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: 7848822
• 负责人: Yizhi Jane Tao
• 金额: $ 32万
• 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/7848822
• 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; Exhibits; Family; Family Picornaviridae; GTP Binding; Genetic Transcription; Genome; Genomics; Hepadnaviridae; Immature B-Lymphocyte; 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; in vitro activity; 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取决于其整个基因组的忠实复制，包括末端序列。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％末端与多肽相连，在RNA病毒或DNA病毒中通常称为RNA病毒或末端蛋白（TP）中的VPG。在那些蛋白质蛋白质的病毒中，有丘陵病毒，蜡膜病毒，腺病毒和肝病病毒引起严重的人类疾病。由于病毒聚合酶通常是针对抗病毒疗法的，因此蛋白质启动的机理研究应为创新抗病毒药物的发展提供有希望的铅。在这里，我们建议将Birnavirus作为范式来阐明病毒蛋白蛋白蛋白培养的RNA合成的结构基础。 Birnaviruses与VPG连接基因组形成了独特的DSRNA病毒家族。蛋白启动和聚合酶催化功能由同一多肽提供，Birnavirus VP1是蛋白质宣传机制结构研究的绝佳模型。研究最细的Birnavirus传染性囊肿病毒（IBDV），通过破坏Fabricius的Bursa中未成熟的B淋巴细胞而引起鸟类的Gumboro疾病。除了与VPG连接的基因组外，成熟的病毒粒子还包含主要的衣壳蛋白VP2和VP3，病毒聚合酶VP1，以及可能的病毒蛋白酶VP4。为此，我们获得了VP1的晶体结构，并表明VP1在体外表现出自由依赖性和聚合酶活性。 VP1的结构显示了一个前所未有的活性位点，该位点由五个基本RNA聚合酶基序成立，该基本酶基序以C-A-B-D-E的排名顺序排列。通过定向的诱变，我们表明致命的聚合酶突变体在自缔合中保持活跃，这表明VP1 gunaylylation和核苷酸聚合被两个独立的活性位点催化。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会揭示病毒蛋白启动的原子细节，而且还增强了我们对聚合酶催化以及Birnavirus家族的进化史的理解。该提议将：（1）阐明蛋白质引发的复制中的鸟苷化机制； （2）阐明末端启动的机理； （3）表征病毒蛋白VP2，VP3和VP4在RNA转录中的功能作用。各种方法，包括生化测定，X射线晶体学，电子显微镜和反向遗传学，将用于解决这些问题。