Structural basis of protein-primed RNA synthesis by birnaviruses

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%末端共价连接到多肽，在RNA病毒中通常称为VPg，在DNA病毒中通常称为末端蛋白（TP）。这些蛋白引发病毒包括小核糖核酸病毒、杯状病毒、腺病毒和肝病毒，它们会引起严重的人类疾病。由于病毒聚合酶经常是抗病毒治疗的靶标，因此对蛋白质启动的机制研究应该为开发创新的抗病毒化合物提供有希望的线索。在这里，我们建议使用birnavvirus作为范例来阐明病毒蛋白引物RNA合成的结构基础。伯纳病毒形成了一个独特的dsRNA病毒家族，其基因组与vpg相关。由于核糖核酸病毒VP1具有相同多肽的蛋白质引物和聚合酶催化功能，因此它是研究蛋白质引物机制的一个很好的模型。传染性法氏囊病病毒（IBDV）是研究得最好的一种病毒，它通过破坏法氏囊中未成熟的b淋巴细胞在禽类中引起Gumboro病。除了与vpg相关的基因组外，成熟病毒粒子还含有主要的衣壳蛋白VP2和VP3，病毒聚合酶VP1，可能还有病毒蛋白酶VP4。为此，我们获得了VP1的晶体结构，并证明VP1在体外具有自胍基化和聚合酶活性。VP1的结构显示了一个前所未有的活性位点，由5个必要的RNA聚合酶基序按C-A-B-D-E排列而成。通过定点诱变，我们发现一个致命的聚合酶突变体在自核苷酸基化中保持活性，这表明VP1的核苷酸基化和核苷酸聚合是由两个独立的活性位点催化的。这些有趣的发现和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，而且还将增强我们对聚合酶催化和birnavvirus家族进化史的理解。本研究将：(1)阐明鸟苷化在蛋白引物复制中的作用机制；(2)阐明终端启动机制；(3)表征病毒蛋白VP2、VP3和VP4在RNA转录中的功能作用。各种各样的方法，包括生化分析、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。

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.