FUNCTION OF THE AD2 DBP HOST-RANGE MUTATION

这项提案的一个主要目标是确定 AD2 DNA结合蛋白(DBP)的氨基端区域。 增强AD2在非洲生长能力的突变体 绿猴肾脏细胞被1000倍单一改变 它们的氨基末端区域的氨基酸。的表型 这些宿主范围突变表明，氨基末端 DBP功能域在晚期腺病毒加工过程中的作用 MRNA.大约12个磷酸化位点发生在 位于宿主范围突变位点附近的氨基末端结构域。舒张压 生产和流产的AD2的磷酸化将被检测 感染以确定差异磷酸化是否 对寄主范围效应负责。AD2 DBP突变体发生改变 将在这些磷酸化位点或其附近构建，以及 这些将被用来确定磷酸化在 特定的站点。磷酸化是翻译后的主要作用 调节蛋白质功能的机制，但调节 这些途径和控制酶还不是很清楚。 检测编码氨基末端的DNA序列 发现了一个开放阅读框架(ORF)。 相反的链可以编码110,000 mW的蛋白质。是这样的 110K蛋白将代表晚期区域4的延伸 1000,000 mW非结构磷蛋白(L4-100K) 氨基末端方向。L4-110K蛋白的信使RNA 已经在感染AD2的HeLa细胞中检测到。第二个目标是 这项建议是为了确定L4-110K蛋白是否是必需的 用于AD2繁殖。推测的110K蛋白质序列为 寄主范围突变；因此，L4-110K基因将 AD2晚期基因调控作用的研究 表达和宿主范围。

A major goal of this proposal is to identify the function of the amino-terminal region of the Ad2 DNA binding protein (DBP). Mutants which enhance the ability of Ad2 to grow in African green monkey kidney cells by 1,000-fold have a single altered amino acid in their amino-terminal region. The phenotype of these host range mutants suggests that the amino-terminal domain of DBP functions in the processing of late adenovirus mRNA. Approximately 12 sites of phosphorylation occur in the amino-terminal domain near the host-range mutation site. DBP phosphorylation will be examined in productive and abortive Ad2 infections to determine if differential phosphorylation is responsible for the host range effect. Ad2 DBP mutants altered at or near these phosphorylation sites will be constructed, and these will be used to determine the function of phosphorylation at particular sites. Phosphorylation is the major postranslational mechanism for regulating protein function, but the regulatory pathways and controlling enzymes are not well understood. Inspection of the DNA sequence that encodes the amino terminus of Ad2 DBP has revealed an open reading frame (ORF) on the opposite strand that could encode a protein of 110,000 MW. Such a 110K protein would represent an extension of the late region 4 1000,000 MW nonstructural phosphoprotein (L4-100K) in the amino-terminal direction. Messenger RNA for a L4-110K protein has been detected in Ad2-infected HeLa cells. A second goal of this proposal is to determine if the L4-110K protein is essential for Ad2 reproduction. The postulated 110K protein sequence is altered in host range mutants; thus, the L4-110K gene will be examined with respect to a role in the regulation of Ad2 late gene expression and host range.