Two conserved histidines (His490 and His621) on the E2 glycoprotein of hepatitis C virus are critical for CD81-mediated cell entry

Hepatitis C virus (HCV) entry is a sequential and multi-step process that includes receptor interactions followed by pH-dependent membrane fusion. Specific and conserved histidine residues on the viral envelope proteins are involved in most pH-induced virus entries. In the case of HCV, some conserved histidines on the E1 and E2 proteins have been investigated in HCV pseudotype particle (HCVpp) systems. However, the roles of these histidines in cell-culture-derived HCV particle (HCVcc) systems remain unclear due to the different aspects of the viral life cycle emphasized by the two systems. In this study, the role of two conserved histidines (His490 and His621, located in domains II and III of E2, respectively) in HCV infection was evaluated in the context of JFH-1-based HCVcc using alanine substitutions. The infectivity of the H490A mutant decreased in spite of comparable initial RNA replication, protein expression and assembly efficiency as WT virus. The H621A mutant did not affect viral protein expression, but exhibited no obvious infectivity; there were fewer core proteins in the culture supernatant compared with WT virus, indicating the partially deficient virus assembly. The HCV receptor CD81-binding ability of the two mutant E2s was assessed further using enzyme immunoassays. The CD81-binding activity of H490A-E2 was reduced, and H621A-E2 was unable to bind to CD81. These data revealed the crucial role played by His490 and His621 in HCV infection, particularly during CD81 binding in cell entry. These results also contributed to the mechanical identification of the histidines involved in pH-dependent HCV entry.