There is overwhelming evidence that CD81 facilitates entry of hepatitis C virus into hepatocytes via interaction with the viral E2 glycoprotein (6
). Definition of specific residues critical for this interaction will greatly assist future development of E2-CD81 targeted entry inhibitors. Antibody blocking experiments, together with molecular modeling, identified several relatively large and discontinuous regions of E2 that were potentially involved in CD81 binding (8
). However, it was unclear whether these regions formed part of the CD81 binding site or were simply in close proximity to the binding site. Specific CD81 residues involved in E2 binding have been defined (13
), whereas reciprocal identification of residues on E2 interacting with CD81 has not previously been attempted. One probable reason for this was the relatively poorly defined CD81 binding regions; hence a large number of residues would need to be mutated to identity those involved in binding. Successful glycoprotein-receptor interaction will exert significant purifying selection on those specific residues involved in binding. Therefore, identification of residues conserved across diverse strains of virus effectively identifies those residues most likely involved in binding, and these residues can then be targeted in mutagenesis experiments. This approach successfully identified human immunodeficiency virus type 1 gp120 residues critical for CD4 binding (37
), residues whose involvement was later confirmed when the crystal structure of gp120 was obtained (28
). However, in the case of HCV, sequence databases contain E1E2 sequences from both functional and nonfunctional viruses; therefore, sites potentially involved in CD81 binding may exhibit amino acid heterogeneity that is contributed by nonfunctional E1E2 clones. To overcome this problem, we first generated a panel of E1E2 clones representing genotypes 1 through 6 and screened these for functionality in the HCVpp assay. We then combined these sequences with those of known infectious clones to generate an alignment of 41 functional and diverse E1E2s. Using this approach we were able to identify a number of residues, located within the putative CD81 binding regions, for alanine replacement mutagenesis. Not only does this approach help define receptor binding at a molecular level, it has the added advantage of identifying those antiviral targets that are conserved across diverse strains of HCV.
Mutations were introduced in the context of full-length E1E2 because the E1E2 heterodimer exhibits superior CD81 binding compared to soluble monomeric E2 (9
). To recover E1E2, the cells have to be lysed with a mixture of detergents, which precludes their use in cell binding assays. However, previous work has shown good correlation between plate-based CD81-LEL binding data and cell-associated CD81-binding data (41
), therefore negating the need to perform cell-based assays.
Before testing the ability of the mutants to bind CD81, we first assessed the effect of each mutation on expression and overall conformation of E2 by probing each mutant protein with a panel of murine monoclonal antibodies. MAb H53, which selectively recognizes correctly folded E2 (10
), showed similar reactivity to the mutant proteins except for some of the mutations introduced between positions 540 and 550, which reduced binding by more than 50%. Similar reductions in binding to these mutants by MAbs H48 and H35 were seen, indicating that the E2 conformation may be sensitive to mutations within this region. Alanine substitution at positions 523 and 530 resulted in a greater-than-90% reduction in binding by MAb H35, while substitution at position 530 had a similar effect on H48 binding. Indeed many of the mutations introduced between positions 523 and 530 diminished the binding of these MAbs. These findings indicate that this region forms part of the epitope recognized by these two MAbs, with residues 523 and 530 and 523 alone as probable contact residues for MAb H35 and MAb H48, respectively. The observed overlap between residues important for CD81, MAb H35, and MAb H48 binding indicates that these MAbs target the CD81 binding site. Importantly, these MAbs have recently been shown to inhibit CD81 binding and neutralize HCVpp infectivity (12
). Similarly, W420A mutation abolished both MAb AP33 (itself a broadly neutralizing antibody [39
]) and CD81 binding, indicating overlap between the CD81 binding site and the AP33 epitope. Finally, mutations at L413, N415, and G418 also reduced the binding of MAb AP33, and we have recently shown that these, together with W420, are probable contact residues (47
Having established that the mutant proteins were correctly folded, we compared their abilities to bind to CD81. We used MAb H53 normalization to ensure that cell lysates contained equivalent amounts of correctly folded E2. In addition, we used subsaturating amounts of E2 so that we could identify mutations that enhanced CD81 binding, as well as those that reduced binding. Alanine substitution at a number of sites resulted in increased CD81 binding. Enhanced CD81 and MAb binding might be due to increased exposure of the receptor and/or antigenic sites. For example, replacement of the threonine at position 534 would lead to the loss of an N-linked glycosylation site, as would the asparagine substitution upstream at position 532. Both of these resulted in moderately increased CD81 binding (Fig. ). It is feasible that removal of a large glycan moiety led to better exposure of the CD81-binding site.
