Residues in a Highly Conserved Claudin-1 Motif Are Required for Hepatitis C Virus Entry and Mediate the Formation of Cell-Cell Contacts

Department of Microbiology and Immunology, Albert Einstein College of Medicine, Bronx, NY 10461, USA.
Journal of Virology (Impact Factor: 4.44). 04/2009; 83(11):5477-84. DOI: 10.1128/JVI.02262-08
Source: PubMed


Claudin-1, a component of tight junctions between liver hepatocytes, is a hepatitis C virus (HCV) late-stage entry cofactor. To investigate the structural and functional roles of various claudin-1 domains in HCV entry, we applied a mutagenesis strategy. Putative functional intracellular claudin-1 domains were not important. However, we identified seven novel residues in the first extracellular loop that are critical for entry of HCV isolates drawn from six different subtypes. Most of the critical residues belong to the highly conserved claudin motif W(30)-GLW(51)-C(54)-C(64). Alanine substitutions of these residues did not impair claudin-1 cell surface expression or lateral protein interactions within the plasma membrane, including claudin-1-claudin-1 and claudin-1-CD81 interactions. However, these mutants no longer localized to cell-cell contacts. Based on our observations, we propose that cell-cell contacts formed by claudin-1 may generate specialized membrane domains that are amenable to HCV entry.

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    • "It has been largely demonstrated that CD81 associates with the tight junction protein CLDN1 to form complexes that are essential to HCV entry (Evans et al., 2007; Cukierman et al., 2009; Harris et al., 2010; Krieger et al., 2010). Although we did not perform quantitative analyses of the interaction between CD81 and CLDN1, we quantified the level of colocalization of these two HCV entry factors and found a significant increase in the presence of EWI-2wint. "
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    ABSTRACT: CD81 is a major receptor for Hepatitis C Virus (HCV). It belongs to the tetraspanin family whose members form dynamic clusters with numerous partner proteins and with one another, forming tetraspanin-enriched areas in the plasma membrane. In our study, we combined single-molecule microscopy and biochemistry experiments to investigate the clustering and membrane behavior of CD81 in the context of cells expressing EWI-2wint, a natural inhibitor of HCV entry. Interestingly, we found that EWI-2wint reduces the global diffusion of CD81 molecules due to a decrease of the diffusion rate of mobile CD81 molecules and an increase in the proportion of confined molecules. Indeed, we demonstrated that EWI-2wint promotes CD81 clustering and confinement in CD81-enriched areas. In addition, we showed that EWI-2wint influences the colocalization of CD81 with Claudin-1 - a co-receptor required for HCV entry. Together, our results indicate that a change in membrane partitioning of CD81 occurs in the presence of EWI-2wint. This study gives new insights on the mechanism by which HCV enters into its target cells, namely by exploiting the dynamic properties of CD81.
    Cellular Microbiology 01/2013; 15(7). DOI:10.1111/cmi.12112 · 4.92 Impact Factor
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    • "Introduction of M32I/K48E changes into claudin-7 (Fig. 1F) re-orientates the loop and allows claudin-7 C64 to interact with CD81 E152 in a similar way to that seen for WT claudin-1. Mutagenesis studies have shown the importance of the highly conserved claudin motif, W30–GLW51–C54–C64 in HCV entry (Cukierman et al., 2009). Our data provide a structural rationale for these observations. "
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    ABSTRACT: Hepatitis C virus (HCV) entry is dependent on host cell molecules tetraspanin CD81, scavenger receptor BI and tight junction proteins claudin-1 and occludin. We previously reported a role for CD81/claudin-1 receptor complexes in HCV entry; however, the molecular mechanism(s) driving association between the receptors is unknown. We explored the molecular interface between CD81 and claudin-1 using a combination of bioinformatic sequence-based modelling, site-directed mutagenesis and Fluorescent Resonance Energy Transfer (FRET) imaging methodologies. Structural modelling predicts the first extracellular loop of claudin-1 to have a flexible beta conformation and identifies a motif between amino acids 62-66 that interacts with CD81 residues T149, E152 and T153. FRET studies confirm a role for these CD81 residues in claudin-1 association and HCV infection. Importantly, mutation of these CD81 residues has minimal impact on protein conformation or HCVglycoprotein binding, highlighting a new functional domain of CD81 that is essential for virus entry.
    Cellular Microbiology 08/2012; 14(12). DOI:10.1111/cmi.12008 · 4.92 Impact Factor
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    ABSTRACT: Hepatitis C virus (HCV) is a small enveloped virus with a positive stranded RNA genome belonging to the Flaviviridae family. The virion has the unique ability of forming a complex with lipoproteins, which is known as the lipoviroparticle. Lipoprotein components as well as the envelope proteins, E1 and E2, play a key role in virus entry into the hepatocyte. HCV entry is a complex multistep process involving sequential interactions with several cell surface proteins. The virus relies on glycosaminoglycans and possibly the low-density lipoprotein receptors to attach to cells. Furthermore, four specific entry factors are involved in the following steps which lead to virus internalization and fusion in early endosomes. These molecules are the scavenger receptor SRB1, tetraspanin CD81 and two tight junction proteins, Claudin-1 and Occludin. Although they are essential to HCV entry, the precise role of these molecules is not completely understood. Finally, hepatocytes are highly polarized cells and which likely affects the entry process. Our current knowledge on HCV entry is summarized in this review.
    Central European Journal of Biology 12/2011; 6(6). DOI:10.2478/s11535-011-0076-y · 0.71 Impact Factor
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