Comprehensive Functional Analysis of N-Linked Glycans on Ebola Virus GP1

Lennemann, Nicholas J.; Rhein, Bethany A.; Ndungo, Esther; Chandran, Kartik; Qiu, Xiangguo; Maury, Wendy

virion transduction was further enhanced by the removal of all NGS within the GP1 core (7G) or the core and MLD (7Gm8G). Consistent with our findings with 7Gm8G, the elimination of N-glycans solely within the MLD (GPm8G) also led to a modest but consistent increase in transduction, suggesting that NGS in both the core and MLD decreased transduction. In total, these results indicated that N-linked glycans are not required for entry into Vero cells but, rather, decrease the efficiency of entry. GP1 N-glycans do not affect virion attachment to Vero cells. As extensive N-linked deglycosylation of GP led to greater transduction, we sought to identify which step(s) in EBOV entry were affected by the loss of N-linked sugars. Removing the GP1 N-glycans within the GP1 core (7G) or throughout GP1 (7Gm8G) did not affect the binding of pseudovirions to the cell surface (see Fig. S3 , top, in the supplemental material) and the binding of all virions tested was decreased to similar levels (~50% reduction) in the presence of a calcium chelator, EGTA (Fig. S3 , middle). This finding was not unexpected, since EBOV entry into Vero cells has been shown to be mediated by the phosphatidylserine receptor, TIM-1, which binds to virion-associated phosphatidylserine in a calcium-dependent manner (16) .