Mutations within a Conserved Region of the Hepatitis C Virus E2 Glycoprotein That Influence Virus-Receptor Interactions and Sensitivity to Neutralizing Antibodies

Cell culture-adaptive mutations within the hepatitis C virus (HCV) E2 glycoprotein have been widely reported. We identify here a single mutation (N415D) in E2 that arose during long-term passaging of HCV strain JFH1-infected cells. This mutation was located within E2 residues 412 to 423, a highly conserved region that is recognized by several broadly neutralizing antibodies, including the mouse monoclonal antibody (MAb) AP33. Introduction of N415D into the wild-type (WT) JFH1 genome increased the affinity of E2 to the CD81 receptor and made the virus less sensitive to neutralization by an antiserum to another essential entry factor, SR-BI. Unlike JFH1_WT, the JFH1_N415D was not neutralized by AP33. In contrast, it was highly sensitive to neutralization by patient-derived antibodies, suggesting an increased availability of other neutralizing epitopes on the virus particle. We included in this analysis viruses carrying four other single mutations located within this conserved E2 region: T416A, N417S, and I422L were cell culture-adaptive mutations reported previously, while G418D was generated here by growing JFH1_WT under MAb AP33 selective pressure. MAb AP33 neutralized JFH1_T416A and JFH1_I422L more efficiently than the WT virus, while neutralization of JFH1_N417S and JFH1_G418D was abrogated. The properties of all of these viruses in terms of receptor reactivity and neutralization by human antibodies were similar to JFH1_N415D, highlighting the importance of the E2 412-423 region in virus entry.Hepatitis C virus (HCV), which belongs to the Flaviviridae family, has a positive-sense single-stranded RNA genome encoding a polyprotein that is cleaved by cellular and viral proteases to yield mature structural and nonstructural proteins. The structural proteins consist of core, E1 and E2, while the nonstructural proteins are p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B (42). The hepatitis C virion comprises the RNA genome surrounded by the structural proteins core (nucleocapsid) and E1 and E2 (envelope glycoproteins). The HCV glycoproteins lie within a lipid envelope surrounding the nucleocapsid and play a major role in HCV entry into host cells (21). The development of retrovirus-based HCV pseudoparticles (HCVpp) (3) and the cell culture infectious clone JFH1 (HCVcc) (61) has provided powerful tools to study HCV entry.HCV entry is initiated by the binding of virus particles to attachment factors which are believed to be glycosaminoglycans (2), low-density lipoprotein receptor (41), and C-type lectins such as DC-SIGN and L-SIGN (12, 37, 38). Upon attachment at least four entry factors are important for particle internalization. These include CD81 (50), SR-BI (53) and the tight junction proteins claudin-1 (15) and occludin (6, 36, 51).CD81, a member of the tetraspanin family, is a cell surface protein with various functions including tissue differentiation, cell-cell adhesion and immune cell maturation (34). It consists of a small and a large extracellular loop (LEL) with four transmembrane domains. Viral entry is dependent on HCV E2 binding to the LEL of CD81 (3, 50). The importance of HCV glycoprotein interaction with CD81 is underlined by the fact that many neutralizing antibodies compete with CD81 and act in a CD81-blocking manner (1, 5, 20, 45).SR-BI is a multiligand receptor expressed on liver cells and on steroidogenic tissue. It binds to high-density lipoproteins (HDL), low-density lipoproteins (LDL), and very low-density lipoproteins (VLDL) (31). The SR-BI binding site is mapped to the hypervariable region 1 (HVR-1) of HCV E2 (53). SR-BI ligands, such as HDL and oxidized LDL have been found to affect HCV infectivity (4, 14, 58-60). Indeed, HDL has been shown to enhance HCV infection in an SR-BI-dependent manner (4, 14, 58, 59). Antibodies against SR-BI and knockdown of SR-BI in cells result in a significant inhibition of viral infection in both the HCVpp and the HCVcc systems (5, 25, 32).Although clearly involved in entry and immune recognition, the more downstream function(s) of HCV glycoproteins are poorly understood, as their structure has not yet been solved. Nonetheless, mutational analysis and mapping of neutralizing antibody epitopes have delineated several discontinuous regions of E2 that are essential for HCV particle binding and entry (24, 33, 45, 47). One of these is a highly conserved sequence spanning E2 residues 412 to 423 (QLINTNGSWHIN). Several broadly neutralizing monoclonal antibodies (MAbs) bind to this epitope. These include mouse monoclonal antibody (MAb) AP33, rat MAb 3/11, and the human MAbs e137, HCV1, and 95-2 (8, 16, 44, 45, 49). Of these, MAbs AP33, 3/11, and e137 are known to block the binding of E2 to CD81.Cell culture-adaptive mutations within the HCV glycoproteins are valuable for investigating the virus interaction(s) with cellular receptors (18). In the present study, we characterize an asparagine-to-aspartic acid mutation at residue 415 (N415D) in HCV strain JFH1 E2 that arose during the long-term passaging of infected human hepatoma Huh-7 cells. Alongside N415D, we also characterize three adjacent cell culture adaptive mutations reported previously and a novel substitution generated in the present study by propagating virus under MAb AP33 selective pressure to gain further insight into the function of this region of E2 in viral infection.