Ribonucleoprotein Structure in Pathogenic Orthobunyaviruses

The orthobunyavirus genus within the Bunyavirales order contains many pathogens of humans and livestock. Their genome consists of three segments of negative-sense, single-stranded RNA and these are encapsidated with polymers of nucleocapsid protein (NP) which binds RNA in a sequence-independent manner to form ribonucleoproteins (RNPs). Formation of RNPs protects the viral genome from the host immune system and has roles in transcription, viral RNA replication and the correct packaging of segments into new virions. Thus, the RNP is an attractive target for the development of antivirals, which is of particular importance as no specific therapies exist for any of the human pathogens of the genus. Within the field there are currently two different hypotheses on the overall structure of the RNP filament, and establishing a definitive model has been hampered by the extreme flexibility and heterogeneity of the filaments. This project aimed to accurately describe the architecture of the orthobunyavirus RNP and produce a model which illuminates its gross structural features, and mechanisms of assembly and RNA binding. Infectious Bunyamwera virus (BUNV) was propagated and purified by ultracentrifugation, allowing the extraction and purification of RNPs from virions, which were then visualized by negative stain electron microscopy (EM). An ensemble of microscopy methods encompassing negative stain EM, single particle cryo-EM and cryo-electron tomography was employed to overcome the extreme heterogeneity of the filaments, and to produce several models of the RNP that permitted the fitting of NP crystal structures and clearly exhibited a helical architecture. This structural information will aid in the development of small molecules which inhibit formation of the orthobunyavirus RNP and which could be investigated further for their therapeutic potential.