VACCINIA VIRUS BIOCHEMICAL GENETICS

痘病毒牛痘，这是活疫苗病毒，用于 根除天花，是一种含有双链DNA的大病毒 一直在利用基因来理解基因组 牛痘病毒已被发现为适当包括牛痘基因的关键 感染细胞的稳态细胞质增加。我们的长远目标 牛痘接种办法的职能组织。我们正在进行的 对一个基本病毒基因网络的遗传分析似乎表明 对晚期病毒转录的控制。网络A18R，当 突变导致晚期病毒RNA和基因G2R水平的合成， 当该基因突变时，会导致晚期病毒蛋白合成减少。 在本应用中，我们建议扩展对该网络的分析 并继续努力开发定向的体外方法 基本痘苗条件致死突变的构建 基因。具体目标是：1)优化和表征病毒 特定的体外转录系统，2)使用生化方案 和适当的突变体，鉴定A18R基因和 确定其在感染中的作用，3)使用生化方案和 适当的突变体，鉴定G2R基因的产物和 确定其在感染中的作用，4)使用遗传方案，确定 基因A18R和G2R之间的关系，并寻找其他基因 参与网络，5)检测丙氨酸扫描诱变耦合 用瞬时显性选择作为体外定向选择的方法 痘苗病毒条件致死突变体的构建。

The poxvirus vaccinia, which is the live vaccine virus used in the eradication of smallpox, is a large double stranded DNA containing virus that replicates in the has been to understand genome using a genetic vaccinia has uncovered be critical for proper includes vaccinia gene increased steady state cytoplasm of infected cells. Our long-range goal the functional organization of the vaccinia approach. Our ongoing genetic analysis of a network of essential viral genes which seem to control of late viral transcription. The network A18R, which when mutated leads to synthesis of levels of late viral RNA, and gene G2R, which when mutated leads to decreased synthesis of late viral proteins. In this application, we propose to extend the analysis of this network and continue our efforts to develop directed in vitro methods for construction of conditionally lethal mutations in essential vaccinia genes. The specific aims are: 1) optimize and characterize a virus specific in vitro transcription system, 2) using biochemical protocols and appropriate mutants, characterize the product of the A18R gene and determine its role in infection, 3) using biochemical protocols and appropriate mutants, characterize the product of the G2R gene and determine its role in infection, 4) using genetic protocols, determine .the relationship between genes A18R and G2R, and seek additional genes involved in the network, and 5) test alanine scanning mutagenesis coupled with transient dominant selection as a method for directed in vitro construction of conditionally lethal mutants of vaccinia.