FUNCTION OF EARLY PROTEINS ENCODED BY ADENOVIRUS TYPE-2

我建议研究2型腺病毒编码的早期蛋白质， 并确定这些蛋白质表达影响的机制 细胞转化、病毒DNA复制和RNA加工。一 方法是基于严格的遗传分析，使用以前的方法 我们实验室开发的，将点突变引入外显子 早期区域IA和IB的结构域(主要是编码序列 负责病毒-RNA调节)，以及腺病毒的转化 基因组。总体而言，人们认为这种特殊的方法将 促进冷敏感或ts腺病毒突变体的产生。 这些条件型腺病毒的快速筛选方法 还将开发突变株。最终，这些突变体将用于 解剖早期病毒的功能并鉴定参与的蛋白质 如上所述。遗传分析的一种补充方法 早期病毒蛋白的研究涉及获得两种类型的 表达载体，一种允许合成病毒的载体 细菌系统中的蛋白质和另一种将在遗传上 旨在允许病毒蛋白在真核细胞中表达。 病毒蛋白质将在细菌系统中合成，通过一种 含有lac启动子的表达载体将主要用于 一种免疫分析的发展。真核表达载体的构建 包含SV40复制起始子和早期启动子的 合成天然形式的早期病毒蛋白的目的 随后的酶分析。在其他实验中，我们将继续 我们对一种新的病毒蛋白的研究似乎编码在 位于病毒染色体早期区域4的一个内含子。因此，这些 实验应该扩大我们对 病毒利用其宿主，以及诱导细胞转化。

I propose to investigate the early proteins encoded by adenovirus type-2, and determine the mechanism by which expression of these proteins effect cellular transformation, viral DNA replication, and RNA processing. One approach is based on a strict genetic analysis, using methods previously developed in our laboratory, to introduce point-mutations into the exon domains of early region IA and IB (the coding sequences primarily responsible for viral-RNA regulation), and transformation of the adenovirus genome. Overall, it is believed that this particular approach will facilitate the creation of either cold-sensitive or ts adenovirus mutants. Rapid screening methods for the selection of these conditional adenovirus mutants will also be developed. Ultimately, the mutants will be used in dissecting early viral functions and in identifying proteins involved in the above aforementioned. A complementary approach to the genetic analysis of early viral proteins involves methods for obtaining two types of expression vectors, one which will allow for the synthesis of the viral proteins in a bacterial system and the other which will be genetically designed to allow expression of the viral proteins in eukaryotic cells. Viral proteins that will be synthesized in the bacterial system, by an expression vector containing the lac promoter, will be used primarily for the development of an immune assay. Construction of a eukaryotic vector containing the SV40 replication origin and early promoter will serve the purpose of synthesizing early viral proteins in their native form for subsequent enzymatic analysis. In additional experiments, we will continue our investigation of a new viral protein that appears to be encoded within an intron located in early region 4 of the viral chromosome. Thus, these experiments should broaden our understanding of mechanisms employed by the virus to exploit its host, as well as induce cellular transformation.