The long-term goal of this research is to understand the pathogenesis of Ebola virus infection at the molecular level. A potential bioterrorism agent, Ebola virus causes severe hemorrhagic disease in humans and is one of the most deadly human pathogens encountered to date, earning classification as a 'Category A Priority Pathogen' in the NIAID Biodefense and Emerging Infectious Disease Research Opportunities Program. Yet, neither therapeutic treatments nor protective vaccines are available to control this often lethal infection. The combination of a reverse genetics system for the generation of Ebola virus entirely from cloned cDNA and access to a biosafety level 4 facility has positioned the applicant to undertake the following specific aims: 1) To understand the regulation of Ebola viral transcription, in which bicistronic minireplicon and reverse genetics strategies will be used to assess the replicative significance of the unique features characterizing the Ebola virus genome, such as overlapping transcriptional stop/start signals and an unusually long intergenic region; 2) To clarify the mechanisms that drive the formation of infectious Ebola virus particles, in which the roles of the VP40 matrix protein and GP glycoprotein in particle formation and budding will be investigated; 3) To define the role of VP24 protein in Ebola viral replication, in which the intracellular localization of VP24, its association with cellular membranes and/or other viral proteins, and its function in virion budding and membrane fusion will be determined; and 4) To identify the factors and mechanisms responsible for the extreme virulence of Ebola virus infection, in which a murine model of Ebola virus infection will be used to screen mutations that may enhance or diminish viral pathogenicity. Moreover, using a wild-type Ebola virus, furin-mediated GP cleavage, or the mucin-like domain in GP, and the secreted glycoprotein, sGP, will also be evaluated for a possible contribution to Ebola virulence. Collectively, these studies will contribute to the deciphering of the molecular basis for the extreme virulence of this pathogen. Ultimately, the information gained could be exploited in the development of antiviral compounds and effective vaccines.
