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

Previous work has shown that macrophages are important cellular targets during the early days of filovirus infection (31) . The effective transduction of murine peritoneal macrophages by N-glycan-deficient GP1-bearing pseudovirions, along with our findings that hMGL-dependent enhancement of transduction was relatively independent of the presence of GP1 N-glycans, supports the idea proposed by others (32) that EBOV entry into macrophages may be mediated by this CLEC. However, additional mechanisms of EBOV entry into macrophages are also likely to be important, particularly in light of our findings that 7Gm8G pseudovirions have~300% better transduction of macrophages than GP, yet 7Gm8G transduces hMGL-expressing cells at~60% of the transduction level of the WT. We and others have identified that TIM family members mediate virus uptake into cells by interacting with phosphatidylserine on the surface of virions (16, 30) , and it is possible that this uptake mechanism is important for EBOV entry into macrophages. Future studies need to explore the role for phosphatidylserine receptors during in vivo filovirus infection. Our findings that deglycosylation of GP pseudovirions enhances the transduction of Vero cells and peritoneal macrophages provides evidence that the N-glycans on GP1 decrease the efficiency of the entry process. The removal of glycans masking the RBD enhanced proteolytic processing of GP but did not result in the ability of unprocessed EBOV GP to bind the C loop of NPC1, consistent with an earlier study demonstrating that the removal of the MLD did not unmask the RBD for NPC1 interaction (23) . This result suggests that there are residues that are critical for NPC1 binding, such as F88 (23) , that are masked by the glycan cap/MLD polypeptide rather than being concealed by the heavy glycan shield. Thus, it is likely that the increased sensitivity of our deglycosylated mutants to proteolytic processing results in more efficient transit through the endosomal compartments, leading to greater transduction efficiency.