MACROMOLECULAR SYNTHESIS IN VIRUS-INFECTED CELLS

拟议的研究计划的总体目标是确定 呼肠孤病毒编码蛋白的功能。六个具体目标是 建议。第一种方法是分离所有呼肠孤病毒的天然形式。 来自感染了强大哺乳动物表达载体的细胞的蛋白质 呼肠孤病毒基因组片段已被克隆到其中。被孤立的人 将检查蛋白质的酶活性和与rna结合的能力。 和其他蛋白质，包括病毒和细胞。第二，瞬时表达 载体将被用来检测呼肠孤病毒蛋白对DNA的影响 复制、转录和蛋白质合成。基因工程 基因组片段将被用来识别功能域。这个 呼肠孤病毒RNA所属蛋白的细胞内定位 已经在重组痘苗病毒株中进行了单独的脂质体感染 包含各种基因组片段。第三，技术将是 开发用于将基因工程基因组片段引入呼肠孤病毒 基因组，以便研究功能改变的影响 病毒表型上的结构域。第四，温度敏感突变体和 反义技术将用于确定在什么阶段无法 单个病毒蛋白的功能阻止了病毒的复制，从而 为病毒蛋白质的功能提供了进一步的线索。第五，选定的SORF 在呼肠孤病毒1型、2型和3型中，细胞受体将在基因中被识别 文库和测序，以确定受体的性质。

The overall aim of the proposed research program is the identify the functions of the proteins encoded by reovirus. Six specific aims are proposed. The first is to isolate the native forms of all reovirus proteins from cells infected with a powerful mammalian expression vector into which the reovirus genome segments have been cloned. The isolated proteins will be examined for enzymic activities and ability to bind to RNA and other proteins, both viral and cellular. Second, transient expression vectors will be used to examine the effect of reovirus proteins on DNA replication, transcription and protein synthesis. Genetically engineered genome segments will be used to identify functional domains. The intracellular location of reovirus proteins into which reovirus RNA species have been lipofected singly and in recombinant vaccinia virus strains containing the various genome segments. Third, techniques will be developed to introduce genetically engineered genome segments into reovirus genomes so as to permit study of the effect of alterations in functional domains on viral phenotype. Fourth, temperature-sensitive mutants and anti-sense technology will be used to determine at what stage inability of individual viral proteins to function arrests virus multiplication, thereby providing further clues to viral protein function. Fifth, selected SORFs in reovirus serotype 1, 2 and 3 cell receptors will be identified in gene libraries and sequenced in order to identify the nature of the receptors.