GENE EXPRESSION OF NEGATIVE STRAND RNA VIRUSES

负链RNA病毒的基因表达

• 批准号: 3140367
• 负责人: Amiya K. Banerjee
• 金额: $ 23.88万
• 依托单位: CLEVELAND CLINIC FOUNDATION
• 依托单位国家: 美国
• 财政年份: 1988
• 资助国家: 美国
• 起止时间: 1988-08-01 至 1993-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3140367
• 关键词: Paramyxoviridae; RNA directed DNA polymerase; Vesiculovirus; antiviral agents; binding proteins; complementary DNA; gene expression; gene mutation; genetic manipulation; genetic mapping; genetic transcription; messenger RNA; molecular cloning; phosphoproteins; protein biosynthesis; protein kinase; ribonucleoproteins; tissue /cell culture; virus RNA; virus diseases; virus genetics; virus protein; virus replication

The long term goal of this proposal is to gain an understanding of the mechanisms by which genome RNA of the negative strand virus group is transcribed and replicated. This group comprises some of the most serious human pathogens in terms of both mortality and morbidity, such as rabies, influenza, mumps, etc. Vesicular stomatitis virus (VSV) - a prototype of negative stranded rhabdoviruses, provides an excellent model system to study transcription and replication in vitro. An RNA-dependent RNA polymerase (L and NS protein) is packaged within the virion which faithfully copies the genome RNA in vitro and in vivo; this enzyme complex, in association with the N protein is also involved in the replication process. Thus, a thorough study of the structure and function of the L and NS proteins, as well as the nucleocapsid protein (N), will be essential to understand the precise mechanism of transcription and replication of VSV. This research proposal is designed to use clonaly expressed NS, N, and L proteins using SP6 transcription system and, by subsequent genetic manipulation of the genes, to study the functions of the domains within the polypeptides in the transcription and replication processes. This approach will lead to a better understanding of the roles of these polypeptides in viral gene expression and the eventual development of possible antiviral drugs. The specific aims of the grant are as follows: (1) Elucidate the functional role of the domains with the NS protein involved in binding to the template and the L protein, phoshorylation, and RNA chain elongation. In addition, determine the domains that govern specificity of interaction between the NS protein, the L protein and the N-RNA template using NS proteins of different serotypes; (2) Determine the functional role of various domains of the N protein involved in binding to the NS protein, and assembly with the genome RNA and replication in vitro; (3) Synthesize biologically active L protein from a cDNA clone and determine the domains involved in transcription. In addition, identify the domains involved in binding to nucleotides, protein kinase and capping activities.

该提案的长期目标是了解 负链基因组RNA的机制 病毒群被转录和复制。 该组包括 一些最严重的人类病原体 死亡率和发病率，如狂犬病、流感、腮腺炎等。 水疱性口炎病毒（VSV）——负链病毒的原型 弹状病毒，提供了一个优秀的模型系统来研究 体外转录和复制。 RNA依赖性RNA 聚合酶（L 和 NS 蛋白）包装在病毒颗粒内， 在体外和体内忠实地复制基因组 RNA；这 与 N 蛋白相关的酶复合物也参与其中 在复制过程中。 因此，对结构的深入研究 L 和 NS 蛋白以及核衣壳的功能 蛋白质（N），对于理解精确机制至关重要 VSV 的转录和复制。 这项研究计划是 设计用于使用 SP6 克隆表达的 NS、N 和 L 蛋白 转录系统，并通过随后的遗传操作 基因，以研究基因内域的功能 转录和复制过程中的多肽。 这 方法将有助于更好地理解这些角色的作用 病毒基因表达中的多肽以及最终的 开发可能的抗病毒药物。 该计划的具体目标 补助金如下： (1) 阐明NS域的功能作用 参与与模板和 L 蛋白结合的蛋白质， 磷酸化和RNA链延长。 此外，确定 控制 NS 之间相互作用的特异性的域 蛋白质、L 蛋白质和使用 NS 蛋白质的 N-RNA 模板 不同血清型； (2)确定功能作用 参与与 NS 结合的 N 蛋白的各个结构域 蛋白质，并与基因组 RNA 组装并在其中复制 体外； (3) 从cDNA合成具有生物活性的L蛋白 克隆并确定参与转录的结构域。 在 此外，确定参与与核苷酸结合的结构域， 蛋白激酶和加帽活性。