STRUCTURE AND FUNCTION OF PARAMYXOVIRUS L PROTEIN

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 它们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的区域映射 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

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.