Visualisation of the bunyavirus RNA synthesis machinery in action

The Bunyavirales order of segmented negative sense RNA viruses comprises some of the most serious human pathogens in existence, including 3 listed by the World Health Organisation as priority pathogens. Their RNA genome associates with two proteins, nucleoprotein (NP) and RNA dependent RNA polymerase (RdRp) to form a ribonucleoprotein complex (RNP); this complex is the bunyaviral RNA synthesis machinery. Previous work demonstrated the RNP complex has inherent flexibility, resulting in conflicting models describing RNP structure. However, our recent work has allowed us to structurally characterise the Bunyamwera virus (BUNV) RNP, by using a variety of microscopy approaches, including negative staining electron microscopy, cryoelectron tomography and atomic force microscopy (1). This work demonstrated that the BUNV RNP presents a helical architecture of the RNP and allowed an atomic model to be generated for the nucleoprotein (NP) arrangement, which in turn allowed NP residues critical for the formation of active RNP to be identified. However, these findings also raised many questions. For example, why are BUNV RNPs loose helices? What is the mechanism responsible for circularising BUNV RNPs? Why is the RdRp not usually observed on BUNV RNPs and what is the structure of the RdRp within the RNP architecture? How representative is this model of other bunyavirus RNPs? This project will build upon this work through investigating the RNPs of members of the Arenaviridae. Members of this family contain human pathogens which are the causative agents of haemorrhagic fever and therapeutic options for such pathogens are limited. Specifically, the RNP of lymphocytic choriomeningitis virus (LCMV) will be investigated, as a representative member of the family. The project will aim to determine the structure of the LCMV RNP (following a similar approach to that employed for BUNV RNP), determine the structure of the LCMV RdRp within the RNP complex, image native RNP's undergoing RNA synthesis and investigate the RNP complex within LCMV viral factories. 1 - Hopkins FR, Álvarez-Rodriguez B, Heath GR, Panayi K, Hover S, Edwards TA, Barr JN, Fontana J. 2022. The Native Orthobunyavirus Ribonucleoprotein Possesses a Helical Architecture. mBio. https://doi.org/10.1128/mbio.01405-22