RNA TUMOR VIRUS--DNA INTEGRATION/GENE EXPRESSION CONTROL

拟议研究的长期目标是了解 RNA肿瘤病毒控制其自身表达的机制 基因组并影响其宿主细胞中基因的表达。 具体目标包括研究逆转录病毒DNA的生物化学 整合、鉴定和分析病毒基因组序列， 控制多聚蛋白和RNA加工。 禽肉瘤/白血病病毒 （ASLV）作为主要模型系统。 然而，比较研究 艾滋病毒感染者也在计划中。 对于整合的研究，实验策略包括分析 从病毒颗粒分离的蛋白质的相关生物化学活性 并通过重组DNA技术在异源系统中产生。 异源系统将被利用，因为它们能够提供 大量成熟的、有活性的蛋白质和功能性基因片段， 可以进行定点诱变。 多种DNA底物 含有推定的病毒整合位点，将使用 敏感的基因检测，以检测重组事件，并评估 研究了体外系统，其中包括细胞提取物和病毒- 感染的和/或纯化的蛋白质和核酸组分。 蛋白质 加工研究将受益于我们不断增长的知识 逆转录病毒蛋白酶的生物化学和三维结构。 突变体将根据这些知识进行设计，并将用于 评价蛋白酶活性对病毒体核心的贡献 形态发生 遗传和生物化学方法也将用于 鉴定和表征参与控制的病毒RNA序列， RNA加工和确定这种控制的机制。 为这些 研究表明，强大的遗传选择可能是由可用的 独特的病毒突变体将提供特殊的优势。 这项工作的结果将有助于了解病毒和 逆转录病毒诱发癌症的细胞生化反应。 在 此外，逆转录病毒代表了一种有前途的工具， 我们对其他情况下所涉及的一般机制的理解， 基因损伤、基因扩增或异常基因 基因重排和控制可能引发恶性肿瘤。

The long term objective of the proposed research is to understand the mechanism by which RNA tumor viruses control the expression of their own genomes and influence the expression of genes in their host cells. Specific aims include studies of the biochemistry of retroviral DNA integration and identification and analysis of viral genomic sequences that control polyprotein and RNA processing. The avian sarcoma/leukosis viruses (ASLV) serve as the primary model systems. However, comparative studies with human immunodeficiency virus (HIV) are also planned. For studies of integration, experimental strategies include analysis of the relevant biochemical activities of proteins isolated from viral particles and produced in heterologous systems through recombinant DNA techniques. The heterologous systems will be exploited for their ability to provide large amounts of mature, active proteins and functional gene fragments that can be subjected to site-directed mutagenesis. A variety of DNA substrates containing putative viral integration sites will be tested in vivo using sensitive genetic assays to detect recombination events and to evaluate the investigated in vitro systems, which include cell extracts and virus- infected and/or purified protein and nucleic components. The protein processing studies will benefit from our growing knowledge of the biochemistry and three-dimensional structure of the retroviral protease. Mutants will be designed based on this knowledge and will be used to evaluate the contribution of protease activity to virion core morphogenesis. Genetic and biochemical approaches will also be used to identify and characterize viral RNA sequences involved in the control of RNA processing and to determine the mechanism of this control. For these studies, powerful genetic selection made possible by the availability of unique viral mutants will provide a special advantage. Results from this work will contribute to an understanding of the viral and cellular biochemical reactions that underlie retrovirus-induced cancer. In addition, the retroviruses represent a promising tool with which to enlarge our understanding of the general mechanisms involved in other instances of oncogenesis where genetic damage, gene amplifications or abnormal gene rearrangements and controls can trigger malignancy.