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， 并确定这些蛋白质的表达影响的机制 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 负责腺病毒的病毒-RNA调节和转化) 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 前面提到的。一种互补的遗传分析方法 早期病毒蛋白包括获得两种类型表达的方法 载体，它将允许在一种 细菌系统和另一个将被基因设计为允许 病毒蛋白在真核细胞中的表达。病毒蛋白 将在细菌系统中通过表达载体合成 含有lac启动子，将主要用于开发 一种免疫检测。含SV40基因真核表达载体的构建 复制起点和早期启动子将服务于 以天然形式合成早期病毒蛋白以供后续 酶分析。 在更多的实验中，我们将继续研究一种新的 病毒蛋白似乎编码在位于早期的内含子中 病毒染色体的第四区域。 因此，这些实验应该扩大我们对机制的理解。 被病毒利用来利用其宿主，以及诱导细胞 转型。

I propose to investigate the early proteins encoded by adenovirus type-2, and determine the mechanism by which expression of these proteins effects 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 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 an 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.