ADENOVIRUS GENE EXPRESSION: GENETICS OF EARLY REGION 4

腺病毒基因表达：早期区域 4 的遗传学

• 批准号: 3139951
• 负责人: Gary W Ketner
• 金额: $ 19.1万
• 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 1996-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3139951
• 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.

人类腺病毒的第4区（E4）位于右端 病毒基因组。 它编码七个蛋白质，其中四种已经 与受感染细胞的调节过程有关。 目标的目标 这里提出的研究是为了确定三个作用机理 E4产品，开放式阅读帧（ORF）3、4和6。ORFS 3和 6是表达病毒晚期基因并作用于A mRNA合成的转录后步骤：缺乏产物的突变体 这些基因正常转录晚期区域，但无法 积累晚期mRNA或其核前体。 蛋白质是 在功能上多余，它们以这种能力的功能是特定的 对于病毒RNA。 这些蛋白质活性的研究将是 三倍。 首先，将使用特定的抗体来确定它 ORF 3和6产品，无论这些产品是否涉及感染 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，将对蛋白质进行测定 在体外与病毒晚期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产品负调节 病毒DNA复制：仅产生ORF 4产品的E4突变体 严重缺乏病毒DNA复制，而缺乏突变体 所有E4正常产生DNA。 表达ORF 4的突变体也抑制 Trans中其他E4缺失突变体的DNA合成。 确定 ORF 4蛋白是否通过对 病毒复制蛋白的合成，它们在E4突变体中的表达 将检查感染。 如果表达的改变 复制蛋白无法解释ORF 4效应，直接 ORF 4产品与复制机械之间的相互作用将 使用ORF 4特异性抗体进行探测。 最后，提取 突变感染的细胞将被分析，以合成病毒的能力 DNA，如果不足，将用于识别有缺陷或缺失的 成分。