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

[00:86952]安妮·西蒙尽管在过去的100年里，RNA病毒一直是深入研究的对象，但作为病毒生命周期中的一个基本过程，人们对其复制仍然知之甚少。参与复制的病毒编码酶，RNA依赖性RNA聚合酶（RdRp），连同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病毒基因组合成的模板。人们对RdRp的启动子知之甚少，迄今为止的许多研究涉及到以trna样结构终止的病毒（例如，溴基花叶病毒）或以多(A)尾部终止的病毒（例如，脊髓灰质炎病毒）。对于基因组RNA以游离羟基终止的病毒，如芜菁皱缩病毒（TCV），我们所知甚少。此外，直到最近才清楚，内部序列，而不仅仅是末端序列，对RdRp启动转录很重要。这些序列可能的作用是(i) RdRp结合序列，或（ii）与末端序列进行RNA-RNA相互作用，将RdRp与病毒基因组结合的序列。由于TCV是最小和最简单的RNA病毒之一，因此它被用作研究参与复制的序列和结构的模型。更重要的是，TCV与非常小的亚病毒rna相关，可用于复制研究。原生质体和体外系统已经被开发用于研究TCV基因组和亚病毒RNA的复制，并且已经确定了几个参与亚病毒RNA satC复制的区域。当使用TCV作为模板时，TCV RdRp经历了一个称为流产起始的过程，该过程产生了能够作为修复satC末端缺失的引物的短RNA序列。初步结果表明，TCV和satC链末端的启动子可能不像之前认为的那样是独立于上游序列的单元，而是可能与内部序列进行特定的相互作用。为了解决这一假设，将通过分析3'单链尾部和上游序列元件在TCV RdRp流产起始中的重要性来确定TCV发夹启动子流产起始的序列/结构要求。这一目标应该允许在TCV基因组上描述流产起始所需的元件。通过将突变TCV的片段替换为具有satC启动子的TCV，并测定其传染性，可以将能够恢复带有satC启动子的TCV复制的突变和位于启动子序列之外的突变进行定位。这一目标应该为阐明与TCV 3'端启动子相互作用的内部顺式作用序列提供另一种方法。此外，将研究在没有3'端碱基的情况下转录所需的satC内部序列的重要性。这些实验的成功完成将为我们理解RdRp的功能及其识别各种线性和结构元件作为互补链合成启动子的能力提供重大进展。

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.