FUNCTION OF THE AD2 DBP HOST-RANGE MUTATION

本提案的一个主要目标是确定 Ad2 DNA结合蛋白（DBP）的氨基末端区域。 增强Ad2在非洲生长能力的突变体 绿色猴肾细胞的1,000倍有一个单一的改变 在其氨基末端区域的氨基酸。 的表型 这些宿主范围突变体表明， DBP结构域在晚期腺病毒加工中的作用 mRNA。 大约12个磷酸化位点发生在 氨基末端结构域附近的主机范围突变位点。 DBP 将在生产性和流产性Ad2中检测磷酸化 感染，以确定差异磷酸化是否 负责宿主范围效应。 Ad2 DBP突变体改变 将在这些磷酸化位点处或附近构建， 这些将用于确定磷酸化的功能， 特定的网站。 磷酸化是主要的翻译后修饰 调节蛋白质功能的机制，但调节 途径和控制酶还没有很好地理解。 检查编码氨基末端的DNA序列 的Ad2 DBP揭示了一个开放的阅读框架（ORF）上， 相反的链，可以编码110，000 MW的蛋白质。 等 110K蛋白代表晚期区域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Ad2感染的HeLa细胞中检测到。 第二个目标是 该建议旨在确定L4 - 110K蛋白是否是必需的， Ad2复制 假设的110K蛋白质序列是 在宿主范围突变体中发生改变;因此，L4 - 110K基因将被 检查了Ad2晚期基因的调节作用 表达式和宿主范围。

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.