Visualisation of the bunyavirus RNA synthesis machinery in action

Bunyavirales目的分段负义RNA病毒包括一些现存的最严重的人类病原体，包括世界卫生组织列为优先病原体的3种。它们的RNA基因组与核蛋白（NP）和RNA依赖性RNA聚合酶（RdRp）两种蛋白质结合形成核糖核蛋白复合物（RNP），该复合物是布尼亚病毒的RNA合成酶。以往的研究表明，RNP复合体具有固有的灵活性，导致描述RNP结构的模型相互冲突。然而，我们最近的工作使我们能够通过使用各种显微镜方法，包括负染色电子显微镜，cryoelectrontomography和原子力显微镜（1），从结构上鉴定Bunyamwera病毒（BUNV）RNP。这项工作表明，BUNV RNP呈现出RNP的螺旋结构，并允许产生核蛋白（NP）排列的原子模型，这反过来又允许识别对活性RNP形成至关重要的NP残基。然而，这些发现也引发了许多问题。例如，为什么BUNV RNP是松散的螺旋？什么是负责循环BUNV RNP的机制？为什么在BUNV RNP上通常观察不到RdRp？RdRp在RNParchitecture中的结构是什么？这种模型对其他布尼亚病毒RNP的代表性如何？本项目将通过研究沙粒病毒科成员的RNP来建立这项工作。该家族成员包含人类病原体，这些病原体是出血热的病原体，并且对这些病原体的治疗选择是有限的。该项目旨在确定LCMV RNP的结构（遵循与BUNV RNP相似的方法），确定RNP复合物内LCMV RdRp的结构，对天然RNP进行RNA合成成像，并研究LCMV病毒工厂内的RNP复合物。1-霍普金斯FR，Álvarez-Rodriguez B，Heath GR，Panayi K，Hover S，Edwards TA，BarrJN，丰塔纳J. 2022。天然正布尼亚病毒核糖核蛋白具有螺旋结构。mBio。https://doi.org/10.1128/mbio.01405-22

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