0022-538X/09/$08.00+0     doi:10.1128/JVI.00246-09
Copyright © 2009, American Society for Microbiology. All Rights Reserved.





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