Mee, C. J., H. J. Harris, M.J. Farquhar, G. Wilson, C. Davis, S.C.D. van IJzendoorn, P. Balfe, and J. A. McKeating. J Virol 83:6211-21. 2009
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Previously, we have shown that epithelial cell polarity effects HCV viral entry (Mee et al, 2008), using a hepatic derived cell line we have gone on to study the role of a liver-like polarized cell phenotype on HCV entry.
Hepatocytes are believed to be the primary reservoir for hepatitis C virus (HCV) replication in vivo and are known to be highly polarized, with tight-junctions (TJ) separating their basolateral (sinusoidal) and apical (canalicular) domains. The hepatoma cell line HepG2 develops polarity over time, resulting in the formation and subsequent re-modeling of bile canalicular (BC) structures where the apical cell surface is separated from the basal membrane via functional TJs. As such, HepG2 cells provide a model system to study the effects of hepatic polarity on HCV entry. Our findings illustrate that complex hepatic polarity alters the localization of HCV receptors and limits HCV entry. This leads us to suggest a role for liver damage, which results in depolarization, in making hepatocytes receptive to viral entry.
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.
Effect of HepG2 polarity on HCV entry. (A) HepG2 cells were grown for 1, 3, or 5 days, fixed in 3% paraformaldehyde, and stained for the BC-expressed marker MRP2. The polarity index was assessed by quantifying the number of MRP2-positive BC per 100 cell nuclei for five fields of view on three replicate coverslips. (B) The BC in polarized HepG2 cells were assessed for both "barrier" and "fence" functions. Cells were incubated with either C6-NBD-SM, to measure fence function, or CMFDA, which measures barrier function. Restriction of C6-NBD-SM to the basal plasma membrane and restriction of CMFDA to the BC indicate that polarized HepG2 cells have functional TJs. (C) HCVcc J6/JFH was used to infect HepG2-CD81 cells at 1, 3, or 5 days postplating. Infected cells were visualized after 72 h (NS5A staining), and infectivity (focus forming units/milliliter) was calculated. **, P < 0.001; ***, P < 0.0001 (t test). (D) HCVpp (white bars) and MLVpp (black bars) infection of HepG2-CD81 cells at 1, 3, or 5 days postplating. Infectivity is expressed relative to HepG2 cells infected immediately after plating _ SD. **, P < 0.001 (t test).
Localization of viral receptors in HepG2 cells and liver tissue. (A) HepG2-CD81 cells were seeded onto glass coverslips at a density resulting in polarized and nonpolarized cells in the population. Cells were dual-stained with antibodies specific for CD81, SR-BI, CLDN1, and OCLN and visualized by confocal microscopy. Bar, 10 μm. (B) Liver sections (3 μm) were cut from paraffin blocks of formalin-fixed tissue and stained with mouse MAbs to CD81, SR-BI, CLDN1, and OCLN. Tissue morphology is shown by arrows, along with sinusoidal endothelium (SE), hepatocytes (H), and cell surfaces (white arrows) on apical canaliculi (A) and basolateral membranes (B).
Effect(s) of HepG2 polarization on CD81 and CLDN1 localization. (A) HepG2 cells transduced to express AcGFP.CLDN1 and DsRED.CD81 were seeded onto glass coverslips. The cells were fixed 1 day postplating, stained for TJs with an antibody to OCLN, and visualized by confocal microscopy. Bars, 20 μm. (B) Quantification of plasma membrane-expressed pools of AcGFP.CLDN1 and DsRED.CD81. Shown are scatter plots of AcGFP.CLDN1 and DsRED.CD81 fluorescent intensity/pixel/cell at plasma membrane locations in polarized and nonpolarized cells. Intensity is presented as arbitrary fluorescence units (AFU).