Occludin stalls HCV particle dynamics apart from hepatocyte tight junctions, promoting virion internalization.

BACKGROUND AND AIMS: Numerous HCV entry factors have been identified, and yet information regarding their spatiotemporal dynamics is still limited. Specifically, one of the main entry factors of HCV is occludin (OCLN), a protein clustered at tight junctions (TJs), away from the HCV landing site. Thus, whether HCV particles slide toward TJs or, conversely, OCLN is recruited away from TJs remain debated. APPROACH AND RESULTS: Here, we generated CRISPR/CRISPR-associated protein 9 edited Huh7.5.1 cells expressing endogenous levels of enhanced green fluorescent protein/OCLN and showed that incoming HCV particles recruit OCLN outside TJs, independently of claudin 1 (CLDN1) expression, another important HCV entry factor located at TJs. Using ex vivo organotypic culture of hepatic slices obtained from human liver explants, a physiologically relevant model that preserves the overall tissue architecture, we confirmed that HCV associates with OCLN away from TJs. Furthermore, we showed, by live cell imaging, that increased OCLN recruitment beneath HCV particles correlated with lower HCV motility. To decipher the mechanism underlying virus slow-down upon OCLN recruitment, we performed CRISPR knockout (KO) of CLDN1, an HCV entry factor proposed to act upstream of OCLN. Although CLDN1 KO potently inhibits HCV infection, OCLN kept accumulating underneath the particle, indicating that OCLN recruitment is CLDN1 independent. Moreover, inhibition of the phosphorylation of Ezrin, a protein involved in HCV entry that links receptors to the actin cytoskeleton, increased OCLN accumulation and correlated with more efficient HCV internalization. CONCLUSIONS: Together, our data provide robust evidence that HCV particles interact with OCLN away from TJs and shed mechanistic insights regarding the manipulation of transmembrane receptor localization by extracellular virus particles.

Characterisation of endogenous Claudin-1 expression, motility and susceptibility to hepatitis C virus in CRISPR knock-in cells.

BACKGROUND INFORMATION: Claudin-1 (CLDN1) is a four-span transmembrane protein localised at cell-cell tight junctions (TJs), playing an important role in epithelial impermeability and tissue homoeostasis under physiological conditions. Moreover, CLDN1 expression is up-regulated in several cancers, and the level of CLDN1 expression has been proposed as a prognostic marker of patient survival. RESULTS: Here, we generated and characterised a novel reporter cell line expressing endogenous fluorescent levels of CLDN-1, allowing dynamic monitoring of CLDN-1 expression levels. Specifically, a hepatocellular carcinoma Huh7.5.1 monoclonal cell line was bioengineered using CRISPR/Cas9 to endogenously express a fluorescent TagRFP-T protein fused at the N-terminus of the CLDN1 protein. These cells were proved useful to measure CLDN1 expression and distribution in live cells. However, the cells were resistant to hepatitis C virus (HCV) infection, of which CLDN1 is a viral receptor, while retaining permissiveness to VSV-G-decorated pseudoparticles. Nonetheless, the TagRFP-CLDN1+/+ cell line showed expected CLDN1 protein localisation at TJs and the cell monolayer had similar impermeability and polarisation features as its wild-type counterpart. Finally, using fluorescence recovery after photobleaching (FRAP) approaches, we measured that the majority of endogenous and overexpressed TagRFP-CLDN1 diffuses rapidly within the TJ, whereas half of the overexpressed EGFP-CLDN1 proteins were stalled at TJs. CONCLUSIONS: The Huh7.5.1 TagRFP-CLDN1+/+ edited cell line showed physiological features comparable to that of non-edited cells, but became resistant to HCV infection. Our data also highlight the important impact of the fluorescent protein chosen for endogenous tagging. SIGNIFICANCE: Although HCV-related studies may not be achieved with these cells, our work provides a novel tool to study the cell biology of TJ-associated proteins and a potential screening strategy measuring CLDN1 expression levels.