BACKGROUND: N-linked glycosylation is a common posttranslational modification found on viral glycoproteins (GPs) and involved in promoting expression, cellular attachment, protection from proteases, and antibody evasion. The GP subunit GP2 of filoviruses contains 2 completely conserved N-linked glycosylation sites (NGSs) at N563 and N618, suggesting that they have been maintained through selective pressures. METHODS: We assessed mutants lacking these glycans for expression and function to understand the role of these sites during Ebola virus entry. RESULTS: Elimination of either GP2 glycan individually had a modest effect on GP expression and no impact on antibody neutralization of vesicular stomatitis virus pseudotyped with Ebola virus GP. However, loss of the N563 glycan enhanced entry by 2-fold and eliminated GP detection by a well-characterized monoclonal antibody KZ52. Loss of both sites dramatically decreased GP expression and abolished entry. Surprisingly, a GP that retained a single NGS at N563, eliminating the remaining 16 NGSs from GP1 and GP2, had detectable expression, a modest increase in entry, and pronounced sensitivity to antibody neutralization. CONCLUSIONS: Our findings support the importance of the GP2 glycans in GP expression/structure, transduction efficiency, and antibody neutralization, particularly when N-linked glycans are also removed from GP1.
BACKGROUND:N-linked glycosylation is a common posttranslational modification found on viral glycoproteins (GPs) and involved in promoting expression, cellular attachment, protection from proteases, and antibody evasion. The GP subunit GP2 of filoviruses contains 2 completely conserved N-linked glycosylation sites (NGSs) at N563 and N618, suggesting that they have been maintained through selective pressures. METHODS: We assessed mutants lacking these glycans for expression and function to understand the role of these sites during Ebola virus entry. RESULTS: Elimination of either GP2 glycan individually had a modest effect on GP expression and no impact on antibody neutralization of vesicular stomatitis virus pseudotyped with Ebola virus GP. However, loss of the N563 glycan enhanced entry by 2-fold and eliminated GP detection by a well-characterized monoclonal antibody KZ52. Loss of both sites dramatically decreased GP expression and abolished entry. Surprisingly, a GP that retained a single NGS at N563, eliminating the remaining 16 NGSs from GP1 and GP2, had detectable expression, a modest increase in entry, and pronounced sensitivity to antibody neutralization. CONCLUSIONS: Our findings support the importance of the GP2 glycans in GP expression/structure, transduction efficiency, and antibody neutralization, particularly when N-linked glycans are also removed from GP1.
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