Astrovirus structure and replication

星状病毒的结构和复制

• 批准号: 8722430
• 负责人: Yizhi Jane Tao
• 金额: $ 19.15万
• 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 2017-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8722430
• 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（由申请人提供）：赤道病毒是小的，不发达的，阳性的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的健康构成严重威胁，以及对野生动物的福祉和经济重要的牲畜。在感染人类的​​四种超义，单链的RNA病毒中（即丘脑病毒，蜡膜病毒，丙型肝炎病毒和星座病毒），肌动病毒的特征最少。甲状腺病毒的〜7kb基因组编码非结构蛋白NSP1A和NSP1AB以及病毒capsid蛋白CP。人星座病毒的低分辨率冷冻EM重建表明，病毒capsid由连续的带壳壳组成，带有30个突出的尖峰。我们的实验室最近确定了星形病毒尖峰的晶体结构，该结构揭示了星形病毒和乙型肝炎病毒（HEV）之间意外的结构同源性。与其他非发电的阳性RNA病毒相比，赤道病毒在几个方面都是独一无二的：（1）病毒感染性需要宿主外胞外蛋白酶对病毒衣壳进行广泛的蛋白水解处理； （2）病毒CP的125AA C末端结构域通过病毒组装后的宿主胱天蛋白酶去除； （3）尽管星体CP在结构上类似于HEV CP，并且HEV RNA具有5'-cap，但尽管Astrovirus CP在结构上类似于Astrovirus CP，但Astrovirus似乎编码了类似于Calicivirus中发现的VPG的VPG。使用重组蛋白，我们已经明确证明了Astrovirus VPG可以被同源病毒RNA依赖性RNA聚合酶（RDRP）尿酸化，并将尿液化位点映射到Tyr-30。为了更好地理解果蝇组装，成熟和复制，我们建议对Astrovirus 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赤道病毒，HEV和蜡膜基本生物学的主要相似性和差异。此外，我们的结果可能在治疗星形病毒方面具有重要的应用 感染。 AIM 1。果皮capsid的组装和成熟。首先，为了确定如何促进病毒组装的星形病毒CP C-C-末端结构域的功能，我们将解决其结构，确定其亚细胞定位，并搜索与促进组装相互作用的细胞蛋白。其次，为了阐明甲状腺病毒成熟的机制，我们将确定胰蛋白酶处理如何改变蛋白质组成，生化特性和果皮capsid的结构构象。 AIM 2。甲状腺病毒RNA复制的机理。我们将通过实验绘制与病毒相关的星形病毒VPG的序列。此外，我们将确定重要的蛋白质氨基酸和病毒RNA的决定因素，用于甲状腺病毒VPG尿苷。为了理解在VPG引发的RNA合成的不同阶段发生的分子事件，我们将确定与核苷酸底物结合的APO RDRP，天然VPG和RDRP-VPG复合物的晶体结构。该复合物的结构将揭示是否将相同的催化机理用于VPG核苷酸化和常规核苷酸聚合。

项目成果

Structure determination of a human virus by the combination of cryo-EM and X-ray crystallography.

• DOI: 10.1007/s41048-016-0027-2
• 发表时间: 2016
• 期刊: Biophysics reports
• 作者: Liu, Zheng;Guu, Tom S Y;Cao, Jianhao;Li, Yinyin;Cheng, Lingpeng;Tao, Yizhi Jane;Zhang, Jingqiang
• 通讯作者: Zhang, Jingqiang