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

Removal of GP1 N-glycans imparts CatB independence and increases protease sensitivity. Endosomal processing of EBOV GP by cathepsin B (CatB) is an important step in EBOV entry that results in the removal of both the MLD and glycan cap, exposing the RBD (17) (18) (19) (20) . Additionally, it has been shown that disruption of the N40 NGS (T42A mutation) leads to CatB independence in the absence of the MLD (21) . As the inclusion of a similar mutation (T42V) in our glycoprotein mutants consistently led to enhanced transduction, we assessed whether the increase in transduction by our mutants correlated with increased CatB independence. Pseudovirion entry mediated by GP1⌬muc, GP, the 6G mutant, containing a fully deglycosylated glycan cap, and the GPm8G mutant containing a MLD that was fully deglycosylated for N-linked glycans were abrogated by treating Vero cells with the CatB inhibitor CA-074, which profoundly blocked CatB activity (Fig. S4A ). These data indicated that these GPs were dependent upon CatB processing for subsequent transduction steps (Fig. 3A ). VSV pseudotyped with the native glycoprotein (VSV-G) served as a control in these studies and was completely resistant to the drug. As anticipated, the mutant with a mutation of T42 in the absence of the MLD was significantly less sensitive to CA-074 ( Fig. 3A , T42V⌬muc); however, we did not observe complete CatB independence as has been previously reported (21) , possibly due to a Val rather than Ala substitution at this site. Assessment of the T42V mutant in the context of GP also demonstrated partial CA-074 independence. The CatB independence of 7G was similar to that of the T42V mutant, and removing all N-linked glycans in GP1 (7Gm8G) enhanced CatB independence slightly more. Our observed trend of enhanced transduction with increasing elimination of GP1 N-linked glycans was more robust than the trend of CatB independence with our mutants. As glycosylation is known to protect from proteases (22), we determined whether removing glycans increased the protease sensitivity of GP. Previous work has shown that treatment with 200 g/ml thermolysin (THL) results in an~20-fold increase in GP-mediated transduction associated with complete removal of the glycan cap and MLD (20, 21) . To initially assess THL sensitivity, we evaluated the transduction efficiency of GP and 7Gm8G following treatment with increasing concentrations of THL up to a high concentration (200 g/ml). 7Gm8G was dramatically more sensitive to low concentrations of THL than GP, providing evidence that the deglycosylated GP was more readily proteolytically processed (Fig. S4B ). Given these results, a panel of NGS mutants was evaluated for the relationship between entry and sensitivity of deglycosylated GP to low concentrations (1.25 g/ml) of protease (Fig. 3B) . A correlation between the number of mutations and the THL sensitivity was observed. The results from 7G and GPm8G indicated that the removal of glycans from the core has a greater effect on protease sensitivity, possibly due to the large number of O-glycans still present in the MLD. However, 7Gm8G showed the greatest protease sensitivity, suggesting an additive effect when the mutations of 7G and GPm8G were combined. These findings indicated that N-glycans in the glycan cap and, to a lesser extent, in the MLD control the efficiency of proteolytic processing, thereby regulating subsequent steps in entry, such as binding to the endosomal receptor, NPC1.