Astrovirus structure and replication

描述（由申请人提供）：星状病毒是一种小型、无包膜、正义RNA病毒，可感染人类、哺乳动物和鸟类，对人类健康、野生动物和具有重要经济意义的牲畜的健康构成严重威胁。在四种感染人类的加义单链RNA病毒（即小核糖核酸病毒、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一种。星状病毒约7kb的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电镜重建显示，病毒衣壳由一个具有30个突出尖峰的连续衣壳组成。我们的实验室最近确定了星状病毒刺突的晶体结构，揭示了星状病毒CPs与戊型肝炎病毒（HEV）之间意想不到的结构同源性。与其他非包膜正义RNA病毒相比，星状病毒在以下几个方面是独特的：(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加工；(2)病毒CP的125aa c端结构域在病毒组装后被宿主半胱天冬酶去除；(3)星状病毒编码的VPg与杯状病毒中的VPg相似，尽管星状病毒的CP在结构上与HEV CP相似，并且HEV RNA有一个5'帽。利用重组蛋白，我们明确地证明了星状病毒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到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的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述星状病毒、HEV病毒和杯状病毒在基础生物学方面的主要异同。此外，我们的研究结果可能在治疗星状病毒方面有重要的应用

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.