title: Influence of N-linked glycans on intracellular transport of hepatitis C virus E1 chimeric glycoprotein and its role in pseudotype virus infectivity.
abstract: We have previously reported a functional role associated with hepatitis C virus (HCV) E1 glycoprotein using vesicular stomatitis virus (VSV)/HCV pseudotype. In this study, we have investigated the role of glycosylation upon intracellular transport of chimeric E1-G, and in infectivity of the pseudotyped virus. Interestingly, surface expressed E1-G exhibited sensitivity to Endoglycosidase H (Endo H) treatment, which was similar to full-length E1, suggesting that additional complex oligosaccharides were not added while E1-G was in transit from the endoplasmic reticulum (ER) to the mammalian cell surface. As a next step, each of the four potential N-linked glycosylation sites located at amino acid position 196, 209, 234, or 305 of the E1 ectodomain were mutated separately (asparagine --> glutamine), or in some combination. FACS analysis suggested that mutation(s) of the glycosylation sites affect the translocation of E1-G to the cell surface to different extents, with no single site being particularly essential. VSV pseudotype virus generated from glycosylation mutants exhibited a decrease in titer with an increasing number of mutations at the glycosylation sites on chimeric E1-G. In a separate experiment, N-glycosidase F treatment of pseudotype generated from the already synthesized E1-G or its mutants decreased virus titer by approximately 35%, and the neutralization activity of patient sera was not significantly altered with N-glycosidase F-treated pseudotype virus. Taken together, our results suggested that E1-G does not add complex sugar moieties during transport to the cell surface and retain the glycosylation profile of its parental E1 sequence. Additionally, the removal of glycans from the E1-G reduced, but does not completely impair, virus infectivity.
authors.affiliation: Department of Internal Medicine, Saint Louis University, 3635 Vista Avenue, St. Louis, MO 63110, USA.
authors.forename: Aster
authors.initials: A
authors.lastname: Beyene
authors.forename: Arnab
authors.initials: A
authors.lastname: Basu
authors.forename: Keith
authors.initials: K
authors.lastname: Meyer
authors.forename: Ranjit
authors.initials: R
authors.lastname: Ray
mesh_terms: D000596:Amino Acids / Q000235:genetics; D000914:Antibodies, Viral / Q000276:immunology; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism / Q000821:virology; D004721:Endoplasmic Reticulum / Q000378:metabolism / Q000821:virology; D006026:Glycoside Hydrolases; D006367:HeLa Cells; D016174:Hepacivirus / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D006526:Hepatitis C / Q000276:immunology; D006801:Humans; D009154:Mutation; D009500:Neutralization Tests; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000494:pharmacology / Q000502:physiology*; D021381:Protein Transport / Q000187:drug effects; D016865:Reassortant Viruses / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D014721:Vesicular stomatitis Indiana virus / Q000235:genetics; D014759:Viral Envelope Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*
chemical_list: D000596:Amino Acids; D000914:Antibodies, Viral; C087035:E1 protein, Hepatitis C virus; D011134:Polysaccharides; D014759:Viral Envelope Proteins; D006026:Glycoside Hydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase