HMee, C. J., J. Grove, H. J. Harris, K. Hu, P. Balfe, and J. A. McKeating. J Virol 82:461-70. 2008
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The recent identification of CLDNs as critical factors important for HCV internalization highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. We demonstrate that HCV can infect both non-polarized and polarized Caco-2 cells, suggesting that CLDN receptor activity may be independent of its TJ function.
The primary reservoir for hepatitis C virus (HCV) replication in vivo is believed to be hepatocytes within the liver. Three host cell molecules have been reported to be important entry factors for receptors for HCV: the tetraspanin CD81, scavenger receptor BI (SR-BI), and the tight-junction (TJ) protein claudin 1 (CLDN1). The recent discovery of a TJ protein as a critical coreceptor highlighted the importance of studying the effect(s) of TJ formation and cell polarization on HCV entry. The colorectal adenocarcinoma Caco-2 cell line forms polarized monolayers containing functional TJs and was found to express the CD81, SR-BI, and CLDN1 proteins. Viral receptor expression levels increased upon polarization, and CLDN1 relocalized from the apical pole of the lateral cell membrane to the lateral cell-cell junction and basolateral domains. In contrast, expression and localization of the TJ proteins ZO-1 and occludin 1 were unchanged upon polarization. HCV infected polarized and nonpolarized Caco-2 cells to comparable levels, and entry was neutralized by anti-E2 monoclonal antibodies, demonstrating glycoprotein-dependent entry. HCV pseudoparticle infection and recombinant HCV E1E2 glycoprotein interaction with polarized Caco-2 cells occurred predominantly at the apical surface. Disruption of TJs significantly increased HCV entry. These data support a model where TJs provide a physical barrier for viral access to receptors expressed on lateral and basolateral cellular domains.
HCV receptor and tight junction protein localization. Caco-2 cells were seeded on glass coverslips under optimised conditions to achieve non-polarized and polarized monolayers. Cells were stained with antibodies specific for: CD81; SR-BI; CLDN1; Occludin-1 and ZO-1. Bound antibodies were visualised with an anti-Mouse-Alexa488 secondary antibody and confocal microscopy. The large panel represents xy sections and the small panel xz sections taken from each of the corresponding xy sections, with the arrow indicating the plane the z-section was taken from. Z-sections ("stacks") were compiled by taking 0.76µm steps through each xy section. Scale bar represents 20µm.
HCV receptor and tight junction protein expression in Caco-2 cells. Protein expression at the cytoplasm and plasma membrane was quantified using Zeiss LSM line-plot analysis software and data expressed as arbitrary fluorescence units (F). Non-polarized (white bars) and polarized (black bars) cells are shown, where the results reflect the mean ± SEM of at least 10 cells from each image. * P <0.01. Scale bar represents 20µm. It is clear that expression of receptors is increased in polarized cells
HCVcc JFH-1 infection of Caco-2 cells. HCVcc JFH-1 infection of non-polarized (white bar) and polarized (black bar) cells was assessed. Larger numbers of infected cells, grouped into bigger foci of infection, were noted in the non-polarized cultures.