A Novel Receptor-induced Activation Site in the Nipah Virus Attachment Glycoprotein (G) Involved in Triggering the

Cellular entry of paramyxoviruses requires the coordinatedactionofboththeattachment(G/H/HN)andfusion(F)glycopro-teins, but how receptor binding activates G to trigger F-mediatedfusionduringviralentryisnotknown.Here,weidentifyareceptor(ephrinB2)-inducedallostericactivationsiteinNipahvirus(NiV)GinvolvedintriggeringF-mediatedfusion.Wefirstgeneratedacon-formational monoclonal antibody (monoclonal antibody 45(Mab45)) whose binding to NiV-G was enhanced upon NiV-G-ephrinB2binding.However,Mab45alsoinhibitedviralentry,anditsreceptorbinding-enhanced(RBE)epitopewastemperature-de-pendent, suggesting that the Mab45 RBE epitope on G may beinvolvedintriggeringF.TheMab45RBEepitopewasmappedtothebase of the globular domain (!6S4/!1H1). Alanine scan mutantswithinthisregionthatdidnotexhibitthisRBEepitopewerealsonon-fusogenic despite their ability to bind ephrinB2, oligomerize, andassociatewithFatwild-type(WT)levels.Althoughcirculardichro-ismrevealedconformationalchangesinthesolubleectodomainofWT NiV-G upon ephrinB2 addition, no such changes weredetected with soluble RBE epitope mutants or short-stalk Gmutants.Additionally,WTG,butnotaRBEepitopemutant,coulddissociatefromFuponephrinB2engagement.Finally,usingabio-tinylated HR2 peptide to detect pre-hairpin intermediate forma-tion, a cardinal feature of F-triggering, we showed that ephrinB2bindingtoWTG,butnottheRBE-epitopemutants,couldtriggerF. In sum, we implicate the coordinated interaction between thebaseofNiV-Gglobularheaddomainandthestalkdomaininmedi-atingreceptor-inducedFtriggeringduringviralentry.