Astrovirus structure and replication

星状病毒的结构和复制

• 批准号: 8430278
• 负责人: Yizhi Jane Tao
• 金额: $ 21.08万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-09-01 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8430278
• 关键词: Active Sites; Adult; Affect; Amino Acids; Antiviral Agents; Astrovirus; Binding; Biochemical; Biological Assay; Biology; Birds; C-terminal; Caliciviridae; Calicivirus; Capsid; Capsid Proteins; Caspase; Cells; Child; Complex; Crystallography; Development; Elderly; Enzymes; Event; Family Picornaviridae; Gastroenteritis; Genome; Grant; Health; Hepatitis E virus; Human; In Vitro; Infection; Laboratories; Livestock; Mammals; Maps; Mediating; Membrane; Metals; Molecular; Molecular Conformation; NMR Spectroscopy; Nonstructural Protein; Nucleotides; Peptide Hydrolases; Perforation; Personal Satisfaction; Phylogenetic Analysis; Polymerase; Polyproteins; Potyviridae; Property; Protein C; Proteins; Proteolysis; Proteolytic Processing; RNA; RNA Viruses; RNA chemical synthesis; RNA replication; RNA-Directed RNA Polymerase; Recombinant Proteins; Recombinants; Research; Resolution; Role; Sampling; Signal Transduction; Site; Site-Directed Mutagenesis; Sorting - Cell Movement; Staging; Structure; Substrate Specificity; Techniques; Translations; Trypsin; Viral; Viral Proteins; Virion; Virus; Virus Assembly; Virus-like particle; Wild Animals; X-Ray Crystallography; extracellular; overexpression; particle; polymerization; protein expression; public health relevance; reconstruction; research study; viral RNA

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.

描述（由申请方提供）：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，对人类健康以及野生动物和重要经济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。星状病毒基因组约7 kb，编码非结构蛋白nsp 1a和nsp 1ab以及病毒衣壳蛋白CP。人类星状病毒的低分辨率冷冻EM重建显示，病毒衣壳由具有30个突出刺突的连续衣壳壳组成。我们的实验室最近确定了星状病毒刺突的晶体结构，这揭示了星状病毒和戊型肝炎病毒（HEV）的CP之间意想不到的结构同源性。与其它无包膜的正义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125 aa C-末端结构域在病毒组装后被宿主半胱天冬酶去除;和（3）尽管星状病毒CP在结构上类似于HEV CP并且HEV RNA具有5 '-帽，但星状病毒似乎编码与杯状病毒中发现的VPg相似的VPg。使用重组蛋白，我们已经明确证明了星状病毒VPg可以通过同源病毒RNA依赖性RNA聚合酶（RdRP）进行尿苷化，并将尿苷化位点映射到Tyr-30。为了更好地了解星形病毒的组装、成熟和复制，我们建议对星形病毒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述星状病毒，戊型肝炎病毒和杯状病毒的基本生物学的主要相似性和差异。此外，我们的研究结果可能在治疗星状病毒方面具有重要应用 感染目标1.星状病毒衣壳的组装与成熟。首先，为了确定星状病毒CP C-末端结构域如何起作用以促进病毒组装，我们将解决其结构，确定其亚细胞定位，并寻找与其相互作用以促进组装的细胞蛋白。其次，为了阐明星状病毒成熟的机制，我们将确定胰蛋白酶处理如何改变星状病毒衣壳的蛋白质组成，生化特性和结构构象。目标2.星状病毒RNA复制的机制。我们将通过实验绘制病毒体相关星状病毒VPg的序列.此外，我们将确定重要的蛋白质氨基酸和病毒RNA决定因素的星状病毒VPg尿苷酸化。为了了解VPg引发的RNA合成的不同阶段期间发生的分子事件，我们将确定载脂蛋白RdRP，天然VPg和与核苷酸底物结合的RdRP-VPg复合物的晶体结构。复合物的结构将揭示是否相同的催化机制用于VPg核苷酸化和常规核苷酸聚合。