Astrovirus structure and replication

描述(申请人提供)：星状病毒是一种小的、无包膜的、正向RNA病毒，可感染人类、哺乳动物和鸟类，对人类健康和野生动物以及经济上重要的牲畜的福祉构成严重威胁。在感染人类的四种正义单链RNA病毒(即微小核糖核酸病毒、杯状病毒、戊型肝炎病毒和星状病毒)中，星状病毒是特征最少的病毒。星状病毒~7kb基因组编码非结构蛋白nsp1a和nsp1ab以及病毒衣壳蛋白CP。对人类星状病毒的低分辨率冷冻-EM重建显示，病毒衣壳由一个连续的衣壳组成，外壳有30个突出的尖刺。我们实验室最近测定了星形病毒尖峰的晶体结构，这揭示了星形病毒的CPS与戊型肝炎病毒(HEV)之间出人意料的结构同源性。与其他无包膜、正义RNA病毒相比，星状病毒在几个方面都是独特的：(1)病毒的感染性需要宿主细胞外蛋白酶对病毒衣壳进行广泛的蛋白水解性处理；(2)病毒CP的125aa C-末端结构域在病毒组装后由宿主半胱氨酸酶去除；以及(3)星状病毒似乎编码与杯状病毒中发现的VPG相似的VPG，尽管星状病毒CP在结构上类似于HEV CP，而HEV RNA具有5‘-帽。利用重组蛋白，我们明确地证明星状病毒VPG可以被同源病毒RNA依赖的RNA聚合酶(RdRP)尿苷化，并将尿苷化位点定位于Tyr-30。为了更好地了解星状病毒的组装、成熟和复制，我们建议对星状病毒CP、VPG和RdRP进行详细的结构和功能分析。我们的研究将有助于描绘出星状病毒、HEV和杯状病毒基础生物学上的主要相似和不同之处。此外，我们的结果可能在治疗星状病毒方面有重要的应用。 感染。目的1.星状病毒衣壳的组装和成熟。首先，为了确定星状病毒CP C-末端结构域如何促进病毒组装，我们将解析其结构，确定其亚细胞定位，并寻找与其相互作用的细胞蛋白来促进组装。其次，为了阐明星状病毒成熟的机制，我们将确定胰酶处理如何改变星状病毒衣壳的蛋白质组成、生化性质和结构构象。目的2.星状病毒RNA复制的机制。我们将在实验中绘制病毒粒子相关的星形病毒VPG的序列。此外，我们还将确定星状病毒VPG尿苷基化的重要蛋白质、氨基酸和病毒RNA决定因素。为了了解在VPG诱导的RNA合成的不同阶段发生的分子事件，我们将确定apo RdRP、天然VPG和与核苷酸底物结合的RdRP-VPG复合体的晶体结构。该复合体的结构将揭示VPG核苷酸聚合和规则核苷酸聚合是否使用相同的催化机理。

DESCRIPTION (provided by applicant): Astroviruses are small, non-enveloped, positive-sense RNA viruses that infect humans, mammals and birds, posing a serious threat to human health and the well being of wild animals and economically important livestock. Among the four plus-sense, single-stranded RNA viruses that infect human (i.e. picornaviruses, caliciviruses, hepatitis E viruses, and astroviruses), astroviruses are the least characterized ones. The ~7kb genome of astrovirus encodes the nonstructural protein nsp1a and nsp1ab and the viral capsid protein CP. A low resolution cryo-EM reconstruction of a human astrovirus shows that the viral capsid consists of a continuous capsid shell with 30 protruding spikes. Our laboratory recently determined the crystal structure of the astrovirus spike, which reveals unexpected structural homology between the CPs of astrovirus and the hepatitis E virus (HEV). Compared to other non-enveloped, positive-sense RNA viruses, astroviruses are unique in several aspects: (1) Virus infectivity requires extensive proteolytic processing of the viral capsid by host extracellulr proteases; (2) The 125aa C-terminal domain of the viral CP is removed by host caspases following viral assembly; and (3) Astroviruses appear to encode a VPg similar to the VPg found in caliciviruses, despite the fact that the astrovirus CP structurally resembles the HEV CP and that HEV RNA has a 5'-cap. Using recombinant proteins, we have unambiguously demonstrated the astrovirus VPg can be uridylated by the cognate viral RNA-dependent RNA polymerase (RdRP) and have mapped the uridylation site to Tyr-30. To provide a better understanding of astrovirus assembly, maturation, and replication, here we propose to carry out detailed structural and functional analyses of the astrovirus CP, VPg and RdRP. Our research will help delineate major similarities and differences in the fundamental biology of astroviruses, HEV, and caliciviruses. Moreover, our results will likely have important applications in treating astrovirus infection. Aim 1. The assembly and maturation of the astrovirus capsid. First, to determine how the astrovirus CP C- terminal domain functions to promote virus assembly, we will solve its structure, determine its subcellular localization, and search for cellular proteins that it interacs with to promote assembly. Second, to elucidate the mechanism of astrovirus maturation, we will determine how trypsin treatment changes the protein composition, biochemical properties and the structural conformation of the astrovirus capsid. Aim 2. The mechanism of astrovirus RNA replication. We will experimentally map the sequence of the virion- associated astrovirus VPg. Furthermore, we will identify important protein amino acid and viral RNA determinants for astrovirus VPg uridylation. To understand the molecular events occurring during the different stages of VPg-primed RNA synthesis, we will determine the crystal structures of an apo RdRP, a native VPg, and an RdRP-VPg complex bound to a nucleotide substrate. The structure of the complex will reveal whether the same catalytic mechanism is used for both VPg nucleotidylation and regular nucleotide polymerization.