Recently, we reported the establishment of monoclonal antibodies (mAbs) recognizing each intact EL domain of OCLN that can strongly prevent HCV infection in vitro and in vivo and these mAbs were applicable for flow cytometric analysis, immunocytochemistry, and cell-based enzyme-linked immunosorbent assay.
Occludin (OCLN), an integral tetra-spanning plasma membrane protein, is a host entry factor essential for hepatitis C virus (HCV) infection, making it a promising host-targeting molecule for HCV therapeutic intervention.
HCV associates with CLDN1 and then OCLN at the tight junction and is internalized via clathrin-mediated endocytosis by an active process requiring EGFR.
Entry of hepatitis C virus (HCV) into hepatocytes is a complex process that involves numerous cellular factors, including the scavenger receptor class B type 1 (SR-B1), the tetraspanin CD81, and the tight junction (TJ) proteins claudin-1 (CLDN1) and occludin (OCLN).
The scavenger receptor class B type I (SR-BI) and claudin-1 (CLDN1), together with human cluster of differentiation (CD) 81 and occludin (OCLN), constitute the minimal set of HCV entry receptors.
We have previously shown that adenoviral or transgenic overexpression of human CD81 and OCLN facilitates HCV uptake into mouse hepatocytes in vitro and in vivo In efforts to refine these models, we constructed knock-in mice in which the second extracellular loops of CD81 and OCLN were replaced with the respective human sequences, which contain the determinants that are critical for HCV uptake.
To explore expression strategy for recombinant TJ proteins possessing integrated and functional extracellular loops, OCLN was here used as a model molecule, and several prokaryotic fusion constructs were designed by docking OCLN extracellular loops (ECLs) to HIV-1 gp41 NHR and CHR six-helical bundle (6HV1); then their biophysical features and anti-HCV activity were evaluated.
The increased mRNA expression for hepatitis C virus (HCV) entry including CD81, Occludin, LDL receptor, and scavenger receptor class B type I in hMSC-derived hepatocytes was also detected, implying its potential to be utilized as an in vitro model to analyze dynamic HCV infection.
It was also demonstrated that the second extracellular loop of OCLN, especially the two cysteine residues, is critical for HCV infection of hepatic cells.
Significant dysregulated expression of HCV receptors (i.e. claudin-1, occludin, tetraspanin CD81, scavenger receptor type B1) has been shown recently during HCV infection.
Overall, while no association between SR-B1, CLDN-1 or CLDN-6 and the susceptibility to HCV was found, CD5 and CD81 expression coincided with virus lymphotropism and that of OCLN with permissiveness of T cell lines but unlikely primary T cells.
Here we demonstrate that transgenic mice stably expressing human CD81 and OCLN also support HCV entry, but innate and adaptive immune responses restrict HCV infection in vivo.
Building on the previous observation that CD81 and occludin comprise the minimal human factors required to render mouse cells permissive to HCV entry in vitro, we attempted murine humanization via a genetic approach.
Following initial engagement with glycosaminoglycans and the low-density lipoprotein receptor, it is thought that HCV entry proceeds via interactions with the tetraspanin CD81, scavenger receptor class B type I (SR-BI), and the tight-junction proteins claudin-1 (CLDN1) and occludin (OCLN), culminating in clathrin-dependent endocytosis of HCV particles and their pH-dependent fusion with endosomal membranes.
Through analysis of chimeras between these OCLN proteins and alanine scanning mutagenesis of the extracellular domains of OCLN, we identified the second half of the second extracellular loop (EC2) and specific amino acids within this domain to be critical for modulating the HCV cell entry factor activity of this protein.