CONTROL OF 2-5A PATHWAY BY TS MUTANT OF VACCINIA

痘苗病毒 TS 突变体对 2-5A 途径的控制

• 批准号: 3145434
• 负责人: RANDALL J. COHRS
• 金额: $ 13.5万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-04-01 至 1993-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3145434
• 关键词: DNA; RNA; RNA splicing; antisense nucleic acid; double stranded RNA; electrophoresis; enzyme complex; glucocorticoids; interferons; microinjections; molecular cloning; natural gene amplification; nucleic acid hybridization; nucleic acid metabolism; pancreatic ribonuclease; plasmids; temperature sensitive mutant; tissue /cell culture; transfection; vaccinia virus; virus genetics; virus replication

RNA turnover is central to the regulation of gene expression; however, the mechanisms involved are not well understood. The temperature sensitive mutant of vaccinia virus, ts22, provides a genetic tool for elucidating mechanism(s) involved in RNA turnover. The ts22 gene regulates the stability of RNA in vaccinia virus infected cells. ts22 virus has an abortive late phenotype. At the non-permissive temperature, ts22 infection proceeds normally in the early stages; however, at 8-10 h post infection, RNA is degraded and virus infection aborted (independent of exogenous interferon treatment). Our results indicate that the degradation of RNA is due to the activation of 2-5A dependent RNase. 2-5A synthetase-RNase pathway, initially discovered as one of the antiviral mechanisms of interferon, has been implicated in controlling RNA turnover during cell growth, hormone status and differentiation. The ability to modulate the 2- 5A pathway would be of value in assessing its role in RNA degradation. We hypothesize that the functional ts22 gene product inactivates the 2-5A system during productive infection. Inactivation of the 2-5A pathway by expression of the functional ts22 gene product, independent of virus infection, should help elucidate the role of 2-5A pathway in cellular RNA turnover. We propose to study the modulation of 2-5A pathway by ts22 gene product. This will be accomplished by determining the growth of ts22 vaccinia virus and rRNA cleavage (an indicator of activation of the 2-5A system) at the non-permissive temperature, in cells in which the 2-5A pathway has been rendered inoperative. Constitutive expression of the functional ts22 gene product will be tested for its ability to inactivate the 2-5A system. The step(s) at which the ts22 gene product inactivates the 2-5A system will be determined. Further use of vectors expressing the wild type ts22 gene or 2-5A synthetase antisense RNA should permit the assessment of the role of the 2-5A system in controlling RNA degradation during cell growth inhibition and we will determine effect of these vectors on the interferon or glucocorticoid induced growth inhibition and reduction in c-myc expression in Daudi cells.

RNA 周转对于基因表达的调节至关重要；然而， 所涉及的机制尚不清楚。 对温度敏感 牛痘病毒突变体 ts22 为阐明 参与 RNA 周转的机制。 ts22 基因调节 痘苗病毒感染细胞中 RNA 的稳定性。 ts22 病毒有一个 失败的晚期表型。 在不允许的温度下，ts22 感染 早期阶段进展正常；然而，在感染后8-10小时， RNA 被降解，病毒感染中止（与外源性无关） 干扰素治疗）。 我们的结果表明 RNA 的降解是 由于 2-5A 依赖性 RNase 的激活。 2-5A合成酶-RNA酶 途径，最初被发现是作为抗病毒机制之一 干扰素，与细胞过程中 RNA 周转的控制有关 生长、激素状态和分化。 调制 2- 的能力 5A 途径对于评估其在 RNA 降解中的作用具有重要价值。 我们 假设功能性 ts22 基因产物使 2-5A 失活 生产性感染期间的系统。 2-5A 途径失活 功能性 ts22 基因产物的表达，独立于病毒 感染，应该有助于阐明 2-5A 途径在细胞 RNA 中的作用 周转。 我们建议研究ts22基因对2-5A途径的调节 产品。 这将通过确定 ts22 的增长来完成 痘苗病毒和 rRNA 裂解（2-5A 激活的指标 系统）在不允许的温度下，在电池中，其中2-5A 路径已失效。 的本构表达式 将测试功能性 ts22 基因产物的失活能力 2-5A系统。 ts22基因产物失活的步骤 2-5A系统将被确定。 表达载体的进一步用途 野生型 ts22 基因或 2-5A 合成酶反义 RNA 应允许 评估 2-5A 系统在控制 RNA 降解中的作用 在细胞生长抑制期间，我们将确定这些载体的效果 对干扰素或糖皮质激素引起的生长抑制和减少 Daudi 细胞中 c-myc 的表达。