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

EBOV GP1 is highly glycosylated, yet limited studies have investigated the role of these sugar chains on GP function to date. In this report, we determined the effects of removing the N-linked glycans present on the EBOV GP1 subunit by examining the impact on protein expression/stability, viral entry, and antiserum/ antibody sensitivity. Surprisingly, the elimination of all N-linked glycans on GP1 had no effect on the expression levels of GP, suggesting that, in the presence of the MLD, N-linked glycans attached to GP1 are not critical for GP folding. However, as our Future studies to test our deglycosylated GPs in the context of recombinant filoviruses to verify our findings are warranted. The loss of GP1 NGS significantly decreased the utilization of four of the five C-type lectins known to be used by EBOV GP for entry, while it enhanced EBOV transduction into Vero cells and peritoneal macrophages. The enhanced levels of transduction were associated with enhanced sensitivity to thermolysin cleavage, but deglycosylation did not alleviate the need for proteolysis prior to NPC1 binding. Finally, the loss of highly conserved glycans on the core of GP1 increased virus sensitivity to antibody neutralization. Therefore, we propose that the strong conservation of individual glycosylation sites in the GP1 core across the Ebola virus genus results, at least in part, from selective pressures to protect the virus against immune responses, despite the fact that these sites collectively can have a negative impact on GP-dependent entry. Modeling N-linked glycans onto the GP1⌬muc structure suggests that the highly conserved RBD of Ebola viruses is effectively masked and protected by glycan cap glycans attached to wellconserved NGS, likely protecting the RBD against selective immune pressures, as previously proposed (6, 8, 9, 15, 18, 37) . However, we provide the first experimental evidence to support this hypothesis. Our results suggest that there are neutralizing epitopes in the GP1 core that are normally masked by N-glycans. Surprisingly, the removal of N-glycans from the MLD, which is highly targeted by neutralizing antibodies (7-9), did not affect antiserum sensitivity. Neutralizing epitopes within the MLD that do not appear to be affected by the loss of N-linked glycans may be obstructed by the large number of O-glycans attached to the MLD. Alternatively, neutralizing epitopes within the MLD may target O-linked glycan sequons. Since filovirus hemorrhagic fevers are acute and often lethal infections in primates, it is likely that antibody-driven positive selection for NGS surrounding conserved regions occurs in nonprimate reservoirs, such as bats. Thus, it is important to understand filovirus infection and persistence in bat populations.