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|The role of glycans in hepatitis C virus infection
|ProQuest Dissertations & Theses
|Source: Dissertation Abstracts International, Volume: 69-12, Section: B, page: 7345.;Advisors: Tatjana Dragic.
|Hepatitis C virus (HCV) causes life-threatening liver disease. There is no vaccine and treatments are suboptimal. HCV entry into hepatocytes is dependent on envelope glycoproteins E1 and E2, the latter being the receptor-binding subunit. E1 and E2 are highly glycosylated. The 15 glycosylation sites on E2 are highly conserved. We and others have shown that L-SIGN binds high-mannose carbohydrates on E2 and mediates HCV capture and trans-infection of hepatoma cells. L-SIGN isoforms encode between 3 and 9 tandem repeats of a 23 residue stretch in the oligomerization domain. We determined these L-SIGN isoforms variably bind the soluble ectodomain of E2 (sE2) and HCV pseudoparticles (HCVpp). These differences were reflected in trans-infection efficiency, which was highest for isoform 7, progressively decreased with decreasing numbers of repeats, and was lowest for isoform 3. These findings provide a molecular mechanism whereby L-SIGN polymorphisms could influence the establishment and progression of HCV infection.;We also identified E2-associated glycans that are critical for viral entry, protein folding and/or neutralization. The removal of any one glycan in position T385, T388, N417 or N532 of HCV E2 caused the virus to become hypersensitive to neutralization by several HCV subtypes (1a, 1b, 2b, 3a, 4e and 5a). Moreover, sE2 comprising mutated glycosylation positions N417A or N532A, bound the HCV entry receptor, CD81, with a higher affinity than wild type sE2. This indicated these glycans mask the highly conserved CD81 receptor binding site, which is a major neutralization epitope.;Based on this work, we hypothesized that immunization of mice with E2 manipulated to contain overexposed functional epitopes will focus the humoral response on the CD81 binding site. In another strategy aimed at exposing E2 functional domains, we treated WT sE2 with DTT to break inter- and intramolecular disulfide bonds. High titers of anti-E2 antibodies were elicited with WT, N417Q/N532Q mutant or DTT-treated sE2. However, only mice immunized with DTT-treated protein neutralized entry of HCVpp expressing the autologous envelope glycoproteins subtype 1a, and cross-neutralized HCVpp subtype 1b. Our findings have implications for vaccine design.
|Appears in Collections:
|Albert Einstein College of Medicine: Doctoral Dissertations
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