CLONING AND EXPRESSION OF INFLUENZA VIRAL RNA SEGMENTS

流感病毒正粘病毒，人类和动物的一个主要群体 病原体，是一组分段包膜负链RNA病毒。 这些病毒在质膜水平上组装和出芽， 特别是在极化上皮细胞的顶侧。的 本项目的长期目标是定义病毒组装的过程， 发芽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。 以来 流感病毒在细胞核内复制， 在质膜上的位置，病毒RNP如何进出细胞膜， 核以及M1的解离和缔合如何促进核 将检查vRNP的进入和退出。 拟议的实验将 确定在去包被过程中M1是否与病毒RNP解离 如果预先存在的M1会干扰核的运输， 传入vRNP。此外，这些实验将确定vRNP是否使 在不存在M1的情况下，从细胞核输出到细胞质中，或 vRNP离开细胞核是否需要M1。此外，域 将描述参与与vRNP相互作用的M1。由于M1是 并置在病毒包膜和RNP之间， M1和跨膜病毒蛋白如HA、NA之间的相互作用 或M2和这些蛋白质中的每一种的结构域参与了 互动将被定义。由于病毒颗粒从顶端出芽， 极化细胞中质膜的区域，存在结构特征 在这些跨膜蛋白中，特别是在NA中，负责 将确定蛋白质靶向顶端质膜。 NA是一种II型膜蛋白。人们对分类知之甚少 II型蛋白的极化转运信号， 特别的。嵌合体构建和缺失以及突变将在本文中描述。 用于定义NA的分选信号。这里提出的实验将 阐明脱膜以及组装和出芽的步骤 并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.