Biochemical characterization of Ebola virus GP

Filoviruses cause sporadic outbreaks of highly lethal hemorrhagic fever throughout central Africa. Virus entry is mediated by the sole viral glycoprotein, GP. Furthermore, GP is the main target for neutralizing antibodies. Thus, a better understanding of GP and its functions is critical for the development of antivirals and vaccines.

GP contains a high number of N- and O-linked glycans, which shield the majority of the protein. These glycans are required for cell surface interactions with C-type lectins that mediate internalization of the virus. We found that GP1, but not GP2, N-linked glycans were required for efficient entry into cells expressing the C-type lectins: L-SIGN, DC-SIGN, and LSECtin expressing cells, but O-linked glycans were sufficient for ASGPRI- and hMGL-dependent entry. However, filoviruses also utilize phosphatidylserine (PS) receptors, which bind PS in the viral membrane, to mediate entry into host cells. We found that all N-linked glycosylation sites in GP1 could be mutated without significant impact on expression. Furthermore, removal of all N-linked glycans increased entry into a PS receptor-dependent cell line and primary murine macrophages. These results correlated with an increase in sensitivity to proteolysis, which is required within the late endosome/lysosome to expose the receptor-binding domain. Surprisingly, removal of N-linked glycans that directly shield the receptor-binding domain did not allow for binding to the intracellular receptor, NPC1. Thus, proteolytic removal of heavily glycosylated domains within the late endosome/lysosome exposes critical receptor-binding residues that are masked by polypeptides and not N-linked glycans. Furthermore, removal of the conserved N-linked glycan on the heptad repeat 1 region in GP2 led to an increase in entry. Conversely, removal of the conserved N-linked glycan on the heptad repeat 2 region decreased entry. Removal of either glycan resulted in a decrease in entry mediated by protease-treated GP. Together, these results suggest N-linked glycans on GP2 are involved in controlling fusion. Interestingly, removal of N-linked glycans masking conserved regions of GP led to a significant increase in convalescent antibody-mediated neutralization. Overall, these results indicate that there is an evolutionary trade-off that results in a decrease in entry efficiency in order to protect virus from the immune system.

Analysis of entry mediated by multiple species of ebolavirus indicated that the residue occupying position 95 is a critical determinant of entry. For Ebola virus (EBOV) GP, Sudan virus (SUDV) GP, and Bundibugyo (BDBV) GP, a lysine at position 95 imparts dependence on the cysteine protease cathepsin B. However, a glutamine at this position alleviates this dependence and is found in some early isolates of SUDV. Furthermore, cathepsin B dependence inversely correlated with an increase in sensitivity to protease-mediated degradation of GP. Mutation of K95 to a glutamine in EBOV GP and BDBV GP led to decreased sensitivity to NPC1 and voltage-operated calcium channel inhibitors. Conversely, mutation of the Q95 to a lysine in SUDV GP decreased sensitivity to NPC1 inhibitors and had no impact on voltage-operated calcium channel inhibitors. However, all proteins regardless of the residue at position 95 required NPC1 for entry. Together these results indicate that a single amino acid polymorphism in GP of ebolaviruses has dramatic impacts on entry factor dependence, suggesting potential differences in entry pathways.