In this study, we demonstrate that several mutations located within a conserved E2 region encompassing residues 412 to 423 influence the viral glycoprotein interaction(s) with cell receptors and neutralizing antibodies. This region carries residues that are critical for recognition of two broadly neutralizing antibodies used here, MAbs AP33 and 3/11 (45
). These residues—L413, N415, G418, and W420 (AP33) and N415, W420, and H421 (3/11)—are well conserved, which is relevant for future vaccine design. However, this requires a better understanding of the epitope-antibody interaction at the structural level. In this respect, studies of viral variants that escape antibody-mediated neutralization should provide useful information and help identify the role of other residues in antigen-antibody interaction.
Three of the five HCV JFH1 E2 mutations (N415D, N417S, and G418D) described in the present study abrogated E2 reactivity to, and virus neutralization by, MAbs AP33 and 3/11. The N415D and N417S mutations arose spontaneously during cell passaging, while the G418D was generated under AP33 selective pressure. Our amino acid sequence alignment shows that N415D and G418D are extremely rare (1 and 0 occurrences, respectively, out of a sample of 1,311 sequences) in natural sequences, whereas N417S, although by no means common, is the major naturally occurring variant (17 occurrences of 27) (Table ). Recently, we described a MAb AP33 neutralization escape variant of a genotype 1a/2a chimeric HCVcc following repetitive rounds of antibody neutralization and amplification in cell culture (17
). This virus contained N415Y and E655G mutations in the E2 glycoprotein. The N415Y mutation alone severely attenuated MAb AP33 (and 3/11) recognition and neutralization, but it did not enhance sensitivity to neutralization by other human anti-E2 MAbs and, interestingly, it substantially reduced viral fitness. Y at position 415 occurs 9 times in natural HCV sequences. Of the 37 variant residues at this position (Table ) it is the third most common, found in genotypes 1a, 1b, 4b, and 6. In contrast, the N415D mutation, although found much rarely in patient isolates, maintained HCVcc fitness, as was the case for the other E2 cell culture-adaptive mutations characterized in the present study. This is likely due to the nature of the substituted amino acid and/or genotypic differences in the E2 glycoprotein. The difference in the E2 sequence could also account for the fact that the repetitive passaging of the genotype 2a JFH1 HCVcc in the presence of a neutralizing concentration of MAb AP33 allowed selection of a virus carrying the E2 G418D substitution. However, the different selection protocols used between the previous (17
) and the present study may, at least in part, be responsible.
We previously showed that alanine replacement of the residue N417 in the HCV genotype 1a H77 E2 moderately reduced MAb AP33 and 3/11 binding (56
). In the present study, a change to serine at this position in the genotype 2a JFH1 strain drastically reduced AP33 and 3/11 binding, rendering JFH1N417S
HCVcc resistant to neutralization by either antibody and highlighting the contribution of N417 to their binding sites on E2. Furthermore, the residue N417 is part of an N-linked glycosylation site (22
), the removal of which from genotype 1a E2 (N417Q) has been shown to increase the sensitivity of HCVpp to antibody neutralization and to increase CD81 binding (22
). The latter observations are also in keeping with our findings. Our data show that the molecular weight of the genotype 2a E2 N417S mutant is identical to the WT glycoprotein (Fig. ); however, it would be inappropriate to conclude on this basis that this site is not used for glycosylation. This is because the N417S change potentially creates a new N glycosylation site over positions 415 to 417 (i.e., a change from NTN to NTS, see Fig. ), which, if utilized, will not alter the migration of the mutant E2 in SDS-PAGE. Clearly, further studies are required to clarify this issue. The N417S change occurs in 17 of the 27 variants at this position (Table ), suggesting that it may be naturally selected.
Bungyoku et al. (10
) previously showed that the E2 T416A mutation in a chimeric J6/JFH1 HCVcc background does not alter virus infectivity in the Huh-7-derived sub line Huh7.5 (7
). In accordance with these data, we find here that the same mutation has no significant affect on HCVcc spread in Huh-7 cells. Furthermore, we previously showed that the T416A mutation in the genotype 1a HCVpp system moderately reduced MAb AP33 and 3/11 recognition (56
), enhanced CD81 binding, and abrogated pseudoparticle infectivity (47
). In contrast, we show here that this mutation in the genotype 2a JFH1 HCVcc enhances E2 reactivity to MAbs AP33 and 3/11 and maintains WT virus infectivity. Together, the different infection systems, viral isolates and/or cell lines used in each study likely account for these inconsistencies. T416A is one of seven variants at a locus subject to positive selection (9
), and although occurring 13 times in our sample, is only a minor component (7% of substitutions) of the extensive variability in this position.
The I422L mutation was first isolated alongside other structural and nonstructural mutations after several rounds of JFH1 HCVcc passaging in Huh-7.5 cells and was shown not to alter virus infectivity, which is in agreement with our findings in Huh-7 cells (27
). Consistent with our previous findings for I422A (56
), the I422L mutation did not affect E2 recognition by MAbs AP33 and 3/11, confirming that this residue is not critical for E2 recognition by either MAb. I422L occurs five times in our sample of 1311 E2 sequences, constituting 13% of the substitutions at this position (Table ).
