Structural Biology of RiboNucleoProtein complexes of pathogenic arena and bunyaviruses

所有负义RNA病毒（nsrv）都将其RNA基因组封装到核糖核蛋白复合物（RNPs）中。RNA是病毒核衣壳(N)蛋白包裹在基因组（和抗基因组）RNA上形成的。RNA必须与其同源聚合酶结合，形成病毒RNA合成的活性模板，从而产生衣壳化的复制产物和未衣壳化的mrna。基因组包装成子代病毒颗粒也需要形成RNP，对于沙粒病毒和布尼亚病毒，病毒组装是通过RNP和病毒糖蛋白之间的直接关联来介导的。因此，RNP的形成对病毒增殖至关重要，因此代表了一个潜在的治疗靶点。除了RNP形成和病毒组装外，沙粒病毒和布尼亚病毒的N蛋白还参与其他关键功能，其中许多与宿主细胞组分的相互作用有关。因此，RNP的形成是一个必须了解的过程，并可能为抗病毒治疗提供靶点。目标：目标1。表达（在大肠杆菌中）并纯化重组N蛋白，这些重组N蛋白来自诸如Oropouche（一种正布尼亚病毒）、Seoul病毒（一种汉坦病毒）、番茄斑疹枯萎病毒（一种Tospovirus）和Lujo病毒（一种沙粒病毒）等病毒和沙粒病毒。这些将用于体外测试，使用RNA结合分析，例如通过荧光各向异性，我们将尝试在有和没有RNA的情况下结晶它们。目标2。在以上每个科的细胞培养物中培养病毒（选择适当的容器水平的代表，允许它们在利兹培养），并从活病毒中纯化RNPs用于EM工作。在将最佳样本放入3D冷冻电镜之前，首先将这些样本进行2D阴性染色。目标3。使用前两个目标的结构数据生成突变体，以测试细胞功能，如迷你基因组测定。反向遗传系统已经用于Bunyamwera病毒（在利兹）、克里米亚刚果出血热病毒和Oropouche病毒。新颖性：直到最近，人们对来自nsrv的N蛋白的高分辨率结构一无所知。我们和其他人一直在使用经典细胞和分子生物学与现代结构生物学技术相结合的方法来研究N蛋白的结构-功能关系，以更好地了解病毒生物学。虽然在了解RNPs方面取得了进展，但与RNPs如何组装以及病毒聚合酶如何复制被封装的RNA有关的几个问题已经出现。我们的目标是用跨学科的方法来回答这些紧迫的问题。及时性：世界不断受到新病毒株的威胁，包括流感、中东呼吸综合征和局部的埃博拉病毒。我们的目的是研究两个分节nsrv家族-布尼亚病毒和沙粒病毒的RNP组装及其结构生物学。这些病毒本身具有高致病性，也与流感等其他病毒密切相关，与埃博拉病毒、腮腺炎病毒、麻疹病毒和狂犬病病毒的关系较远。RNP结构和功能的信息将为我们提供诸如控制病毒基因表达和逃避宿主免疫等基本机制的信息，从而可能为新的抗病毒药物的开发提供途径。

All negative sense RNA viruses (NSRVs) encapsidate their RNA genomes into ribonucleoprotein complexes (RNPs). RNPs are formed as viral Nucleocapsid (N) protein wraps around the genomic (and anti-genomic) RNA. RNPs must associate with their cognate polymerase to form active templates for viral RNA synthesis, resulting in generation of encapsidated replication products and unencapsidated mRNAs. RNP formation is also required for genome packaging into progeny virus particles, and for arenaviruses and bunyaviruses, virus assembly is mediated through direct association between the RNP and viral glycoproteins. RNP formation is thus essential for virus multiplication and therefore represents a potential therapeutic target.In addition to RNP formation and virus assembly, the N proteins of arenaviruses and bunyaviruses are implicated in other critical functions, many of which relate to interactions with components of the host cell. RNP formation is therefore a process that it is essential to understand, and may provide a target for anti-viral therapy.Objectives: Objective 1. Express (in E. coli) and purify recombinant N protein from example bunya and arena viruses such as Oropouche (an Orthobunyanvirus), Seoul virus (a Hantavirus), tomato spotted wilt virus (a Tospovirus), and Lujo virus (an Arenavirus). These will be used for in vitro testing using RNA binding assays e.g. by fluorescence anisotropy, and we will attempt to crystallise them both with and without RNA. Objective 2. Grow viruses in cell culture from each of the above families (choosing representatives of the appropriate containment level to allow them to be cultured at Leeds), and purify RNPs from live virus for EM work. Initially these will be characterized by 2D negative stain before taking the best examples into 3D cryo-EM.Objective 3. Use the structural data derived from the first two objectives to generate mutants to test function in cells such as mini-genome assays. Reverse genetics systems are already in use for Bunyamwera virus (in Leeds), Crimean Congo Hemorrhagic Fever virus and Oropouche virus.Novelty: Until very recently, nothing was known about the high-resolution structures of N proteins from NSRVs. We, and others, have been using a combination of classical cell and molecular biology with modern structural biology techniques to investigate N protein structure-function relationships to better understand virus biology. While progress in being made towards understanding RNPs, several questions have emerged relating to how RNPs are assembled, and how the encapsidated RNA is replicated by the viral polymerase. We aim to use an interdisciplinary approach to answer these pressing questions.Timeliness: The world is under constant threat from emergence of new virus strains including influenza, MERS and topically, Ebola virus. We aim to study the structural biology of RNP assembly and its architecture in two families of segmented NSRVs - Bunyaviruses and Arenaviruses. These are highly pathogenic in their own right and also are closely related to others such as influenza, and more distantly to Ebola, Mumps, Measles and Rabies viruses. Information of RNP structure and function will inform us on basic mechanisms such as control of viral gene expression and evading host immunity, and may therefore suggest routes to new antivirals.