Molecular basis for Rift Valley fever phlebovirus NSs protein function

Rift Valley fever phlebovirus (RVFV) is one of the 8 priority pathogens identified by the WHO that urgently require strategies for the prevention of epidemics and treatment of the disease caused. RVFV relies on its non-structural NSs protein for pathogenicity. NSs is imported into the cell nucleus where it forms characteristic filaments and suppresses the interferon response by interfering with the TFIIH complex, shutting down global transcription, and by specifically inhibiting IFN expression. The modes of action of NSs are poorly understood. The first structure for NSs provided a molecular basis for filament formation and will now inform dissection of NSs functions and interactions. We will use cryo-EM and X-ray microscopy to resolve NSs filament structures. We will use correlative fluorescence and EM to home in on filaments, and various sectioning approaches to obtain medium to near-atomic resolution structures. Crystallography and NMR will be used to obtain the missing structure of the N-terminal domain of NSs, which has been shown to be functionally important. Comparative structural analyses of NSs proteins of two other human-pathogenic phleboviruses will provide insights into the molecular determinants of NSs localisation and polymerisation. Structural work will be complemented by functional analyses using a recombinant viruses, and transient expression systems. We will use a variant of NSs that lacks the ability to form filaments to assess their role in interferon suppression. Using a covalent tagging system for pull-downs we will identify NSs binding partners in cytosol and nuclei that might be required for filament formation and import into the nucleus. Our data show a direct interaction of a soluble NSs variant with the TFIIH complex, which we will characterise using structural biology and functional assays. This project will generate fundamental insights into molecular mechanisms underpinning pathogenesis of RVFV and other phleboviruses.