Abstract: Project Overview for Structural and Functional Studies of Ebola Virus RNA synthesis Ebolaviruses (EBOVs) are non-segmented negative-strand RNA viruses (NNSVs) and Category A Priority biodefense pathogens that cause frequent lethal hemorrhagic fever, including the devastating and ongoing outbreak in West Africa. Approved anti-EBOV therapeutics are lacking. EBOV replicates with high efficiency in vivo while effectively evading host innate antiviral immunity. The unrestrained replication and associated inflammation leads to death. A complete understanding of EBOV pathogenesis therefore requires understanding how the viral RNA dependent RNA polymerase (RDRP) complex interacts with host factors. The overarching goal of this program project proposal is to understand the mechanisms of RDRP function by defining the interactions among its components and with the host proteome and by defining signals that regulate its function. Toward this goal Project 1 will identify cis- and trans-acting factors that impact EBOV RDRP activity. Project 2 will define a structural basis for the EBOV RDRP complex, identify regulatory mechanisms, and determine species and strain specificities, and Project 3 will use genome-wide RNAi and proteomic strategies to identify host factors that modulate RDRP function. Project 1 provides functional context into which a subset of Project 3 “hits” can be interpreted, while Project 3 will likely identify host factors relevant to the findings of Project 1. Project 2 will provide a structural framework that will be used to integrate findings from both Projects 1 and 3. Together, these studies will provide mechanistic insights into how protein-protein and protein-vRNA interactions control EBOV RDRP function. Research projects are supported by an Administrative Core, a Protein Production and Protein Interaction Core, and critically, a Biosafety Level 4 (BSL4) Core. Core B will generate unique reagents, including monoclonal and synthetic antibodies. Core C (BSL4) will provide unique capabilities to obtain materials from and perform tests using wild type and mutant EBOVs as well as pathogenesis evaluation. The work will be performed by highly productive and collaborative investigators with expertise in every aspect of the proposed studies, including biochemistry, EBOV pathogenesis, high throughput screening and data analysis, immunology, proteomics, structural biology, and virology. These studies will provide unprecedented insights into the components of the EBOV RDRP and in its interaction with the host as well as species and strain differences that affect the RDRP complex. We also expect to identify specific viral and host factor interactions as novel therapeutic targets.