HiPathBunya

The Bunyavirales is a very large and diverse order of segmented negative stranded viruses comprising more than 500 species classified in ten families. Spread worldwide, arthropods and rodents are their natural reservoirs and man is occasionally infected resulting in severe diseases including meningitis, encephalitis and haemorrhagic fevers. Their ongoing geographical expansion concerns Europe: significant outbreaks of Crimean-Congo Haemorrhagic fever virus (CCHFV) have occurred in Greece, Turkey, Kosovo, Albania and Spain. The future spread of some of these infectious agents from their traditional geographical niches due to vector species redistribution arising through climate change is a potential threat, emphasising the need to understand their mechanism of function and develop drugs to counteract them. In this context, we propose to address in the HiPathBunya project two critical steps of bunyavirus viral cycle: replication, that gives rise to full-length copies of the genome, and transcription by cap-snatching, that gives rise to translation competent viral mRNAs. These reactions are catalysed by the viral polymerase (L) which interacts with the 3’ and 5’ ends of viral RNA segments and with nucleoproteins (NP) to form ribonucleoprotein particles (RNP) that are the structural units of transcription and replication. To achieve a depth understanding of the biosynthetic mechanisms of viral RNA synthesis, we propose an integrative structural and functional study of the replication machinery of two evolutionarily distant and highly pathogenic bunyaviruses: Hantaan (Hantavirus) and CCHFV (Nairovirus). We first aim at understanding how the structure and dynamic of the polymerase enable all the activities necessary for replication and transcription. For that purpose, we propose to determine the structure of full-length Hantaan and CCHFV polymerases by X-ray crystallography and cryo-EM. We expect to reveal new domains and functions shedding light on ignored aspects of bunyavirus replication. We will then analyse polymerase-promoters binding mode and derive a detailed biochemical analysis of bunyavirus replication/transcription. This will pave the way towards a structural analysis of the polymerase in different functional states. We will them aim at analysing replication in the functional context of RNP using a multi-scale multi-resolution approach. We have recently determined a high-resolution cryo-EM structure of recombinant helical nucleocapsids of Hantaan virus. We will pursue this analysis to determine the exact position of RNA inside the nucleocapsid. Then, studies on the interaction with the L protein will be carried out in order to understand how the L protein gains access to the RNA template. Our integrated view of replication will be completed by the immuno-fluorescence and cellular EM analysis of infected cells sections which will reveal RNP organization in vivo. Finally, we will aim at defining the dynamics of the L proteins during transcription and replication in isolation and in the RNP context. We will use high speed atomic force microscopy on replicating and transcribing samples, also including cellular factors that could be essential for the RNP functions. This will be done with the L protein isolated and with the native RNPs. Like this, the high-resolution structural data will acquire a new dimension by revealing the movements necessary to accomplish the replication and transcription processes. Taken together these approaches will enable to derive a structure-based, mechanistic model to explain how segmented, negative-strand RNA viruses perform transcription and replication and how the switch between the two modes of operation is mechanistically achieved and regulated. It will be of crucial impact for the development of broad spectrum anti-virals.