Filovirus nucleocapsid assembly, caught in the act, by in situ structural biology

Filoviruses emerge nearly annually and cause hemorrhagic fever with high mortality, and frequent unpredictable outbreaks. Development of broad-spectrum antiviral agents is needed and will be guided by a clear understanding of the structures and assemblies of essential viral machinery. The filovirus nucleocapsid forms the core of the virion and is essential for replication and transcription of the viral genome and host immune suppression. The viral replication and nucleocapsid assembly occur in viral replication factories in host cytoplasm. Assembly of these nucleocapsids in replication factories and their subsequent transport to the cell surface, and packaging into virions are all critical steps in the filovirus life cycle. Much about these essential processes, however, remains unknown, because the assembly processes happen inside cells, and traditional structural biology approaches involved use of purified, often recombinant, samples outside cells. Thus, we do not yet have structural understandings of what filovirus assembly, transport, matrix-interaction and virion-recruitment processes look like. For example, structures are available for the recombinant N-terminal half of the EBOV nucleoprotein (NP) coiled with cellular RNA, but we don’t yet understand the structure of the C-terminal half of NP that recruits the other NC proteins, nor do we understand how other viral proteins assemble to form complete nucleocapsid structure or how the nucleocapsid interacts with the virus matrix. A new technology, in situ cryo-electron tomography, now allows us to carry out structural biology analyses inside cells and allows us to collect previously inaccessible information about the structure of the assembling nucleocapsid inside infected cells. The goal of this project is to apply cryo-focused ion beam (FIB) milling-enabled in situ cryo-electron tomography (cryo-FIB-ET) to understand how the Ebola virus nucleocapsid is assembled inside cells and how the structures change before and after virion incorporation. Study of these complexes inside cells, from both transfected and infected cells, will provide us our first information about the assembly process of virus replication factories inside cells and complete structural model of filovirus nucleocapsid structure. The fundamental structural organization and critical protein interfaces revealed in this study are likely conserved among all filoviruses. Hence, the proposed work will provide insight into the fundamental structural principles of filovirus assembly. The knowledge and expertise gained here may also be relevant for understanding the structures of other negative-strand RNA viruses that have a similar protein structural organization.