Summary Ebolavirus (EboV) is a highly pathogenic enveloped virus that causes outbreaks of zoonotic infection in Africa. The clinical symptoms are manifestations of the massive production of pro-inflammatory cytokines in response to infection1 and in many outbreaks, mortality exceeds 75%. The unpredictable onset, ease of transmission, rapid progression of disease, high mortality and lack of effective vaccine or therapy have created a high level of public concern about EboV2. Here we report the identification of a novel benzylpiperazine adamantane diamide-derived compound that inhibits EboV infection. Using mutant cell lines and informative derivatives of the lead compound, we show that the target of the inhibitor is the endosomal membrane protein Niemann-Pick C1 (NPC1). We find that NPC1 is essential for infection, that it binds to the virus glycoprotein (GP), and that the anti-viral compounds interfere with GP binding to NPC1. Combined with the results of previous studies of GP structure and function, our findings support a model of EboV infection in which cleavage of the GP1 subunit by endosomal cathepsin proteases removes heavily glycosylated domains to expose the N-terminal domain3–7, which is a ligand for NPC1 and regulates membrane fusion by the GP2 subunit8. Thus, NPC1 is essential for EboV entry and a target for anti-viral therapy.

Nipah virus (NiV) is a deadly emerging paramyxovirus. The NiV attachment (NiV-G) and fusion (NiV-F) envelope glycoproteins mediate both syncytium formation and viral entry. Specific N-glycans on paramyxovirus fusion proteins are generally required for proper conformational integrity and biological function. However, removal of individual N-glycans on NiV-F had little negative effect on processing or fusogenicity and has even resulted in slightly increased fusogenicity. Here, we report that in both syncytium formation and viral entry assays, removal of multiple N-glycans on NiV-F resulted in marked increases in fusogenicity (>5-fold) but also resulted in increased sensitivity to neutralization by NiV-F-specific antisera. The mechanism underlying the hyperfusogenicity of these NiV-F N-glycan mutants is likely due to more-robust six-helix bundle formation, as these mutants showed increased fusion kinetics and were more resistant to neutralization by a fusioninhibitory reagent based on the C-terminal heptad repeat region of NiV-F. Finally, we demonstrate that the fusogenicities of the NiV-F N-glycan mutants were inversely correlated with the relative avidities of NiV-F's interactions with NiV-G, providing support for the attachment protein "displacement" model of paramyxovirus fusion. Our results indicate that N-glycans on NiV-F protect NiV from antibody neutralization, suggest that this "shielding" role comes together with limiting cell-cell fusion and viral entry efficiencies, and point to the mechanisms underlying the hyperfusogenicity of these N-glycan mutants. These features underscore the varied roles that N-glycans on NiV-F play in the pathobiology of NiV entry but also shed light on the general mechanisms of paramyxovirus fusion with host cells.

The cytoplasmic tails of the envelope proteins from multiple viruses are known to contain determinants that affect their fusogenic capacities. Here we report that specific residues in the cytoplasmic tail of the Nipah virus fusion protein (NiV-F) modulate its fusogenic activity. Truncation of the cytoplasmic tail of NiV-F greatly inhibited cell-cell fusion. Deletion and alanine scan analysis identified a tribasic KKR motif in the membraneadjacent region as important for modulating cell-cell fusion. The K1A mutation increased fusion 5.5-fold, while the K2A and R3A mutations decreased fusion 3-to 5-fold. These results were corroborated in a reversepseudotyped viral entry assay, where receptor-pseudotyped reporter virus was used to infect cells expressing wild-type or mutant NiV envelope glycoproteins. Differential monoclonal antibody binding data indicated that hyper-or hypofusogenic mutations in the KKR motif affected the ectodomain conformation of NiV-F, which in turn resulted in faster or slower six-helix bundle formation, respectively. However, we also present evidence that the hypofusogenic phenotypes of the K2A and R3A mutants were effected via distinct mechanisms. Interestingly, the K2A mutant was also markedly excluded from lipid rafts, where ϳ20% of wild-type F and the other mutants can be found. Finally, we found a strong negative correlation between the relative fusogenic capacities of these cytoplasmic-tail mutants and the avidities of NiV-F and NiV-G interactions (P ‫؍‬ 0.007, r 2 ‫؍‬ 0.82). In toto, our data suggest that inside-out signaling by specific residues in the cytoplasmic tail of NiV-F can modulate its fusogenicity by multiple distinct mechanisms.

, we found that virus-specific differences in the requirement for cathepsin B are correlated with sequence polymorphisms at residues 47 in GP1 and 584 in GP2. We applied these findings to the analysis of additional ebolavirus isolates and correctly predicted that the newly identified ebolavirus species Bundibugyo, containing D47 and I584, is cathepsin B dependent and that ebolavirus Zaire-1995, the single known isolate of ebolavirus Zaire that lacks D47, is not. We also obtained evidence for virusspecific differences in the role of cathepsin L, including cooperation with cathepsin B. These studies strongly suggest that the use of endosomal cysteine proteases as host factors for entry is a general property of members of the family Filoviridae. E bdaviruses and the closely related marburgvirus comprise the family Filoviridae (6,8,9,16). Several lines of recent investigation have elucidated key steps in the pathway for ebolavirus entry into cells. Ebolavirus particles attach to cells through the binding of their glycoprotein (GP) to cell surface receptors or lectins, such as TIM-1 and DC-SIGN, expressed on the plasma membrane (1,22,27,29,37). Membrane-bound particles are taken up into cells by a macropinocytosis-like mechanism and transported to late endosomes/lysosomes (LE/LY) (20,30,31,34), which contain essential entry host factors. We previously showed that cleavage of ebolavirus Zaire-Mayinga (EBOV-May) GP by endosomal cysteine proteases is required for infection (7). More recent work has revealed a second host factor in LE/LY that is broadly required by filoviruses: Niemann-Pick C1 (NPC1) (5, 10), a multipass transmembrane protein that resides in the limiting membrane (44). According to a recently proposed model, virus GP is cleaved by endosomal cysteine proteases and binds to NPC1 (10).Several studies have examined the role of protease cleavage in more detail for EBOV-May. They show that cathepsin L functions in concert with cathepsin B to cleave the GP1 subunit of virus GP (7,35). Structural and functional studies reveal that proteases remove the heavily glycosylated carboxyl-terminal domain of GP1 to expose a more conserved domain that is closely associated with GP2 (12,19,25) and that is proposed to contain the binding site for the filovirus receptor (4,13,24,28). Further, we recently showed that cleaved, but not uncleaved, GP1 binds to purified LE/LY membranes in an NPC1-dependent manner and coimmunoprecipitates with NPC1 (10). We identified small molecules that target NPC1, inhibit infection, and block the binding of cleaved GP1 to NPC1-containing membranes (10), strongly suggesting that the conserved N-terminal domain of GP1 is a ligand for NPC1. Taken together, these previous findings suggest a model in which proteolytic cleavage of GP to remove the carboxylterminal domain of GP1 and expose its N-terminal domain may be functionally analogous to the role of CD4 binding to HIV gp120 to displace highly variable loops and create the coreceptorbinding site (18). Our recent studies show that NPC1 expression...