STRUCTURAL STUDIES OF VIRAL STRUCTURES

病毒结构的结构研究

• 批准号: 8171499
• 负责人: Yizhi Jane Tao
• 金额: $ 1.34万
• 依托单位: CORNELL UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8171499
• 关键词: Acute; Adenoviruses; Antiviral Agents; Antiviral Therapy; Architecture; Binding; Binding Sites; Birnavirus; Blood capillaries; Borna disease virus; Calicivirus; Capsid; Complex; Computer Retrieval of Information on Scientific Projects Database; DNA; Data; Data Set; Double Stranded RNA Virus; Double-Stranded RNA; Drosophila genus; Electrostatics; Family; Family Picornaviridae; Freezing; Funding; Genome; Genomics; Grant; Home environment; Human; Influenza; Influenza A virus; Institution; Laboratories; Length; Link; Modeling; Molecular; Mononegavirales; Nucleoproteins; Partitiviridae; Penicillium; Polymerase; Proteins; RNA; RNA Binding; RNA Viruses; Reovirus; Research; Research Personnel; Resolution; Resources; Rhabdoviridae; Ribonucleoproteins; Roentgen Rays; Source; Structure; Surface; Temperature; Totivirus; United States National Institutes of Health; Viral; Viral Proteins; Virus; Virus Diseases; base; capillary; human disease; influenza A virus nucleoprotein; influenzavirus; interest; member; polypeptide; reconstruction; respiratory; viral RNA

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. (1) DXV VP1: The viability of all viruses is dependent upon faithful replication of their entire genome, including terminal sequences. Many DNA and RNA viruses use protein primers, and produce genomic DNA/RNA molecules with the 5` end covalently linked to a polypeptide, often called VPg or terminal protein (TP). Birnaviruses form a unique family of dsRNA viruses with a VPg-linked genome. Here we propose to use birnaviruses as a paradigm to elucidate the structural basis of viral protein priming in genome replication. The drosophila X virus (DXV) in the birnavirus family encodes polypeptide VP1 that has both the polymerase and viral primer function. We have now grown DXV VP1 crystals that diffract to 3.5 angstrom at home. Our plan is to collect a high-resolution native data set at CHESS and also to prepare several heavy atom derivatives. Because a variety of viruses that cause human diseases (e.g., picornaviruses, caliciviruses, adenoviruses and heptadnaviruses) initiate genome replication by protein priming, the proposed research will provide promising leads for developing new antiviral compounds, as viral polymerases are often targets for antiviral therapy. (2) Influenza nucleoprotein: Influenza A viruses, which cause highly contagious, acute respiratory illnesses in humans, are a group of negative-strand (-) RNA viruses. Like other (-)RNA viruses, the genome of influenza A viruses, eight segments in total, is encapsidated in the form of ribonucleoprotein (RNP) complexes. The nucleoprotein (NP), the major protein component of RNPs, binds along the entire length of each genomic RNA segment, forming the double-helical RNP structure found in mature virus. The crystal structure of influenza A virus NP has recently been determined in our laboratory, and it shows an overall fold and topology vastly different from those of rhabdoviruses and borna disease virus, both members of Mononegavirale order. Based on calculated electrostatic potential, the RNA binding site appears to be located on the outer surface of NP oligomers, suggesting that viral RNA is likely to be exposed on the exterior of viral RNP complexes. In contrast, NP from rhabdoviruses binds to RNA at the interior of their oligomeric complexes. To determine how influenza virus NP binds RNA, we have now obtained several forms of crystals of NP bound to RNA. These crystals are thin-plates that are only 0.2mm wide. Our plan is to collect a high-resolution data for the complex and to solve the crystal structure using molecular replacement. (3) Penicillium stoloniferum Virus F (PsV-F): PsV-F is a double-stranded RNA virus in the Partitiviridae family. Previous studies on many dsRNA viruses suggest that these viruses contain an intact core capsid that protects the dsRNA genome and serves as the machinery for producing viral mRNAs during virus infection. In the past several years, X-ray crystal structures of members in the reovirus, birnavirus and totivirus family have been determined, and they show interesting similarities as well as distinct features in their viral capsid structure and function. As a dsRNA virus with only two dsRNA segments, it is important to determine whether PsV-F is similar capsid architecture as other dsRNA with multiple RNA segments. We have already obtained crystals of PsV-F that diffract to at least 3.6 angstrom at home. These crystals cannot be frozen, however. Our plan is to collect a complete data set at room temperature from capillary-mounted crystals. The crystal structure will be solved using EM reconstruction as molecular replacement models.

