Hepatitis C virus (HCV) is a major human pathogen that causes serious liver disease, including cirrhosis and hepatocellular carcinoma. The primary target cells of HCV are hepatocytes, and entry is restricted by interactions of the envelope glycoproteins, E1 and E2, with cellular receptors. E1 and E2 form noncovalently linked heterodimers and are heavily glycosylated. Glycans contribute to protein folding and transport as well as protein function. In addition, glycans associated with viral envelopes mask important functional domains from the immune system and attenuate viral immunogenicity. Here, we explored the role of N-and O-linked glycans on E2, which is the receptor binding subunit of the HCV envelope. We identified a number of glycans that are critical for viral entry. Importantly, we showed that the removal of several glycans significantly increased the inhibition of entry by sera from HCV-positive individuals. Only some of the glycans that affected entry and neutralization were also important for CD81 binding. Our results show that HCV envelopeassociated glycans play a crucial role in masking functionally important regions of E2 and suggest a new strategy for eliciting highly neutralizing antibodies against this virus.Hepatitis C virus (HCV) is a single-stranded positive RNA flavivirus that causes serious liver disease in humans (2,21,29,47). Approximately 15% of newly infected individuals clear the virus, and an estimated 170 million people worldwide are persistently infected with HCV (11, 31). Persistence probably results from antigenic variability that allows the virus to escape host immunity (39). It is presently unclear what roles the different arms of the immune system play in the outcome of HCV infection. Several groups have shown that the clearance of HCV infection correlates with a strong, early cytotoxic-T-lymphocyte response (11,31,54). Both neutralizing and nonneutralizing antibodies are elicited by natural HCV infection, but their roles in viral clearance remain contested (4, 9, 10, 33). High antibody titers elicited by the vaccination of chimpanzees with recombinant HCV envelope glycoproteins correlate with protection or a delay in disease onset after challenge with live virus (1,16,45). Furthermore, individuals negative for HCV RNA but positive for HCV antibodies are 12 times more likely to clear a second infection than individuals infected for the first time (35). These studies support the premise that neutralizing antibodies are an essential component of protective immunity, but its correlates remain to be identified.The infectious HCV particle is enveloped by a lipid membrane comprising E1-E2 heterodimers that specifically restrict viral tropism to hepatocytes (8,27,30,47,53). Based on analogies to alphaviral envelope glycoproteins as well as structural modeling, it has been postulated that E1 serves as the fusogenic subunit and that E2 acts as the receptor binding subunit of the HCV envelope (22,24,32,42,60). Alternatively, HCV E1 may facilitate E1-E2 folding into a metastable conformat...
