STRUCTURAL STUDIES OF VIRAL STRUCTURES

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 (1)DVVP 1：所有病毒的生存能力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，并产生基因组DNA/RNA分子，其5 '端共价连接到通常称为VPg或末端蛋白（TP）的多肽。 双RNA病毒形成了一个独特的双链RNA病毒家族，其基因组与VP g相连。 在这里，我们建议使用双RNA病毒作为一个范例，以阐明在基因组复制的病毒蛋白引发的结构基础。 双RNA病毒科中的果蝇X病毒（Drosophila X virus，DXV）编码具有聚合酶和病毒引物功能的多肽VP 1。 我们现在已经在家里生长出了能在3.5埃下工作的DXV VP 1晶体。 我们的计划是在CHESS收集高分辨率的原生数据集，并准备几个重原子衍生物。 因为引起人类疾病的各种病毒（例如，由于小核糖核酸病毒、杯状病毒、腺病毒和嗜七核糖核酸病毒通过蛋白质引发启动基因组复制，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)流感病毒核蛋白：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（总共8个片段）以核糖核蛋白（RNP）复合物的形式被包裹。 核蛋白（NP）是RNP的主要蛋白组分，其沿着每个基因组RNA片段的整个长度结合，形成在成熟病毒中发现的双螺旋RNP结构。 最近在我们的实验室中已经确定了流感A病毒NP的晶体结构，并且它显示出与弹状病毒和博尔纳病病毒（两者都是单负病毒目的成员）的总体折叠和拓扑结构截然不同的总体折叠和拓扑结构。 基于计算的静电势，RNA结合位点似乎位于NP寡聚体的外表面上，表明病毒RNA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，我们现在已经获得了几种形式的NP结合RNA的晶体。 这些晶体是只有0.2毫米宽的薄板。 我们的计划是收集复杂的高分辨率数据，并使用分子置换来解决晶体结构。 (3)匍匐青霉病毒F（PsV-F）：PsV-F是偏体病毒科中的双链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衣壳，其保护dsRNA基因组并在病毒感染期间充当产生病毒mRNA的机制。 在过去的几年中，在呼肠孤病毒，双RNA病毒和全病毒家族的成员的X射线晶体结构已被确定，它们显示出有趣的相似性，以及在其病毒衣壳结构和功能的独特功能。 PsV-F是一种只有两个dsRNA片段的dsRNA病毒，因此确定PsV-F是否具有与其他具有多个RNA片段的dsRNA相似的衣壳结构是重要的。 我们已经在国内获得了至少3.6埃的PsV-F晶体。 然而，这些晶体不能被冷冻。 我们的计划是收集一个完整的数据集在室温下从毛细管安装的晶体。 晶体结构将使用EM重建作为分子置换模型来求解。

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.