ADENOVIRUS GENE EXPRESSION--GENETICS OF EARLY REGION 4

腺病毒基因表达--早期4区遗传学

• 批准号: 3139952
• 负责人: Gary W Ketner
• 金额: $ 18.43万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-08-01 至 1996-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3139952
• 关键词: Adenoviridae; DNA binding protein; DNA replication; RNA binding protein; RNA biosynthesis; antibody specificity; crosslink; gel electrophoresis; gene deletion mutation; gene expression; genetic mapping; laboratory rabbit; molecular weight; mutant; nucleic acid sequence; open reading frames; posttranscriptional RNA processing; protease inhibitor; protein biosynthesis; protein sequence; protein structure function; small nuclear RNA; transfection; virus RNA; virus genetics; virus infection mechanism; virus protein

Early region 4 (E4) of human adenoviruses is located at the right end of the viral genome. It encodes seven proteins, four of which have been implicated in regulatory processes in infected cells. The goal of the studies proposed here is to determine the mechanisms of action of three E4 products, those of open reading frames (ORFs) 3, 4, and 6. ORFs 3 and 6 are required for the expression of viral late genes and act at a post-transcriptional step in mRNA synthesis: mutants lacking the products of these genes transcribe the late region normally, but fail to accumulate late mRNA or its nuclear precursors. The proteins are functionally redundant, and their functions in this capacity are specific for viral RNA. Studies of the activities of these proteins will be threefold. First, it will be determined, using antibodies specific for the ORF 3 and 6 products, whether those products associate in infected cells with viral RNA or with structures known to be involved in gene expression (such as snRNPs). Second, the proteins will be assayed for the ability to bind to viral late RNA in vitro. Finally, the phenotype of ORF 3-,ORF 6- mutants will be duplicated in an in vitro system to permit its biochemical dissection. The E4 ORF 4 product negatively regulates viral DNA replication: an E4 mutant that produces only the ORF 4 product is profoundly deficient in viral DNA replication, while mutants lacking all of E4 produce DNA normally. Mutants expressing ORF 4 also inhibit DNA synthesis by other E4 deletion mutants in trans. To determine whether the ORF 4 protein inhibits replication via an effect on the synthesis of viral replication proteins, their expression in E4 mutant infections will be examined. If alterations of the expression of replication proteins cannot account for the ORF 4 effect, direct interactions between the ORF 4 product and the replication machinery will be probed using ORF 4-specific antibodies. Finally, extracts from mutant-infected cells will be assayed for the ability to synthesize viral DNA, and if deficient, will be used to identify the defective or missing components.

人类腺病毒的早期区域 4 (E4) 位于 病毒基因组。 它编码七种蛋白质，其中四种已被 参与受感染细胞的调节过程。 的目标 这里提出的研究是为了确定三种作用机制 E4 产品，开放阅读框 (ORF) 3、4 和 6 的产品。ORF 3 和 6 是病毒晚期基因表达所必需的，并以 mRNA 合成中的转录后步骤：缺乏产物的突变体 这些基因的晚期区域正常转录，但未能 积累晚期 mRNA 或其核前体。 这些蛋白质是 功能上是多余的，并且他们在这个方面的功能是特定的 对于病毒RNA。 对这些蛋白质活性的研究将 三重。 首先，将使用特异性抗体来确定 ORF 3 和 6 产品，这些产品是否与感染相关 具有病毒RNA或已知参与基因的结构的细胞 表达（例如 snRNP）。其次，将检测蛋白质 体外与病毒晚期RNA结合的能力。 最后，表型 ORF 3-、ORF 6-突变体将在体外系统中复制，以允许 它的生化解剖。 E4 ORF 4 产品负向调节 病毒DNA复制：仅产生ORF 4产物的E4突变体 病毒DNA复制严重缺陷，而突变体缺乏病毒DNA复制 所有E4均正常产生DNA。 表达 ORF 4 的突变体也会抑制 其他 E4 缺失突变体的 DNA 反式合成。 确定 ORF 4蛋白是否通过影响 病毒复制蛋白的合成及其在 E4 突变体中的表达 将检查感染情况。 如果改变表达式 复制蛋白不能解释 ORF 4 效应，直接 ORF 4产物和复制机器之间的相互作用将 使用 ORF 4 特异性抗体进行探测。 最后，摘录自 将检测突变体感染的细胞合成病毒的能力 DNA，如果有缺陷，将用于识别缺陷或缺失 成分。