The Primed Ebolavirus Glycoprotein (19-Kilodalton GP1,2): Sequence and Residues Critical for Host Cell Binding▿ †

Entry of ebolavirus (EBOV) into cells is mediated by its glycoprotein (GP1,2), a class I fusion protein whose structure was recently determined (J. E. Lee et al., Nature 454:177-182, 2008). Here we confirmed two major predictions of the structural analysis, namely, the residues in GP1 and GP2 that remain after GP1,2 is proteolytically primed by endosomal cathepsins for fusion and residues in GP1 that are critical for binding to host cells. Mass spectroscopic analysis indicated that primed GP1,2 contains residues 33 to 190 of GP1 and all residues of GP2. The location of the receptor binding site was determined by a two-pronged approach. We identified a small receptor binding region (RBR), residues 90 to 149 of GP1, by comparing the cell binding abilities of four RBR proteins produced in high yield. We characterized the binding properties of the optimal RBR (containing GP1 residues 57 to 149) and then conducted a mutational analysis to identify critical binding residues. Substitutions at four lysines (K95, K114, K115, and K140) decreased binding and the ability of RBR proteins to inhibit GP1,2-mediated infection. K114, K115, and K140 lie in a small region modeled to be located on the top surface of the chalice following proteolytic priming; K95 lies deeper in the chalice bowl. Combined with those of Lee et al., our findings provide structural insight into how GP1,2 is primed for fusion and define the core of the EBOV RBR (residues 90 to 149 of GP1) as a highly conserved region containing a two-stranded β-sheet, the two intra-GP1 disulfide bonds, and four critical Lys residues

Multiple Cationic Amphiphiles Induce a Niemann-Pick C Phenotype and Inhibit Ebola Virus Entry and Infection

<div><p>Ebola virus (EBOV) is an enveloped RNA virus that causes hemorrhagic fever in humans and non-human primates. Infection requires internalization from the cell surface and trafficking to a late endocytic compartment, where viral fusion occurs, providing a conduit for the viral genome to enter the cytoplasm and initiate replication. In a concurrent study, we identified clomiphene as a potent inhibitor of EBOV entry. Here, we screened eleven inhibitors that target the same biosynthetic pathway as clomiphene. From this screen we identified six compounds, including U18666A, that block EBOV infection (IC₅₀ 1.6 to 8.0 µM) at a late stage of entry. Intriguingly, all six are cationic amphiphiles that share additional chemical features. U18666A induces phenotypes, including cholesterol accumulation in endosomes, associated with defects in Niemann–Pick C1 protein (NPC1), a late endosomal and lysosomal protein required for EBOV entry. We tested and found that all six EBOV entry inhibitors from our screen induced cholesterol accumulation. We further showed that higher concentrations of cationic amphiphiles are required to inhibit EBOV entry into cells that overexpress NPC1 than parental cells, supporting the contention that they inhibit EBOV entry in an NPC1-dependent manner. A previously reported inhibitor, compound 3.47, inhibits EBOV entry by blocking binding of the EBOV glycoprotein to NPC1. None of the cationic amphiphiles tested had this effect. Hence, multiple cationic amphiphiles (including several FDA approved agents) inhibit EBOV entry in an NPC1-dependent fashion, but by a mechanism distinct from that of compound 3.47. Our findings suggest that there are minimally two ways of perturbing NPC1-dependent pathways that can block EBOV entry, increasing the attractiveness of NPC1 as an anti-filoviral therapeutic target.</p> </div

CADs do not disrupt the interaction of 19 kDa GP and NPC1.

<p>(A) NPC1-FLAG-enriched LE/Lys membranes from CHO NPC1 cells were disrupted and then incubated with inhibitors for 30 min at RT: mock (4% DMSO), E64d (10 µM), compound 3.47 (13 µM), clomiphene (242 µM), Ro 48-8071 (174 µM), and U18666A (800 µM); each inhibitor was used at a concentration 100 fold over its IC₅₀ for inhibition of infection. The samples were then incubated with 3 µg uncleaved (GP) or cleaved (GP_{19 kDa}) EBOV GP ectodomains for 1 hr at RT. Samples were then lysed, and incubated overnight with anti-FLAG beads. Bound NPC1 and GP were then eluted from beads, and run on an SDS-PAGE gel. The gel was then transferred, blotted for both NPC1 and EBOV GP, and imaged for fluorescent signal. As predicted, uncleaved GP (∼130 kDa) did not co-precipitate with NPC1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056265#pone.0056265-Cote1" target="_blank">[20]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056265#pone.0056265-Miller1" target="_blank">[24]</a>. (B) The intensities of the GP, GP_{19 kDa}, and NPC1 bands from each sample of the blot shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0056265#pone-0056265-g007" target="_blank">Fig. 7A</a> were quantified and GP or GP_{19 kDa} was normalized to its respective NPC1 band signal. The experiment was conducted four times with similar results, and a representative experiment is shown.</p