CONSTRUCTION AND ANALYSIS OF RETROVIRUS MUTANTS

本提案所述实验的长期目标是 增加我们对逆转录病毒复制的理解， 称为RNA肿瘤病毒。 这些代理人，广泛分布在自然界中， 在动物和人类中引起各种白血病、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 可能的结构和功能的病毒基因产物编码的 莫洛尼鼠白血病病毒，一种典型的复制能力 逆转录病毒 为实现这一目标，将采取三种主要方法。 首先，将使用病毒基因组的克隆DNA拷贝的诱变， 在选定的区域进行特定的改变，这些改变的效果 病毒生命周期中的突变将通过直接分析 病毒蛋白质和核酸。 病毒gag，pol， 和env基因，以及各种顺式作用区域，将在 这边 这些实验将确定每一个基因中的基本序列， 基因或区域，并将进一步定义它们在复制中的作用。 第二，将开展一项重大努力， 病毒突变体。 我们会制造出一系列具有复制能力的 回复突变体病毒来自几个突变的父母，每一个产生的凭借 抑制突变以补偿原始基因的影响， 突变 这些回复突变的定位和分析将有助于 定位病毒基因产物中相互作用的结构域， 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物进行生化检测 在细菌中表达为融合蛋白。将开发快速检测方法， 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 现有的突变体小组，以帮助定位这些需要的区域， 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.