CLONING AND EXPRESSION OF INFLUENZA VIRAL RNA SEGMENTS

流感病毒正粘病毒是人和动物的主要群体 病原体，是一组被分段包膜的负链RNA病毒。 这些病毒在质膜水平上组装和发芽， 特别是在极化的上皮细胞的顶端。这个 该项目的长期目标定义了病毒组装和 萌芽中。拟议项目的具体目标是审查 M1和NA在组装和出芽过程中的作用。自.以来 流感病毒的复制发生在细胞的细胞核和萌芽阶段 放在质膜上，病毒RNP是如何进出质膜的 原子核以及M1的解离和缔合如何促进原子核 将检查vRNP的进出情况。拟议的实验将 确定M1在去涂层过程中是否与病毒RNP解离 如果先前存在的M1会干扰核运输 传入vRNP。此外，这些实验将确定vRNP是否会 在没有M1的情况下，从细胞核输出到细胞质或 如果vRNP退出原子核需要M1。此外，域 将描述与vRNP相互作用的M1。由于M1是 位于病毒被膜和RNP之间的蛋白质-蛋白质 M1与HA、NA等跨膜病毒蛋白的相互作用 或M2以及这些蛋白中每一个的结构域参与了 互动将被定义。因为病毒颗粒从顶端发芽 极化细胞中的质膜结构域，结构特征 在这些跨膜蛋白中，特别是在NA中，负责 将蛋白质定位到根尖质膜，将被确定。 NA是一种II型膜蛋白。人们对这种分类知之甚少 I型蛋白和NA的极化运输信号 很特别。嵌合结构、缺失和突变将是 用于定义NA的分类信号。这里提出的实验将 阐明去涂层以及组装和萌芽所涉及的步骤 这将有助于设计抗病毒药物来阻断这些步骤 在病毒复制中。

Influenza viruses orthomyxoviruses, a major group of human and animal pathogens, are a group of segmented enveloped negative strand RNA viruses. These viruses assemble and bud at the level of plasma membrane, specifically on the apical side of the polarized epithelial cells. The long term goal of this project define the processes of viral assembly and budding. The specific objectives in the proposed project are to examine the functions of M1 and NA in the assembly and budding processes. Since influenza virus replication occurs in the cell's nucleus and budding takes place at the plasma membrane, how the viral RNP gets in and out of the nucleus and how dissociation and association of M1 facilitates the nuclear entry and exit of vRNP will be examined. Proposed experiments will determine if M1 becomes dissociated from the viral RNP during uncoating and if the preexisting M1 will interfere with the nuclear transport of the incoming vRNP. In addition, these experiments will determine if vRNP made in the absence of M1 is exported out of the nucleus into the cytoplasm or if M1 is needed for vRNP's exit from the nucleus. Furthermore the domains of M1 involved in interaction with vRNP will be delineated. Since M1 is juxtaposed between the viral envelope and RNP, the protein-protein interactions between M1 and the transmembrane viral proteins like HA, NA or M2 and the domains of each of these proteins involved in the interactions will be defined. Since viral particles bud from the apical domain of plasma membrane in polarized cells, structural features present in these transmembrane proteins, particularly in NA, responsible for targeting the protein to the apical plasma membrane, will be determined. NA is a type II membrane protein. Little is known about the sorting signals for polarized transport of type II protein in general and NA in particular. Chimeric constructions and deletions and mutations will be used to define sorting signals of NA. Experiments proposed here will elucidate the steps involved in uncoating as well as assembly and budding and will be useful in designing the antiviral agents to block these steps in viral replication.