Structural basis of protein-primed RNA synthesis by birnaviruses

双RNA病毒蛋白引发的RNA合成的结构基础

• 批准号: 7418192
• 负责人: Yizhi Jane Tao
• 金额: $ 33.54万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-06-01 至 2012-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7418192
• 关键词: Active Sites; Address; Adenoviruses; Antiviral Agents; Antiviral Therapy; Aspartate; Binding; Biochemical; Biological Assay; Birds; Birnavirus; Bursa of Fabricius; Calicivirus; Capsid Proteins; Catalysis; Complex; DNA Viruses; DNA-Directed RNA Polymerase; Development; Disease; Double Stranded RNA Virus; Drosophila genus; Electron Microscopy; Endopeptidases; Exhibits; Family; Family Picornaviridae; GTP Binding; Genetic Transcription; Genome; Genomics; Guanosine Monophosphate; Hepadnaviridae; Immature B-Lymphocyte; In Vitro; Infectious bursal disease virus; Infectious pancreatic necrosis virus; Length; Link; Mass Spectrum Analysis; Methods; Mutagenesis; Nucleotides; Peptide Hydrolases; Polymerase; Proteins; RNA; RNA Viruses; RNA chemical synthesis; Recording of previous events; Research; Research Personnel; Role; Site-Directed Mutagenesis; Structural Models; Structure; Viral; Viral Proteins; Virion; Virus; Viviparous-1 protein; X-Ray Crystallography; base; human disease; in vitro Assay; innovation; member; mutant; particle; polymerization; polypeptide; positional cloning; programs; reconstruction

DESCRIPTION (provided by applicant): The viability of all viruses is dependent upon faithful replication of their entire genome, including terminal sequences. Many viruses use protein primers and produce full-length genomic DNA/RNA molecules with the 5% end covalently linked to a polypeptide, often called VPg in RNA viruses or terminal protein (TP) in DNA viruses. Among those protein-priming viruses are picornaviruses, caliciviruses, adenoviruses and hepadnaviruses that cause serious human diseases. As viral polymerases are often targeted for antiviral therapy, mechanistic studies of protein priming should provide promising leads in the development of innovative antiviral compounds. Here we propose to use birnaviruses as a paradigm to elucidate the structural basis of viral protein-primed RNA synthesis. Birnaviruses form a unique family of dsRNA viruses with a VPg-linked genome. With protein priming and polymerase catalytic functions provided by the same polypeptide, birnavirus VP1 serves as an excellent model for structural studies of the protein-priming mechanism. Infectious bursal disease virus (IBDV), the best studied birnavirus, causes Gumboro disease in avian species by destroying immature B-lymphocytes in the Bursa of Fabricius. In addition to a VPg-linked genome, mature virion contains the major capsid proteins VP2 and VP3, the viral polymerase VP1, and possibly the viral protease VP4. To this end, we have obtained a crystal structure of VP1 and have shown that VP1 exhibits both self-guanylylation and polymerase activities in vitro. The structure of VP1 shows an unprecedented active site formed by five essential RNA polymerase motifs arranged in the permuted order of C-A-B-D-E. Through site-directed mutagenesis, we showed that a fatal polymerase mutant remains active in self-guanylylation, suggesting that VP1 gunaylylation and nucleotide polymerization are catalyzed by two separate active sites. These intriguing discoveries, and our other preliminary results, indicate that structure-function studies of VP1 will not only unveil the atomic details of viral protein priming, but also enhance our understanding of polymerase catalysis as well as the evolutionary history of the birnavirus family. This proposal will: (1) elucidate the mechanism of guanylylation in protein-primed replication; (2) elucidate the mechanism of terminal priming; and (3) characterize the functional roles of viral proteins VP2, VP3 and VP4 in RNA transcription. A diverse array of methods, encompassing biochemical assays, X-ray crystallography, electron microscopy and reverse genetics, will be utilized in addressing these questions.

描述（由申请人提供）：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。许多病毒使用蛋白质引物并产生全长基因组 DNA/RNA 分子，其 5% 末端与多肽共价连接，通常在 RNA 病毒中称为 VPg，在 DNA 病毒中称为末端蛋白 (TP)。这些蛋白质启动病毒包括引起严重人类疾病的小核糖核酸病毒、杯状病毒、腺病毒和嗜肝DNA病毒。由于病毒聚合酶通常是抗病毒治疗的目标，因此蛋白质引发的机制研究应该为创新抗病毒化合物的开发提供有希望的线索。在这里，我们建议使用双RNA病毒作为范例来阐明病毒蛋白引发的RNA合成的结构基础。 Birnaviruses 形成一个独特的 dsRNA 病毒家族，具有 VPg 连接的基因组。由于同一多肽提供蛋白质启动和聚合酶催化功能，双RNA病毒VP1成为蛋白质启动机制结构研究的优秀模型。传染性法氏囊病病毒 (IBDV) 是研究最深入的双病毒，它通过破坏法氏囊中未成熟的 B 淋巴细胞而导致鸟类感染 Gumboro 病。除了 VPg 连接的基因组外，成熟病毒体还含有主要衣壳蛋白 VP2 和 VP3、病毒聚合酶 VP1，可能还含有病毒蛋白酶 VP4。为此，我们获得了VP1的晶体结构，并表明VP1在体外表现出自身鸟苷酸化和聚合酶活性。 VP1 的结构显示出前所未有的活性位点，由按 C-A-B-D-E 排列顺序排列的五个必需 RNA 聚合酶基序形成。通过定点诱变，我们发现致命的聚合酶突变体在自身鸟苷酸化中仍然具有活性，这表明 VP1 鸟苷酸化和核苷酸聚合是由两个独立的活性位点催化的。这些有趣的发现以及我们的其他初步结果表明，VP1 的结构功能研究不仅将揭示病毒蛋白启动的原子细节，还将增强我们对聚合酶催化以及双RNA病毒家族进化史的理解。该提案将：（1）阐明蛋白质引发的复制中鸟苷酸化的机制； (2)阐明末端启动机制； (3) 表征病毒蛋白 VP2、VP3 和 VP4 在 RNA 转录中的功能作用。将利用多种方法来解决这些问题，包括生化测定、X射线晶体学、电子显微镜和反向遗传学。