Structural basis of protein-primed RNA synthesis by birnaviruses

描述（由申请方提供）：所有病毒的生存能力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，其中5%末端与多肽（在RNA病毒中通常称为VP g或在DNA病毒中称为末端蛋白（TP））共价连接。在这些蛋白质引发病毒中，小核糖核酸病毒、杯状病毒、腺病毒和嗜肝DNA病毒引起严重的人类疾病。由于病毒聚合酶通常是抗病毒治疗的靶点，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病毒作为一个范例，以阐明病毒蛋白引发的RNA合成的结构基础。双RNA病毒形成具有VPg-连锁基因组的独特dsRNA病毒家族。由于蛋白质引发和聚合酶催化功能由相同的多肽提供，双RNA病毒VP 1作为蛋白质引发机制的结构研究的一个很好的模型。传染性法氏囊病病毒（IBDV）是目前研究最多的双RNA病毒，通过破坏法氏囊中未成熟的B淋巴细胞而引起禽类的传染性法氏囊病。除了与VPg相连的基因组外，成熟的病毒体还含有主要的衣壳蛋白VP 2和VP 3、病毒聚合酶VP 1以及可能的病毒蛋白酶VP 4。为此，我们获得了VP 1的晶体结构，并表明VP 1在体外表现出自我鸟苷酰化和聚合酶活性。VP 1的结构显示了一个前所未有的活性位点，由五个基本的RNA聚合酶基序以C-A-B-D-E的排列顺序形成。通过定点突变，我们发现，一个致命的聚合酶突变体仍然活跃在自我鸟苷酰化，这表明VP 1 gunaylylation和核苷酸聚合是由两个独立的活性位点催化。这些有趣的发现和我们的其他初步结果表明，VP 1的结构-功能研究不仅将揭示病毒蛋白引发的原子细节，而且还将增强我们对聚合酶催化以及双RNA病毒家族进化历史的理解。这项建议将：（1）阐明蛋白质引发复制中鸟苷酰化的机制;（2）阐明末端引发的机制;（3）表征病毒蛋白VP 2、VP 3和VP 4在RNA转录中的功能作用。一系列不同的方法，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.