RP01 Project Summary/Abstract for Biochemical and structural mechanisms at the filoviral-host interface The recent filoviral outbreaks, including the 2013-2016 Ebola virus (EBOV) outbreak in West Africa that introduced the virus to other continents, highlight the imminent threat to global health posed by filoviruses and the urgent need for basic and translational efforts. The current COVID-19 pandemic further illustrates the significance of understanding infectious diseases. While recent efforts to develop countermeasures have resulted in vaccine candidates and some therapeutics, filoviruses remain a considerable threat to human health and key questions are still outstanding. There are substantial gaps in our understanding of host-viral interactions that contribute to disease, including how viral proteins limit host responses and assemble into infectious particles. Work in Research Project 1 (RP01) will use proteomic, biochemical, and structural methods to identify and validate molecular mechanisms at the host-viral interface in cellular networks to address this gap. We will address these longstanding mechanistic questions using biochemical and hybrid structural methods, including mass spectrometry, NMR, X-ray crystallography, small angle X-ray scattering (SAXS), and cryoelectron microscopy (cryo-EM), cryoelectron tomography (cryo-TM) to characterize filoviral nucleocapsid (NC) interactions and to define high impact filoviral-host factor interactions modulating PTMs that impact the viral replication cycle, viral NC assembly, and egress. Our strong publication record, preliminary results from ongoing studies, and access to unique facilities and resources support these efforts. We are uniquely positioned due to our highly productive and collaborative team with complementary expertise and a prior record of co-authored studies with investigators in RP02, RP03, Core B, and Core C. Our Aims are: Aim 1. Determine the structural basis and dynamics of the filoviral nucleocapsid (NC) and define NC-host interactions; Aim 2. Develop Protein-protein interaction (PPI) maps that include post-translational modifications (PTMs) from virally infected cells for EBOV and MARV; and Aim 3. Define the molecular mechanisms for PPIs that contribute to filoviral infection defined by this project, RP02 and RP03. At the completion, we expect to define filoviral interaction with host factors that contribute to filoviral infection. Our arsenal of tools from mass spectrometry, biochemistry, and structural biology, together with work from the Research Projects and Scientific Cores within the PPG, enable us to clearly define each contribution to filoviral infection and identify new targets for antivirals.