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.

该子项目是利用该技术的众多研究子项目之一 资源由 NIH/NCRR 资助的中心拨款提供。子项目及 研究者 (PI) 可能已从 NIH 的另一个来源获得主要资金， 因此可以在其他 CRISP 条目中表示。列出的机构是 对于中心来说，它不一定是研究者的机构。 (1) DXV VP1：所有病毒的生存能力都取决于其整个基因组（包括末端序列）的忠实复制。 许多 DNA 和 RNA 病毒使用蛋白质引物，并产生 5' 末端与多肽（通常称为 VPg 或末端蛋白 (TP)）共价连接的基因组 DNA/RNA 分子。 Birnaviruses 形成一个独特的 dsRNA 病毒家族，具有 VPg 连接的基因组。 在这里，我们建议使用双RNA病毒作为范例来阐明基因组复制中病毒蛋白启动的结构基础。 双RNA病毒家族中的果蝇X病毒(DXV)编码具有聚合酶和病毒引物功能的多肽VP1。 我们现在已经在家中培育出了衍射强度为 3.5 埃的 DXV VP1 晶体。 我们的计划是在 CHESS 收集高分辨率的原生数据集，并准备几种重原子衍生物。 由于引起人类疾病的多种病毒（例如小核糖核酸病毒、杯状病毒、腺病毒和七DNA病毒）通过蛋白质启动启动基因组复制，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线索，因为病毒聚合酶通常是抗病毒治疗的靶点。 （2）流感核蛋白：甲型流感病毒是一组负链（-）RNA病毒，可引起人类高度传染性的急性呼吸道疾病。 与其他 (-)RNA 病毒一样，甲型流感病毒的基因组共有八个片段，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，沿着每个基因组 RNA 片段的整个长度结合，形成成熟病毒中发现的双螺旋 RNP 结构。 我们实验室最近确定了甲型流感病毒NP的晶体结构，其整体折叠和拓扑结构与弹状病毒和博纳病病毒（这两种病毒均属于单链病毒目）有很大不同。 根据计算的静电势，RNA结合位点似乎位于NP寡聚物的外表面，这表明病毒RNA很可能暴露在病毒RNP复合物的外部。 相反，来自弹状病毒的 NP 在其寡聚复合物的内部与 RNA 结合。 为了确定流感病毒 NP 如何结合 RNA，我们现在获得了几种形式的 NP 与 RNA 结合的晶体。 这些晶体是薄板，宽度仅为 0.2 毫米。 我们的计划是收集该配合物的高分辨率数据并使用分子置换来解析晶体结构。 (3)青霉菌病毒F(PsV-F)：PsV-F是部分病毒科的双链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衣壳，可保护 dsRNA 基因组，并作为病毒感染期间产生病毒 mRNA 的机制。 在过去的几年中，呼肠孤病毒、双病毒和totivirus家族成员的X射线晶体结构已被确定，它们在病毒衣壳结构和功能方面表现出有趣的相似性和独特特征。 作为仅具有两个 dsRNA 片段的 dsRNA 病毒，确定 PsV-F 是否与其他具有多个 RNA 片段的 dsRNA 具有相似的衣壳结构非常重要。 我们已经在家中获得了衍射至少3.6埃的PsV-F晶体。 然而，这些晶体不能被冷冻。 我们的计划是在室温下从毛细管安装的晶体收集完整的数据集。 将使用电磁重建作为分子替代模型来求解晶体结构。