Initiation of (-)-Strand Synthesis in Turnip Crinkle Virus Associated RNAs

芜菁皱纹病毒相关 RNA 中 (-)-链合成的起始

• 批准号: 0086952
• 负责人: Anne Simon
• 金额: $ 43.02万
• 依托单位: University of Maryland, College Park
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-03-01 至 2006-08-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0086952&HistoricalAwards=false
• 关键词: Initiation; Strand; Synthesis; Turnip; Crinkle

0086952 Anne SimonAlthough RNA viruses have been the subjects of intensive investigations for the past 100 years, replication, a fundamental process in the life cycle of a virus, remains poorly understood. The viral-encoded enzyme involved in replication, the RNA-dependent RNA polymerase (RdRp), together with possible proteins from the host, must recognize specific viral RNAs, initiate complementary strand synthesis and then use these complementary strands as templates for synthesis of the viral genome. Promoters for RdRp are poorly understood, and much of the research to date involves viruses terminating with either tRNA-like structures (e.g., Brome mosaic virus) or poly(A) tails (e.g., Poliovirus). Much less is known about viruses whose genomic RNA(s) terminate with a free hydroxyl group such as turnip crinkle virus (TCV). In addition, it is only recently becoming clear that internal sequences, and not just end sequences, are important for initiation of transcription by RdRp. Possible roles for such sequences are (i) RdRp-binding sequences, or (ii) sequences that engage in RNA-RNA interactions with end sequences to bind the RdRp to the viral genome. TCV is used as a model to study sequences and structures involved in replication since it is among the smallest and simplest of the RNA viruses. More importantly, TCV is associated with very small subviral RNAs that can be used in replication studies. Protoplast and in vitro systems have been developed to study replication of TCV genomic and subviral RNAs and several regions involved in replication of the subviral RNA satC have been identified. The TCV RdRp undergoes a process called abortive initiation when using TCV as template, which produces short RNA sequences able to function as primers for repair of deletions of satC ends. Preliminary results suggest that promoters at the ends of TCV and satC strands may not be functioning as units independent of upstream sequence as previously thought, but rather may be involved in specific interactions with interior sequences. To address this hypothesis, the sequence/structural requirements for abortive initiation from the TCV hairpin promoter will be determined by analyzing the importance of the 3' single-stranded tail and an upstream sequence element in abortive initiation by the TCV RdRp. This objective should permit the delineation of an element required for abortive initiation on the TCV genome. Mutations that are able to restore replication of TCV with the promoter from satC and that are outside the promoter sequence will be mapped by replacing segments of the mutant TCV into TCV with the promoter of satC and assaying for infectivity. This objective should provide another means for elucidating internal cis-acting sequences that interact with the 3' terminal promoter of TCV. In addition, the importance of an interior sequence in satC required for transcription in the absence of the 3' end bases will be investigated. Successful completion of these experiments should provide significant advances in our understanding of the function of RdRp's and their ability to recognize a wide variety of linear and structural elements as promoters for complementary strand synthesis.

0086952安妮·西蒙尽管RNA病毒在过去的100年里一直是深入研究的对象，但对病毒生命周期中的基本过程复制仍然知之甚少。 病毒编码的参与复制的酶，RNA依赖性RNA聚合酶（RdRp），以及可能来自宿主的蛋白质，必须识别特定的病毒RNA，启动互补链合成，然后使用这些互补链作为模板合成病毒基因组。 对RdRp的启动子知之甚少，迄今为止的许多研究涉及以tRNA样结构终止的病毒（例如，雀麦花叶病毒）或聚（A）尾（例如，脊髓灰质炎病毒）。 对于其基因组RNA以游离羟基终止的病毒，如芜菁皱缩病毒（TCV），了解得更少。 此外，直到最近才变得清楚的是，内部序列，而不仅仅是末端序列，对于RdRp的转录起始是重要的。 此类序列的可能作用是（i）RdRp结合序列，或（ii）参与RNA-RNA相互作用的序列，其与末端序列结合RdRp至病毒基因组。 TCV是RNA病毒中最小和最简单的一种，因此被用作研究复制过程中所涉及的序列和结构的模型。 更重要的是，TCV与可用于复制研究的非常小的亚病毒RNA相关。 已经开发了原生质体和体外系统来研究TCV基因组和亚病毒RNA的复制，并且已经鉴定了参与亚病毒RNA satC复制的几个区域。 当使用TCV作为模板时，TCV RdRp经历称为失败起始的过程，其产生能够作为引物用于修复satC末端缺失的短RNA序列。 初步结果表明，TCV和satC链末端的启动子可能不像以前认为的那样作为独立于上游序列的单元发挥作用，而是可能参与与内部序列的特定相互作用。 为了解决这一假设，将通过分析3'单链尾和上游序列元件在TCV RdRp的失败起始中的重要性来确定从TCV发夹启动子的失败起始的序列/结构要求。这一目标应允许描绘的TCV基因组上的流产启动所需的元素。 能够用来自satC的启动子恢复TCV复制并且在启动子序列之外的突变将通过用satC的启动子将突变TCV的片段替换到TCV中并测定感染性来定位。这一目的将为阐明与TCV 3'末端启动子相互作用的内部顺式作用序列提供另一种手段。 此外，将研究在3'端碱基不存在的情况下转录所需的satC中的内部序列的重要性。这些实验的成功完成应该提供显着的进步，我们的理解的功能的RdRp的和他们的能力，以识别各种各样的线性和结构元件作为启动子的互补链合成。