MACROMOLECULAR SYNTHESIS IN VIRUS-INFECTED CELLS

拟议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 提出了 首先是分离所有呼肠孤病毒的天然形式 来自用强大的哺乳动物表达载体感染的细胞的蛋白质 呼肠孤病毒基因组片段已经被克隆到其中。 的分离的 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 载体将用于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 基因组片段将用于鉴定功能结构域。 的 呼肠孤病毒RNA种类进入呼肠孤病毒蛋白的细胞内定位 已经单独和在重组牛痘病毒株中进行脂质体转染 包含各种基因组片段。 第三，技术将 将基因工程基因组片段引入呼肠孤病毒 基因组，以便允许研究功能改变的影响， 病毒表型的结构域。 第四，温度敏感突变体和 反义技术将被用来确定在什么阶段不能 单个病毒蛋白发挥功能可以阻止病毒增殖， 为病毒蛋白功能提供了进一步的线索。 第五，选定的SORF 在呼肠孤病毒血清型1、2和3中，将在基因中鉴定细胞受体 文库并测序以鉴定受体的性质。

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.