Role of N-Linked Glycans in the Functions of Hepatitis C Virus Envelope Proteins Incorporated into Infectious Virions

Abstract

Hepatitis C virus (HCV) envelope glycoproteins are highly glycosylated, with generally 4 and 11 N-linked glycans on E1 and E2, respectively. Studies using mutated recombinant HCV envelope glycoproteins incorporated into retroviral pseudoparticles (HCVpp) suggest that some glycans play a role in protein folding, virus entry, and protection against neutralization. The development of a cell culture system producing infectious particles (HCVcc) in hepatoma cells provides an opportunity to characterize the role of these glycans in the context of authentic infectious virions. Here, we used HCVcc in which point mutations were engineered at N-linked glycosylation sites to determine the role of these glycans in the functions of HCV envelope proteins. The mutants were characterized for their effects on virus replication and envelope protein expression as well as on viral particle secretion, infectivity, and sensitivity to neutralizing antibodies. Our results indicate that several glycans play an important role in HCVcc assembly and/or infectivity. Furthermore, our data demonstrate that at least five glycans on E2 (denoted E2N1, E2N2, E2N4, E2N6, and E2N11) strongly reduce the sensitivity of HCVcc to antibody neutralization, with four of them surrounding the CD81 binding site. Altogether, these data indicate that the glycans associated with HCV envelope glycoproteins play roles at different steps of the viral life cycle. They also highlight differences in the effects of glycosylation mutations between the HCVpp and HCVcc systems. Furthermore, these carbohydrates form a "glycan shield" at the surface of the virion, which contributes to the evasion of HCV from the humoral immune response.

Global Mapping of O-Glycosylation of Varicella Zoster Virus, Human Cytomegalovirus, and Epstein-Barr Virus

“…Viruses such as HIV-1, HCV, and Hendra virus exploit N-glycans to shield themselves from the host immune response (23,25,115,116). In a similar way, O-glycans have also been suggested to shield immunodominant epitopes in herpesviruses (91,92).…”

Entry and replication of recombinant hepatitis C viruses in cell culture

“…Secondly, HCVpp presumably assemble as retroviruses, in post-Golgi compartments or at the plasma membrane with a rather unspecific envelope protein incorporation. As a consequence, substantial differences between HCVpp and HCVcc were found concerning their viral glycoproteins in term of glycosylation pattern and oligomerization [152,[161][162][163]. Moreover, the retroviral capsid precludes the use of HCVpp as a model to study uncoating.…”

Hepatitis c virus and host cell lipids: An intimate connection

“…29,30 By analogy to the related flaviviruses, it is assumed that E1 and E2 initially form heterodimers, but might rearrange into trimeric complexes required for entry. [31][32][33][34] The p7 protein, composed of two TM regions that are interconnected by a short cytoplasmic loop, 35 is a viroporin able to form hexa-and heptameric complexes serving as ion channels and required for virus assembly and release. 36,37 NS2 is a dimeric integral membrane protein, composed of two functionally and topologically distinct domains: a highly hydrophobic, N-terminal, membrane anchor domain and a C-terminal cysteine protease domain, which liberates the C-terminus of NS2 from NS3.…”