Removal of other N-linked glycosylation sites had variable effects on CD81 binding and infectivity. Increased migration rates of E2 in immunoprecipitation assays confirmed that all of the glycosylation sites were modified by the addition of glycans. Loss of the glycosylation site at 423 resulted in an E2 protein that was capable of noncovalent heterodimer formation and of CD81 binding. However, this mutant was unable to confer infectivity in the HCVpp assay. By contrast, loss of the glycosylation sites at 417, 532, and 540 did not abolish HCVpp infectivity, although infectivity conferred by E2 mutated at amino acids 417 and 532 was less than 50% of that conferred by wild-type E1E2. While these findings are in general agreement with a recently published analysis of the effect of N glycosylation on HCVpp infectivity (21
), we observed a more marked reduction in HCVpp infectivity associated with loss of the glycosylation sites at 417 and 423. This is most likely due to differences in the nature of the amino acid replacement; we used alanine substitution whereas Gofford et al. used a more conservative glutamine residue.
A number of residues in the first putative CD81 binding region, which is located immediately downstream of the first hypervariable region (HVR1), reduced CD81 binding by greater than 50%. In particular, substitution W420A reduced binding by greater than 1 log10
, while replacement at residues 421 and 422 both reduced binding by more than 50%. This finding is at odds with previously published data where MAb 3/11, whose epitope maps to this region of E2, only partially blocked CD81 binding (17
). Based on this observation the authors argued that antibodies to this region did not directly block the CD81 binding site, but rather exerted their inhibitory effect through steric hindrance. However, an equally plausible argument to explain their findings is that CD81 may have a much greater affinity for E2 than the 3/11 MAb used in their analysis (17
Surprisingly, none of the mutations introduced into the second putative CD81 binding region (residues 474 to 495) reduced CD81 binding. This region had been implicated in CD81 binding, as MAb 6/41a, which recognizes a linear epitope between residues 480 to 493, was apparently capable of blocking E2 binding to CD81-LEL and vice versa (17
). However, in our previous work we did not observe any CD81-E2 blocking effect for this MAb (38
). In addition, MAb 6/41a was also unable to block the infectivity of HCVpp carrying autologous H77 E1E2 proteins (24
). These observations, together with our current mutagenesis experiments, suggest that this region is not directly involved in CD81 binding. However, it is still possible that more variable residues within this and the other regions under analysis, while not directly mediating binding, may influence CD81 binding affinity. This notion is supported by domain-swapping experiments that showed that the second hypervariable region (HVR2), encompassing residues 474 to 478, is capable of modulating CD81-E2 interaction (43
The third putative binding region analyzed in our studies contained a number of residues that were pivotal in CD81 binding. In particular, substitutions Y527A, W529A, G530A, and D535A resulted in greater-than-90% reductions in CD81 binding, without affecting the overall conformation of the E2 protein. This region contains other sites that had indirectly been implicated in CD81 binding. For example, insertional mutations at P545 inhibit E1E2-mediated cell fusion, and this was proposed to be due to loss of CD81 binding (27
). However, our data show that this residue per se is not directly involved in CD81 interaction.
Residues in CD81 LEL shown to be critical for binding to E2 include a phenylalanine residue at position 186 (13
). This residue is located on the head subdomain of CD81 and forms part of a low-polarity patch with other additional solvent-exposed aliphatic hydrophobic residues, for example, isoleucine at positions 181 and 182 and leucine at position 185 (26
). These data, together with our mutational analyses, suggest that E2-CD81 binding is mediated by interaction of predominantly hydrophobic residues present on both molecules.
The effects of mutagenesis on HCVpp infectivity were interesting. Firstly, some mutations that reduced (but did not abrogate) CD81 binding had minimal impact on HCVpp infectivity. This suggests that suboptimal binding conferred by each E1E2 complex at a cell surface does not adversely affect entry. This is in agreement with previous studies that showed that E2 molecules that had different CD81 binding affinities were all capable of conferring comparable HCVpp infectivity (54
). Secondly, while there was good concordance between loss of CD81 binding and abrogation of HCVpp infectivity, many mutations that had minimal, if any, effect on CD81 binding resulted in a large reduction in HCVpp infectivity. We showed that loss of infectivity was unlikely due to unsuccessful formation of the E1E2 noncovalent heterodimer, the supposed functional form of the viral glycoproteins (14
), or inefficient HCVpp incorporation. Abrogation of HCVpp infectivity conferred by some of the mutations has previously been shown. HCV entry is a multifactorial process (6
); therefore, it is possible that some of the mutations interfere with other steps in the viral entry process, for example with SR-BI binding or the fusion process. It will be important to confirm these findings in alternative infection systems. Introduction of these mutations into the recently described in vitro infectious clone JFH-1 (31
) will help resolve this issue.
In conclusion, these studies have identified a number of conserved E2 residues that are critical for CD81 binding and thus provide significant new insights into this important step in the virus life cycle. In addition, these data will prove important in the future design and targeting of small-molecule inhibitors to block CD81 binding, particularly if the detailed structure of the E2 molecule can be resolved.