Anne K Schwarz, Joe Grove, Ke Hu, Christopher J Mee, Peter Balfe and Jane A McKeating.
J. Virol. 83:12407-14. 2009.
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Recent evidence has shown that a number of host cell molecules acting together are the key for HCV entry: these are the tetraspanin CD81, the scavenger receptor class B member I (SR-BI), the tight junction (TJ) protein Claudin (CLDNs 1, 6 or 9), and, most recently, occludin. HCV enters cells via a pH and clathrin dependent endocytic pathway, however the exact role(s) played by each of the 4 host cell molecules in the entry process are unclear.
In this study we demonstrate that cellular contact increases SR-BI and CLDN1 expression levels and promotes HCV internalization. We found that CLDN1 over expression in sub-confluent cells was unable to recapitulate this effect, whereas increased SR-BI expression enhanced virus internalization rates and led to accelerated escape from neutralizing antibodies. These observations illustrate the critical and rate-limiting role of SR-BI in HCV infection.
Hepatitis C virus (HCV) entry occurs via a pH- and clathrin dependent endocytic pathway and requires a number of cellular factors including CD81, the tight junction proteins Claudin-1 (CLDN1) and occludin, and the scavenger receptor (SR-BI). HCV tropism is restricted to the liver, where hepatocytes are tightly packed. Here we demonstrate that SR-BI and CLDN1 expression is modulated in confluent human hepatoma cells, with both receptors being enriched at cell-cell junctions. Cellular contact increased HCVpp and HCVcc infection and accelerated the internalization of cell-bound HCVcc, suggesting that the cell contact modulation of receptor levels may facilitate the assembly of receptor complexes required for virus internalization. CLDN1 overexpression in sub-confluent cells was unable to recapitulate this effect, whereas increased SR-BI expression enhanced HCVpp entry and HCVcc internalization, demonstrating a rate-limiting role for SR-BI in HCV internalization.
Cell density and receptor expression.
A. Sub-confluent and confluent cells were stained with antibodies specific for CD81, CLDN1 and SR-BI. B. Linear profile histograms, where arrows indicate plasma membrane staining. C. Receptor expression at the plasma membrane (black bars) and cytoplasm (white bars) at low (left) and high (right) density. D. Western blot analysis of protein expression in sub-confluent (SC) and confluent (C) Huh-7.5 cells. E. CD81, CLDN1, SR-BI, OCLN and the tight junction protein ZO-1 expression was quantified in Huh-7.5 cells when sub-confluent (SC-white bars), confluent (C - gray bars) or post confluent (+ 2 days, black bars). Receptor expression at the various time points is plotted relative to confluent cells.
CLDN1 and of SR-BI show a clear increase in expression as cell density increases, whereas the other proteins tested do not.
CLDN1 and SR-BI overexpression in Huh-7.5 cells.
A. Parental and pTRIP transduced cells were plated at a standard seeding density and 26h later stained with antibodies specific for CLDN1 or SR-BI. B. Receptor expression levels at the plasma membrane (black bars) and cytoplasm (white bars) were determined by linear profile plot analysis. C. Parental or transduced cells at the same seeding density were infected with HCVcc (JFH-1) at an approximate multiplicity of infection of 0.3 for 1h. and the levels of infection determined by staining for NS5A+ foci at 48h post infection. Overexpression of SR-BI leads to an increase in infection.
Effect of CLDN1 and SR-BI overexpression on JFH-1 entry kinetics
Huh-7.5 cells over-expressing CLDN1 (A) or SR-BI (B) were incubated with HCVcc (JFH-1) at an approximate multiplicity of infection of 0.3 for 1h on ice. Entry was initiated by elevating the temperature to 37°C, after the times indicated infection was neutralized with an anti-E2 antibody (C1). NS5A+ foci were enumerated 48h post infection, and the percentage of C1-resistant particles calculated relative to the infectivity measured at the 2h time point.
Viral entry and escape from neutralization is clearly faster in the SR-BI over-expressing cells.