Ebola virus is among the deadliest viruses known and has caused on ongoing and sustained epidemic in Western Africa 2013-2015. The mysteries behind Ebola virus RNA transcription, replication and viral assembly remain mysteries in part, because no high- resolution structural models yet exist by which we may interpret these processes. The mission of this project is to define the structural and biochemical mechanisms governing Ebola virus RNA synthesis and viral assembly. The viral nucleocapsid is a multi-protein-RNA complex at the core of each virion that serves as a molecular machine to synthesize and encapsidate new copies of the viral RNA genome. The viral proteins in this machine have both positive and negative influences on viral RNA synthesis and control the balance of transcription and replication. Understanding the structures, protein-protein interactions, and assemblies of this nucleocapsid machine is key to development of antivirals. However, current low resolution models of the nucleocapsid present conflicting models. Here, we propose complementary crystallographic analysis of each complex that builds the nucleocapsid (NP-RNA, NP-VP35, NP-VP24, NP-VP30 and NP-VP40), combined with high-resolution electron microscopy reconstructions of assembled nucleocapsids. The EM reconstructions will be achieved using state-of-the-art instrumentation, innovative techniques, and high-quality purified samples, and the structural findings will be supported by biochemical, biophysical and functional analysis. Importantly, crystals are in hand for nearly all proposed complexes and functional assays are operational in the laboratory. The resulting structures of the viral nucleocapsid and its component assemblies will transform our understanding of the assembly and replication of Ebola and emerging filoviruses. Further, the results of this proposal will provide roadmaps for functional analysis of these viruses as well as strategies for development of antivirals to defend human populations against them.
Ebola virus can be up to 90% lethal and is responsible for the ongoing epidemic in Western Africa. This project will provide molecular images of the viral nucleocapsid, will illuminate how the virus assembles and replicates, and will provide blueprints for development of antiviral drugs.
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