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.

Fetal bovine serum inhibits hepatitis C virus attachment to host cells.

Fetal bovine serum (FBS), used normally as a basic cell culture supplement, inhibits influenza virus growth. However, the role of FBS in the regulation of hepatitis C virus (HCV) infection has not been studied extensively and remains largely unclear. We adopted the established cell-cultured HCV (HCVcc) isolated from the JFH-1 strain and two sets of solutions (cDMEM7.4 and cDMEM6.8; RHMNB6.8 and RHMN6.8) to investigate the effect of FBS on HCV infection. Our data indicate that FBS blocks HCV infection in a dose-dependent manner. The infectivity of HCV diluted in the RHMNB solution was more susceptible to the addition of FBS than that diluted in the cDMEM solution. In addition, FBS-mediated blocking of HCV infection occurred at the step of virus attachment to the target cells, suggesting that FBS contains factors that interfere with the early steps in HCV infection.

The Arg719 residue at the C-terminal end of the stem region in hepatitis C virus JFH-1 E2 glycoprotein promotes viral infection.

Hepatitis C virus (HCV) envelope glycoprotein E2 is involved in virus assembly and initial entry into host cells. The tertiary organization of the E2 ectodomain is mainly composed of domains I-III, followed by the stem (ST) region and transmembrane (TM) domain. The ST region is critical for reorganizing the envelope glycoproteins during the membrane fusion process. While this region is relatively flexible, the physicochemical properties of its amino acid residues are conserved. Whether and how this physicochemical conservation is required for HCV infection is still unclear. The last residue of the E2 ST region evolved to be either an arginine or lysine among different HCV strains, suggesting that the residues confer different functions during HCV infection. To address this possibility, we constructed an R719K point mutant in the JFH-1 strain (genotype 2a) in the context of the cell-culture derived HCV (HCVcc) system. Compared with wild-type (wt) HCV, the R719K mutant exhibited decreased growth, and its extracellular and intracellular infectivity were also significantly decreased at 48 and 72 h post-electroporation. Correspondingly, less RNA and HCV core protein was observed in the supernatant for the R719K mutant, as well as less efficient RNA replication and protein expression. These findings indicate that the 719th residue of arginine on E2 is critical to promote HCV replication and infection. The data provide new clues for the biochemical function of E2, which is required for efficient HCV assembly and infection.