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.
Ebola virus and other filoviruses are the cause of an invariably fatal hemorrhagic fever in humans in equatorial Africa, and are agents of concern for bioterrorism. In order to infect humans, these viruses must break into the cytoplasm of human cells, where they can commandeer the resources needed to produce more viruses. This invasion process used by filoviruses is unusual in its complexity, and coopts many of our own proteins and pathways. Previous work by us and others has shed light on this mechanism, but the final steps in invasion that allow the virus to reach the cytoplasm still remain mysterious. Using new tools that allow us to `catch the virus in the act', we will uncover exactly how filoviruses escape into the cytoplasm, and how they exploits our cells during this process. Our work will provide information crucial to the design and development of antiviral drugs that block the ability of filoviruses to infect humans.