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.

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 (1)DXV VP1：所有病毒的生存都依赖于其整个基因组的忠实复制，包括末端序列。许多DNA和RNA病毒使用蛋白质引物，产生基因组DNA/RNA分子，其5‘端与一种多肽共价连接，通常被称为VPG或终端蛋白(TP)。鸟核病毒形成了一个独特的dsRNA病毒家族，其基因组与VPG相连。在这里，我们建议使用鸟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。果蝇X病毒(DXV)属于鸟病毒家族，其编码的多肽VP1既具有聚合酶功能，又具有病毒引物功能。我们现在已经在国内生长出了衍射率为3.5埃的DXV VP1晶体。我们的计划是在国际象棋上收集一个高分辨率的原始数据集，并准备几个重原子衍生品。由于引起人类疾病的各种病毒(如小核糖核酸病毒、杯状病毒、腺病毒和七核糖核酸病毒)通过蛋白质启动来启动基因组复制，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八个片段，以核糖核蛋白(RNP)复合体的形式被包裹。核蛋白(NP)是RNPs的主要蛋白质成分，沿着每个基因组RNA片段的全长结合，形成成熟病毒中存在的双螺旋RNP结构。最近我们实验室测定了甲型流感病毒NP的晶体结构，它的整体折叠和拓扑结构与横纹病病毒和博尔纳病病毒的晶体结构有很大的不同，这两种病毒都属于单重目。根据计算的静电势，RNA结合位置似乎位于NP低聚物的外表面，这表明病毒RNA可能暴露在病毒RNP复合体的外部。相反，来自弹状病毒的NP在其寡聚复合体的内部与RNA结合。为了确定流感病毒NP如何与RNA结合，我们现在已经获得了几种形式的NP与RNA结合的晶体。这些晶体是只有0.2毫米宽的薄板。我们的计划是收集该络合物的高分辨率数据，并使用分子置换来解决晶体结构问题。 (3)匍匐青霉病毒F(Penicillium stoloniferum Virus F，PSV-F)：PSV-F是Partitiviridae科的双链RNA病毒。以前对许多dsRNA病毒的研究表明，这些病毒包含一个完整的核心衣壳，保护dsRNA基因组，并在病毒感染期间作为产生病毒mRNAs的机械。在过去的几年里，呼肠孤病毒、小鸟病毒和轮状病毒家族成员的X射线晶体结构已经被确定，它们在病毒衣壳结构和功能上既有有趣的相似之处，也有明显的特点。作为一种只有两个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是很重要的。我们已经在国内获得了至少3.6埃衍射的PSV-F晶体。然而，这些晶体不能被冻结。我们的计划是在室温下从毛细管安装的晶体中收集完整的数据集。晶体结构将使用EM重构作为分子置换模型来求解。