CONSTRUCTION AND ANALYSIS OF RETROVIRUS MUTANTS

本提案中描述的实验的长期目标是 增加我们对逆转录病毒复制的理解，以前 被称为核糖核酸肿瘤病毒。这些毒剂在自然界中分布广泛， 在动物和人身上引起各种白血病、肿瘤和其他疾病。 这项工作的具体目标是尽可能详细地确定 可能编码的病毒基因产物的结构和功能 Moloney鼠白血病病毒，一种复制能力强的原型病毒 逆转录病毒。为了实现这一目标，将采取三种主要方法。 首先，病毒基因组的克隆DNA拷贝的突变将被用于 在选定的区域进行特定的更改，以及这些更改的影响 病毒生命周期上的突变将通过直接分析 病毒蛋白质和核酸。病毒Gag的几个结构域，Poll， 和env基因，以及各种顺式作用区域，将在 这边请。这些实验将确定每个序列中的基本序列 基因或区域，并将进一步确定它们在复制中的作用。 第二，将启动一项重大工作，对 病毒变种人。我们将生成一系列具有复制能力的 来自几个突变亲本的回复病毒，每个都是由于 抑制补偿原始基因影响的突变 突变。绘制和分析这些回复突变将有助于 定位相互作用的病毒基因产物的结构域， 并可能有助于识别与病毒DNA和RNA结合的蛋白质。 第三，将对三种病毒基因产品进行生化检测 在细菌中以融合蛋白的形式表达。快速检测技术将被开发用于 筛选这些蛋白质的各种活性，如DNA结合、tRNA 结合和核酸酶活性。在每种情况下，都将应用这些测试 以帮助定位这些基因所需的区域 活动。

The long-term objective of the experiments described in this proposal is to increase our understanding of the replication of retroviruses, formerly called RNA tumor viruses. These agents, widely distributed in nature, cause a variety of leukemias, tumors and other diseases in animals and man. The specific goal of this work is to determine in as much detail as possible the structure and functions of the viral gene products encoded by the Moloney murine leukemia virus, a prototypical replication-competent retrovirus. Three major approaches will be taken toward that goal. First, mutagenesis of cloned DNA copies of the viral genome will be used to make specific alterations in selected regions, and the effects of these mutations on the viral life cycle will be determined by direct analyses of viral proteins and nucleic acids. Several domains of the viral gag, pol, and env genes, as well as various cis-acting regions, will be analyzed in this way. These experiments will determine the essential sequences in each gene or region, and will further define their roles in replication. Second, a major effort will be initiated to carry out reversion analysis of virus mutants. We will generate a series of replication-competent revertant viruses from several mutant parents, each arising by virtue of suppressing mutations that compensate for the effects of the original mutation. Mapping and analysis of these reversion mutations will help to locate domains of the viral gene products that interact with one another, and may help identify proteins that bind to viral DNA and RNA. Third, biochemical tests will be applied to three viral gene products expressed as fusion proteins in bacteria. Rapid assays will be developed to screen these proteins for various activities, such as DNA binding, tRNA binding, and nuclease activity. In each case, these tests will be applied to existing panels of mutants to help localize the regions needed for these activities.