STRUCTURE AND FUNCTION OF PARAMYXOVIRUS L PROTEIN

副粘病毒L蛋白的结构和功能

• 批准号: 2069442
• 负责人: Griffith D. Parks
• 金额: $ 9.24万
• 依托单位: WAKE FOREST UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-08-01 至 1998-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2069442
• 关键词: DNA directed RNA polymerase; Paramyxovirus; RNA biosynthesis; RNase protection assay; complementary DNA; density gradient ultracentrifugation; gene deletion mutation; genetic models; genetic transcription; genome; immunoprecipitation; nucleocapsid; posttranscriptional RNA processing; protein structure function; recombinant proteins; simian virus; site directed mutagenesis; structural genes; transfection; virion; virus RNA; virus genetics; virus infection mechanism; virus protein; virus replication

The proposed research will focus on the structure and function of the Large (L) protein of the paramyxovirus family of non-segmented negative-strand RNA viruses. The paramyxoviruses are a diverse group of infectious agents responsible for a variety of medically and economically important diseases of humans and animals. By comparison to the more abundant paramyxovirus proteins, our understanding of the structure and functions of the L protein is incomplete. The 250 kDa L protein is a multifunctional polypeptide which possesses the catalytic sites involved in various steps in viral RNA synthesis. Because L catalytic activities depend on interactions of the L polypeptide with other viral proteins, the first two goals of this research are to identify the domains of L which direct two classes of essential protein-protein interactions. First, a cDNA clone which expresses the paramyxovirus SV5 L polypeptide-will be employed to map the regions of L which direct interactions with the second viral polymerase subunit protein P. Second, cDNA-derived mutant L polypeptides will be assayed for their ability to bind to the genomic nucleocapsid structure and to be incorporated into progeny virions. The mapping of regions of L involved in these two types of protein-protein interactions will be important, because they will provide the first identification of essential structural domains for this extraordinary multifunctional polypeptide. Moreover, these data will set the stage for a rational mutational approach to identifying the catalytic regions of the L protein. As a third goal addressing the functional domains of L, a recently-developed in vivo replication system will be employed to analyze the SV5 genomic sequences which direct L polymerase functions. Specifically, a model cDNA-derived dicistronic viral genome will be used in a mutational analysis of the intercistronic sequences which modulate L protein functions during viral mRNA transcription. The information gained from these experiments on intercistronic sequences will fill a major gap in our understanding of the signals controlling the various L protein activities. Together, these experiments on the L protein are focused on setting the foundation for the future identification of the catalytic domains of this multifunctional polypeptide and of the cis-acting genomic sequences which regulate its enzymatic functions.

拟议的研究将集中在结构和功能， 非分节段副粘病毒家族的大（L）蛋白 负链RNA病毒。 副粘病毒是一种多样的 一组传染性病原体，负责各种医学上的 和经济上重要的人类和动物疾病。 通过 与更丰富的副粘病毒蛋白相比，我们的 了解L蛋白的结构和功能， 不完整 250 kDa L蛋白是一种多功能多肽 其具有参与各种步骤的催化位点， 病毒RNA合成。 因为L催化活性取决于 L多肽与其他病毒蛋白的相互作用， 本研究的前两个目标是确定L 其指导两类必需的蛋白质-蛋白质相互作用。 首先，表达副粘病毒SV 5 L的cDNA克隆 多肽-将被用来映射区域的L， 与第二病毒聚合酶亚基蛋白P的相互作用。 其次，将测定cDNA衍生的突变体L多肽的 它们结合基因组核衣壳结构和 被整合到后代病毒体中。 L区域的映射 参与这两种蛋白质相互作用的蛋白质 重要的是，因为它们将提供第一个识别 这一非凡的多功能的重要结构域 多肽。 此外，这些数据将为 用理性突变法鉴定催化区域 L蛋白质。 作为第三个目标， 结构域的L，最近开发的体内复制系统将 利用SV 5基因组序列分析， 聚合酶功能。 具体来说，一个模型cDNA衍生的 双顺反子病毒基因组将用于以下突变分析： 调节L蛋白功能的顺反子间序列 在病毒mRNA转录过程中。 从这些获得的信息 对顺反子间序列的实验将填补 我们对控制各种L蛋白的信号的理解 活动 总之，这些关于L蛋白的实验 重点是为今后确定 该多功能多肽的催化结构域和 调节其酶促的顺式作用基因组序列 功能协调发展的