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制中的重要性才刚刚开始 得到充分的赞赏。 RNA 中的病毒持久性尤其令人费解 不产生原病毒（整合DNA拷贝）的病毒。 那里 是动物、植物、昆虫和真菌的双链RNA病毒 表现出毅力。 真菌病毒均一且 永久持续：它们没有自然的传染周期。 他们的 因此，复制必须受到细胞的调节，因为它们既不 杀死细胞也不会感染受感染细胞的所有后代。 之中 真菌病毒，酿酒酵母，或酵母病毒，ScV， 是最简单、最好理解的。 酵母是一个理想的系统，其中 解决持久性的本质：其复杂的易用性 分子和经典遗传学是其他真核生物无法比拟的。 在 ScV 中，只有一种必需的病毒 dsRNA，L1，其已知的 序列，并且只有一种主要衣壳多肽（cap 基因产物）。 L1只有两个大的开放阅读框，cap和pol。聚合蛋白， 由逆转录病毒样移码事件表达的包含RNA依赖的 RNA 聚合酶 (RDRP)。 在本申请中，我们试图了解 (1) ScV移码机制及其可能的关系 细胞解旋酶，以及 (2) pol 开放中的功能域 阅读框架。 ScV 移码系统，第一个采用该技术的系统 显示移码与核糖体暂停相关，将 用于详细了解 RNA 结构之间的关系 和移码事件。 将创建更多突变体来测试 ScV 移码假结的更完整模型，这些将是 通过适当的体外和体内移码分析 异源环境，以及通过脚跟印实验 确定移码区域中暂停核糖体的位置。 这 移码区域的结构也将直接分析 对单链和双链特异性核酸酶的敏感性。 的作用 移码中的细胞解旋酶将通过过度表达来确定 产生β-半乳糖苷酶的细胞中的解旋酶基因 移码事件。 cap-pol融合蛋白未经加工；它的所有活动都是 以融合蛋白的形式在病毒颗粒中表达。 这些， 包括 RNA 结合域、核苷酸磷酸水解酶 (NPH)、 和 RDRP，将通过定点诱变进一步定义 cDNA 的表达。 NPH 活动似乎仅限于 75 个氨基酸区域，当表达为 β-半乳糖苷酶时 融合蛋白保留其活性。 缺乏这种活性的突变体将 构建全尺寸 cDNA 克隆，可用于 体内互补测试，以确定病毒功能 国家公共卫生局的。 同样，RNA 结合活性似乎是 定位于 pol 的 100 个氨基酸区域。 假定基因座 在 pol 开放阅读框中与细胞蛋白质相互作用， 已被识别。 这些将以系统的方式改变 定义它们在体内的功能。