The unprecedented 2014–2015 Ebola virus (EBOV) outbreak in West Africa has highlighted the urgent, unfulfilled need for vaccines and therapeutics to prevent and treat infections by EBOV and other filoviruses. However, their development is challenged by crucial gaps in our understanding of the virus-host molecular interactions that underpin filovirus infection, disease, and transmission. Recent work on the entry mechanism of filoviruses has revealed that these agents are prototypic members of a group of `late-penetrating' viruses that extensively exploit host endo/lysosomal factors and pathways to invade cells. During the previous grant period, we found that host endosomal cysteine proteases (cysteine cathepsins) program the viral glycoprotein GP to bind the endosomal cholesterol transporter Niemann-Pick C1 (NPC1), which we showed to be an essential intracellular entry receptor. Despite these and other discoveries, however, the culminating steps in filovirus entry—GP-mediated fusion between viral and cellular lipid bilayers, and cytoplasmic escape of the viral nucleocapsid core—remain enigmatic. The overarching goals of this renewal application are to define the molecular mechanism of filovirus membrane fusion, and to identify new filovirus entry host factors with antiviral potential. To fulfill these objectives, the principal investigator has assembled a multidisciplinary team that includes a field leader in the chemical biology of proteases, a leading expert on the biogenesis and functions of endosomes and lysosomes, and a virologist who will extend findings with surrogate viruses to authentic filoviruses. Together we will: (1) Define viral and cellular requirements for filovirus membrane fusion, dissect steps in the fusion reaction, and identify the elusive filovirus fusion trigger factor(s). (2) Determine the roles of newly- identified host factors in viral trafficking to membrane fusion sites, and employ proteomic approaches to uncover novel host factors involved in filovirus membrane fusion.