Structural Biology of RiboNucleoProtein complexes of pathogenic arena and bunyaviruses

所有负义RNA病毒（NSRV）都将其RNA基因组折叠成核糖核蛋白复合物（RNP）。RNP形成为病毒核衣壳（N）蛋白包裹基因组（和反基因组）RNA。RNP必须与它们的同源聚合酶结合以形成用于病毒RNA合成的活性模板，从而产生双核苷酸化的复制产物和未双核苷酸化的mRNA。RNP的形成也是基因组包装到子代病毒颗粒中所必需的，并且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缔合介导的。因此，RNP的形成是病毒增殖所必需的，因此代表了一个潜在的治疗靶点。除了RNP的形成和病毒组装，沙粒病毒和布尼亚病毒的N蛋白还涉及其他关键功能，其中许多涉及与宿主细胞组分的相互作用。因此，RNP的形成是一个过程，它是必不可少的了解，并可能提供一个抗病毒治疗的目标。Express（in E.大肠杆菌）中纯化重组N蛋白，并从示例性布尼亚病毒和竞技场病毒如奥罗普什病毒（一种正布尼亚病毒）、首尔病毒（一种汉坦病毒）、番茄斑萎病毒（一种番茄斑萎病毒）和卢霍病毒（一种沙粒病毒）中纯化重组N蛋白。这些将用于使用RNA结合测定法（例如通过荧光各向异性）的体外测试，并且我们将尝试在有和没有RNA的情况下使它们结晶。目标2.在细胞培养物中从上述每个家族中培养病毒（选择适当控制水平的代表，使其能够在利兹培养），并从活病毒中纯化RNP用于EM工作。最初，这些将通过2D负染色来表征，然后将最好的例子带入3D冷冻EM。使用前两个目标的结构数据来产生突变体，以测试细胞中的功能，如微型基因组测定。反向遗传学系统已经用于Bunyamwera病毒（在利兹）、克里米亚刚果出血热病毒和奥罗波什病毒。新奇：直到最近，人们对NSRV 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。虽然在理解RNP方面取得了进展，但出现了一些与RNP如何组装以及病毒聚合酶如何复制核糖核酸有关的问题。我们的目标是使用跨学科的方法来回答这些紧迫的问题。时间：世界不断受到新病毒株的威胁，包括流感，MERS和局部，埃博拉病毒的出现。我们的目的是研究RNP组装的结构生物学和它的架构在两个家庭的分段NSRV-布尼亚病毒和沙粒病毒。这些病毒本身具有高致病性，与其他病毒如流感密切相关，与埃博拉病毒、腮腺炎病毒、麻疹病毒和狂犬病病毒关系较远。RNP结构和功能的信息将告知我们的基本机制，如控制病毒基因表达和逃避宿主免疫，因此可能会建议新的抗病毒药物的途径。

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.