An exploration of conditions proposed to trigger the Ebola virus glycoprotein for fusion

Fénéant, Lucie; Szymańska-de Wijs, Katarzyna M.; Nelson, Elizabeth A.; White, Judith M.

Ebolaviruses continue to inflict horrific disease and instill fear. The 2013–2016 outbreak in Western Africa caused unfathomable morbidity and mortality (over 11,000 deaths), and the second largest outbreak is on-going in the Democratic Republic of the Congo. The first stage of an Ebolavirus infection is entry, culminating in delivery of the viral genome into the cytoplasm to initiate replication. Among enveloped viruses, Ebolaviruses use a complex entry pathway: they bind to attachment factors on cell surfaces, are engulfed by macropinocytosis, and traffic through the endosomal system. En route, the receptor binding subunit of the glycoprotein (GP) is reduced from ~130 to ~19 kDa by cathepsins. This event allows cleaved GP (GP(cl)) to bind to Niemann-Pick C1 (NPC1), its endosomal receptor. The virus then fuses with a late endosomal membrane, but how this occurs remains a subject of debate. An early, but standing, observation is that entry of particles bearing GP(cl) is inhibited by agents that raise endosomal pH or inhibit cysteine proteases, suggesting the need for an additional factor(s). Yet, some have concluded that NPC1 is sufficient to trigger the fusion activity of GP(cl). Here, we re-examined this question using sensitive cell-cell and pseudovirus-cell fusion assays. We did not observe detectable GP(cl)-mediated fusion with NPC1 or its GP(cl) binding domain at any pH tested, while robust fusion was consistently observed with GP from lymphocytic choriomeningitis virus at low pH. Addition of proposed fusion-enhancing factors—cations (Ca(++) and K(+)), a reducing agent, the anionic lipid Bis(Monoacylglycero)Phosphate, and a mixture of cathepsins B and L—did not induce detectable fusion. Our findings are in line with the earlier proposal that an additional factor is required to trigger the full fusion activity of GP(cl) after binding to NPC1. We discuss caveats to our study and what the missing factor(s) might be.