该副本是利用众多研究子项目之一 由NIH/NCRR资助的中心赠款提供的资源。子弹和 调查员（PI）可能已经从其他NIH来源获得了主要资金， 因此，可以在其他清晰的条目中表示。列出的机构是 对于中心，这不一定是调查员的机构。 （1）DXV VP1：所有病毒的生存能力取决于其整个基因组的忠实复制，包括末端序列。 许多DNA和RNA病毒都使用蛋白质引物，并与5`末端共价连接到多肽，通常称为VPG或末端蛋白（TP），并产生基因组DNA/RNA分子。 Birnaviruses与VPG连接基因组形成了独特的DSRNA病毒家族。 在这里，我们建议使用birnavirus作为范式来阐明基因组复制中病毒蛋白启动的结构基础。 Birnavirus家族中的果蝇X病毒（DXV）编码具有聚合酶和病毒引物功能的多肽VP1。 现在，我们已经种植了DXV VP1晶体，它们在家中衍射为3.5埃斯特罗姆。 我们的计划是在国际象棋中收集一个高分辨率的本机数据集，并准备几种重型原子衍生物。 因为多种引起人类疾病的病毒（例如，picornaveruses，pahiciviruses，palicivires，腺病毒和亨氏病毒）启动了蛋白质启动的基因组复制，因此拟议的研究将为开发新的抗病毒化合物提供有希望的铅，因为病毒聚合酶通常是抗病毒疗法的靶标。 （2）流感核蛋白：流感病毒，引起人类高度传染性的急性呼吸道疾病，是一组阴性链（ - ）RNA病毒。 与其他（ - ）RNA病毒一样，流感A病毒的基因组总共八个片段也以核糖核蛋白（RNP）复合物的形式封装。 核蛋白（NP）是RNP的主要蛋白质成分，沿每个基因组RNA段的整个长度结合，形成了成熟病毒中发现的双螺旋RNP结构。 流感A病毒NP的晶体结构最近在我们的实验室中确定，它显示了与Mononegavirale Order的成员的总体折叠和拓扑结构与Rhabdovirus和Borna病毒病毒大致不同。 基于计算的静电电势，RNA结合位点似乎位于NP低聚物的外表面，这表明病毒RNA可能会暴露在病毒RNP复合物的外部。 相比之下，来自色齿病毒的NP与寡聚复合物内部的RNA结合。 为了确定流感病毒NP如何结合RNA，我们现在获得了与RNA结合的几种形式的NP晶体。 这些晶体是仅0.2mm宽的薄板。 我们的计划是为复合物收集高分辨率数据，并使用分子置换来解决晶体结构。 （3）杆菌病毒F（PSV-F）：PSV-F是Partitiviridae家族中的双链RNA病毒。 先前对许多DSRNA病毒的研究表明，这些病毒含有完整的核心封闭状态，可保护DSRNA基因组，并用作病毒感染期间产生病毒mRNA的机械。 在过去的几年中，已经确定了eo病毒，Birnavirus和Totivirus家族中成员的X射线晶体结构，并且它们在病毒式衣壳结构和功能中表现出有趣的相似性以及独特的特征。 作为仅具有两个DSRNA段的DSRNA病毒，重要的是确定PSV-F是否与具有多个RNA段的其他DSRNA相似。 我们已经获得了衍射的PSV-F晶体至少在家中备份3.6埃斯特罗姆。 但是，这些晶体不能冷冻。 我们的计划是从毛细管安装的晶体以室温收集完整的数据集。 将使用EM重建作为分子替代模型来解决晶体结构。