The Molecular Mechanism Underlying Species Specific Of CD81 Mediated HCV Entry

Hepatitis C virus (HCV) infection is the leading cause of chronic liver diseases, with over 170 million infected individuals worldwide. HCV replication and spread present highly restricted host tropism,only human and chimpanzee are the nature host of HCV. The unavailability of the small animal model is a major barrier in HCV research due to the mechanism underlying receptor-mediated HCV entry is not fully elucidated.After decades of efforts, at least four HCV entry receptors or co-receptors were identified, including CD81, scavenger receptor class B member 1 (SR-B1), Claudin-1 and Occludin. The cell surface protein CD81 participates a variety of physiological processes, among which the best known role is the indispensible entry-level receptor for HCV. It has been reported that CD81-LEL mediates the self-dimerization and the direct binding to HCV E2. However, the molecular mechanism for the high species restriction is still unclear.Up to date, the only available structural information about HCV co-receptors is focused on hCD81-LEL. The crystal structure of hCD81-LEL displayed a helical, "mushroom-shaped" domain comprising five a-helices. Helices A and E constitute the "stalk" supporting the "head" comprised of B, C and D helices. Four invariant cysteines residues form two tetraspanin signature disulfide bridges stablizing the overall folding. To shed light on the mechanism underlying CD81 mediated HCV entry, we determined the crystal structures of HCV non-permissive CD81-LELs, the mCD81-LEL and agmCD81-LEL. Despite the overall folding shared with hCD81-LEL, we identified an intramolecular bond between 188-196 residues that connected the highly flexible D-helix to the stable E-helix at the stalk domain, a feature unavailable in hCD81. To investigate the impact of the distinct structural feature, we performed mutagenesis study showing that the introduction of the 188-196 bond to hCD81-LEL could impair the E2 binding and abrogate hCD81 mediated HCV entry and infection. Intriguingly, the introduction of residue 186F to HCV non-permissive CD81s alone could neither rescue the E2 binding capability nor the HCVpp entry efficiency and HCVcc infectivity, the disruption of the natural 188-196 bond was prerequisite. Collectively, our results reveal a combination of molecular determinants essential for CD81 mediate HCV entry, which include both the functionality of F186 and a finely tuned conformation dynamics of the surface loops of CD81-LEL. Our findings provide the novel insights into the highly restricted HCV tropism and offer a guideline of selecting CD81 orthologs supporting HCV entry, which could contribute to the establishment of the small animal model.