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 细胞转化、病毒DNA复制和RNA加工。 一 这种方法是基于严格的遗传分析，使用以前的方法 在我们实验室开发的，将点突变引入外显子 早期区域IA和IB的结构域（编码序列主要是 负责病毒-RNA调节和转化） 基因组 总的来说，相信这种特殊的方法将 促进冷敏感或TS腺病毒突变体的产生。 快速筛选这些条件腺病毒的方法 还将开发突变体。 最终，变种人将被用于 剖析病毒的早期功能， 上述。 一种补充的方法，以遗传分析， 早期病毒蛋白质包括获得两种类型表达的方法 载体，一个将允许合成的病毒蛋白质在一个 另一个是基因设计， 病毒蛋白在真核细胞中的表达。 的病毒蛋白 将在细菌系统中通过表达载体合成 含有lac启动子的，将主要用于开发 免疫分析 SV 40真核表达载体的构建 复制起点和早期启动子将用于 以天然形式合成早期病毒蛋白， 酶分析 在另外的实验中，我们将继续研究一种新的 一种病毒蛋白，似乎编码在位于早期 病毒染色体的第4区。 因此，这些实验应该扩大我们对机制的理解， 利用病毒来利用其宿主，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.