STRUCTURE AND FUNCTION OF PARAMYXOVIRUS L PROTEIN

拟议的研究将集中在结构和功能上。 副粘病毒非节段家族的L大蛋白 负链RNA病毒。副粘病毒是一种多样化的 一组感染性病原体，负责各种医学上的 以及对人类和动物具有重要经济意义的疾病。通过 与更丰富的副粘病毒蛋白相比，我们的 对L蛋白的结构和功能的了解是 不完整。L蛋白是一种多功能多肽 它拥有参与不同步骤的催化中心 病毒RNA合成。因为L的催化活性取决于 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的领域 它指导了两类基本的蛋白质-蛋白质相互作用。 一、表达副粘病毒SV5型L的cDNA克隆 多肽-将用于绘制L指导的区域 与病毒第二聚合酶亚基蛋白P的相互作用。 第二，cdna衍生的突变体L多肽将被检测 它们与基因组核衣壳结构结合的能力和 被合并到后代病毒粒子中。L的地域测绘 参与这两种类型的蛋白质相互作用的将是 很重要，因为它们将提供第一个标识 这一非凡多功能的基本结构域 多肽。此外，这些数据将为 确定催化区的合理突变方法 L的蛋白质。作为第三个目标，解决功能 新近开发的体内复制系统L的域将 被用来分析指导L的SV5基因组序列 聚合酶起作用。具体地说，一种模型cDNA源 双顺反子病毒基因组将用于突变分析 调控L蛋白功能的顺反子间序列 在病毒信使核糖核酸转录期间。从这些信息中获得的信息 对跨顺反子序列的实验将填补 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蛋白质的实验是 重点是为未来识别 该多功能多肽的催化结构域和 调控其酶活性的顺式作用基因组序列 功能。

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.