EXPRESSION OF THE YEAST VIRAL DSRNA GENOME

酵母病毒 DSRNA 基因组的表达

• 批准号: 3270998
• 负责人: JEREMY A BRUENN
• 金额: $ 13.32万
• 依托单位: STATE UNIVERSITY OF NEW YORK AT BUFFALO
• 依托单位国家: 美国
• 财政年份: 1977
• 资助国家: 美国
• 起止时间: 1977-05-01 至 1997-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3270998
• 关键词: RNA binding protein; RNA virus; Saccharomyces cerevisiae; chromatography; double stranded RNA; electrophoresis; frameshift mutation; fusion gene; gene complementation; gene expression; genetic mapping; genetic translation; helicase; host organism interaction; hydrolase; nucleic acid structure; open reading frames; protein structure function; radiotracer; reporter genes; site directed mutagenesis; virus RNA; virus genetics; virus protein

The importance of viral persistence in pathogenesis is just beginning to be fully appreciated. Viral persistence is especially puzzling in RNA viruses that do not produce proviruses (integrated DNA copies). There are double-stranded RNA viruses of animals, plants, insects, and fungi that exhibit persistence. The fungal viruses are uniformly and permanently persistent: they have no natural infectious cycle. Their replication must therefore be regulated by the cell, since they neither kill the cell nor fail to infect all progeny of an infected cell. Among the fungal viruses, the Saccharomyces cerevisiae , or yeast virus, ScV, is the simplest and best understood. Yeast is an ideal system in which to address the nature of persistence: the ease of its sophisticated molecular and classical genetics is unmatched in other eucaryotes. In ScV, there is only one essential viral dsRNA, L1, with a known sequence, and only one major capsid polypeptide (the cap gene product). L1 has only two large open reading frames, cap and pol. The pol protein, expressed by a retrovirus-like frameshift event contains an RNA-dependent RNA polymerase (RDRP). In the present application, we seek to understand (1) the mechanism of frameshifting in ScV and its possible relationship to cellular helicases, and (2) the functional domains in the pol open reading frame. ScV frameshifting, the first system in which it has been shown that frameshifting is correlated with a ribosomal pause, will be exploited to understand in detail the relationship between RNA structure and the frameshifting event. Further mutants will be created to test more complete models for the ScV frameshift pesudoknot and these will be analyzed by frameshifting in vitro and in vivo in appropriate heterologous contexts, as well as by heelprinting experiments to determine the location of paused ribosomes in the frameshift region. The structure of the frameshift region will also be analyzed directly by sensitivity to single and double strand specific nucleases. The role of cellular helicases in frameshifting will be ascertained by overexpression of helicase genes in cells producing beta-galactosidase by virtue of a frameshift event. The cap-pol fusion protein is not processed; all of its activities are expressed in viral particles in the form of the fusion protein. These, including the RNA binding domain, the nucleotide phosphohydrolase (NPH), and the RDRP, will be further defined by site-directed mutagenesis and expression of cDNAs. The NPH activity appears to have been localized to a 75 amino acid region, which when expressed as a beta-galactosidase fusion protein retains its activity. Mutants lacking this activity will be constructed in full-sized cDNA clones that can be used for complementation tests in vivo, in order to determine the viral function of the NPH. Similarly, the RNA binding activity appears to have been localized to a 100 amino acid region of pol. Putative loci for interaction with cellular proteins, within the pol open reading frame, have been identified. These will be altered in a systematic way to define their functions in vivo.

病毒持久性在发病机理中的重要性才刚刚开始 完全赞赏。 病毒持续性在RNA中特别令人困惑 不产生原病毒（集成DNA拷贝）的病毒。 那里 是动物，植物，昆虫和真菌的双链RNA病毒 那表现出持久性。 真菌病毒是统一的， 永久持久：它们没有自然的感染周期。 他们的 因此，复制必须由细胞调节，因为它们都不 杀死细胞也无法感染感染细胞的所有后代。 之中 真菌病毒，酿酒酵母或酵母菌病毒SCV， 是最简单，最好的理解。 酵母是一个理想的系统 解决持久性的本质：其精致的便利性 分子和经典遗传学在其他桉树中是无与伦比的。 在SCV中，只有一个必需的病毒dsRNA，L1，已知 序列，只有一个主要的衣壳多肽（CAP基因产物）。 L1只有两个大开放式阅读框，帽子和pol。 pol蛋白， 由逆转录病毒样截图事件表示的依赖RNA RNA聚合酶（RDRP）。 在本应用中，我们试图了解 （1）SCV中的帧速率机制及其可能的关系 到细胞解旋酶，以及（2）POL开放中的功能域 阅读框架。 SCV Frameshifting，它是第一个系统 表明框架与核糖体暂停相关，将是 利用详细了解RNA结构之间的关系 和架空活动。 将创建进一步的突变体进行测试 SCV移码段Pesudoknot的更完整的型号，这些模型将是 通过在适当的体外和体内分析 异源环境，以及通过脚后跟实验 确定暂停的核糖体在移状区域的位置。 这 还将直接分析移码区域的结构 对单链特异性核酸酶的敏感性。 的作用 通过过表达将确定帧速率中的细胞解旋酶 由于A 边框事件。 Cap-Pol融合蛋白未加工；它的所有活动都是 以融合蛋白的形式以病毒颗粒表达。 这些， 包括RNA结合结构域，核苷酸磷酸化酶（NPH）， RDRP将由位置定向的诱变和 cDNA的表达。 NPH活动似乎已定位到 一个75个氨基酸区域，当该区域表示为β-半乳糖苷酶时 融合蛋白保留其活性。 缺乏这种活动的突变体将 以全尺寸的cDNA克隆来构建 为了确定病毒功能，体内互补测试 NPH。 同样，RNA结合活性似乎已经 位于POL的100个氨基酸区域。 推定的基因座 在POL开放阅读框内与细胞蛋白的相互作用， 已经确定了。 这些将以系统的方式改变 在体内定义其功能。