Structural Biology of RiboNucleoProtein complexes of pathogenic arena and bunyaviruses

所有负义RNA病毒(NSRV)都将其RNA基因组封装成核糖核蛋白复合体(RNPs)。RNPs是由病毒核衣壳(N)蛋白包裹在基因组(和反基因组)RNA上形成的。RNPs必须与它们的同源聚合酶结合，形成病毒RNA合成的活性模板，从而产生有囊化的复制产物和未囊化的mRNAs。RNP的形成也是基因组包装成后代病毒颗粒所必需的，对于ArenaVirus和BunyaVirus，病毒组装是通过RNP和病毒糖蛋白之间的直接结合来调节的。因此，RNP的形成对病毒的繁殖至关重要，因此是一个潜在的治疗靶点。除了RNP的形成和病毒组装外，ArenaVirus和BunyaVirus的N蛋白还参与其他关键功能，其中许多涉及与宿主细胞成分的相互作用。目的：1.在大肠杆菌中表达和纯化兔病毒和Arena病毒的重组N蛋白，如Oropouche病毒(Oropouche)、汉坦病毒(HantaVirus)、番茄斑萎病病毒(TospoVirus)和卢霍病毒(Lujo Virus)。这些将用于使用RNA结合分析的体外测试，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。目的2.在上述每个家族的细胞培养物中培养病毒(选择合适的遏制水平的代表以允许它们在利兹培养)，并从活病毒中提纯RNP用于EM工作。首先，这些将通过2D负染进行表征，然后将最好的例子纳入3D冷冻-EM。目的3.使用前两个目标获得的结构数据来产生突变体，以测试细胞中的功能，如微基因组分析。反向遗传学系统已经被用于治疗布尼安韦拉病毒(利兹)、克里米亚刚果出血热病毒和奥罗波什病毒。新奇的是：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蛋白结构-功能关系，以更好地了解病毒生物学。虽然在理解RNPs方面取得了进展，但也出现了一些问题，涉及RNPs是如何组装的，以及封装的RNA是如何被病毒聚合酶复制的。我们的目标是用跨学科的方法来回答这些紧迫的问题。时效性：世界不断受到新病毒株的威胁，包括流感、MERS和局部的埃博拉病毒。我们的目标是研究两个节段型NSRV-BunyaVirus和ArenaVirus中RNP组装的结构生物学及其结构。这些病毒本身具有很高的致病性，也与流感等其他病毒密切相关，更远的是与埃博拉、腮腺炎、麻疹和狂犬病病毒有关。RNP结构和功能的信息将为我们提供有关控制病毒基因表达和逃避宿主免疫等基本机制的信息，因此可能为开发新的抗病毒药物提供途径。

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.