The reduced sensitivity of these mutants to inhibition by the anti-SR-BI antibody was an unexpected result. In keeping with these observations, we found that all of the mutants studied here were insensitive to HDL-mediated enhancement of virus infection. The exact mechanism by which the HDL-SR-BI association facilitates HCV entry is currently unknown. Although no interaction between HDL and HCVpp particles has been demonstrated in culture medium, the possibility of an association occurring at a postbinding stage cannot be discounted (58
). More importantly, the binding of HDL to HCVcc virions has yet to be investigated. Also, it has been postulated that the lipid transfer events resulting from HDL-SR-BI binding, known to be essential for regulating the properties of cells membranes, may affect the fusion efficiency of the HCV envelope with cell membranes (58
). SR-BI was first identified as a putative HCV receptor based on its ability to bind soluble, truncated E2 (sE2) via HVR1 (53
). However, sE2 may not fully mimic E2 structures on the HCV virion (11
) and an interaction between SR-BI and the E1E2 heterodimers has yet to be confirmed. In addition, the initial binding of serum HCV to SR-BI was found not to be mediated by HVR-1 or indeed other regions of the E2 glycoprotein. Instead, the association of VLDL with virus particles appeared to play a critical role in the primary interaction with SR-BI (40
). Thus, there is much uncertainty as to how HCV utilizes this receptor during virus entry. In the absence of definitive assays that can measure an interaction between SR-BI with full-length E1E2 or indeed HCVcc virions, it is difficult to decipher the effects caused by our E2 mutations to the entry process via this receptor. However, the siRNA knockdown experiment shows that SR-BI is not dispensable for the mutant virus entry.
Substitution of N415, T416, and N417 resulted in increased E2-CD81 binding, whereas the binding of E2 I422L mutant to CD81 was unaltered. Moreover, each mutant virus, including JFH1G418D, exhibited a significantly greater sensitivity to neutralization by hCD81-LEL, suggesting an increased affinity of the mutated glycoproteins for CD81. This suggests that the adaptive mutations improve the accessibility of CD81 binding residues of the E2 present on mature virions. The heightened inhibition of these mutants by a range of human anti-HCV glycoprotein antibodies (and by the anti-E2 rodent MAbs AP33 and 3/11 in the case of mutants T416A and I422L) supports this theory.
Other studies have identified cell culture-adaptive mutations within the E2 glycoprotein (13
). In particular, the mutation G451R has been extensively characterized (18
). This single mutation increases the buoyant density of the virus, as well as its ability to bind CD81. G451R also reduces SR-BI dependency and increases virus sensitivity to neutralization by E2 specific antibodies, indicating the greater availability of epitopes on the mutant particle. The mutations characterized in the present study have very similar phenotypes to G451R, suggesting that the E2 mutations selected in HCVcc may have arisen in response to similar selective pressures. For example, to persist during long-term culture subtle alterations to E2 conformation may enhance virus-receptor interactions and maintain spread. In line with this, it has been shown that during persistent infection of JFH1 in cell culture certain cell populations emerge that are less permissive to HCV infection due to a decrease in the cell surface expression of CD81 (65
). Although this possibility was not investigated in our study or the others (10
), the increased affinity of each mutant to CD81 (Fig. ) may assist viral spread in cells presenting less CD81.
The E2 region studied here lies immediately C-terminal to the HVR-1, in which positive selection is active. In contrast, the region of interest has only a single site detected as positively selected—that at position 416 (9
). Consistent with this, the survey of 1311 E2 sequences reported here shows that 182 of them are variants (ca. 14%) at position 416. At the remaining sites of interest, substitution is much rarer, never higher than 4% of the total sample (Table ). In addition, the specific mutations described here occur relatively rarely in natural HCV sequences (Table ). For instance, the G-to-D change at position 418 is not found at all in our sample of 1,311 sequences. Indeed, this position is extremely conserved, with only two substitutions found in the full-length E2 sequences sampled (Tables and ). Both of those substitutions are G to S and occur in closely related sequences (not shown). The N415D change is similarly very rare in our sample, occurring only once.
The I-to-L substitution at position 422 is also rare (Table ). Where substitution does occur naturally at position 422, it is confined to relatively hydrophobic residues (Table ), suggesting that a selective constraint for hydrophobicity applies at this position. In contrast, the N417S change constitutes 63% of all variants found at that position, occurring 17 times (Table ). The T416A change is intermediate in frequency, constituting 7% of all naturally occurring substitutions at that position. The naturally occurring variants at all positions are found in a wide range of genotypes (Table ). For instance, position 416 has T to A substitution in genotypes 1a, 3a, and 6 and position 417 has N-to-S substitutions in 1a, 1b, 3a, and 2/5 recombinants. The substitutions produced in the present study at positions 415 to 417 and 422 are spontaneous occurrences in long-term cell passage, where selective conditions may be very different to those found in the natural host. This is a plausible explanation for the relative rarity of these substitutions in naturally occurring sequences.
Broadly neutralizing MAbs to this conserved region hold great promise as therapeutics. Moreover, the epitope recognized by these MAbs can be considered a valid lead for future vaccine design. However, an in-depth understanding of how the neutralizing antibodies interact with E2 is necessary, for both effective vaccine design and understanding the role of the epitope in receptor interaction and virus entry. An additional challenge for vaccine design is the emergence of viral variants during the course of infection that escape antibody neutralization. In vivo
, the prevalence of antibodies reactive to the E2 region 412-423 (QLINTNGSWHIN) is <2.5% (55
). Therefore, there is no great selective pressure acting on this region driving the emergence of neutralization escape mutations. If such mutants do arise spontaneously, they are likely to confer either reduced virus fitness (17
) or an increased vulnerability to neutralization by circulating antibodies targeting various glycoprotein regions, thereby eliminating these variants from the virus pool. In summary, our data contribute to further defining the role of key residues within the HCV E2 412-423 region that influence virus-receptor interactions and antibody-mediated neutralization.