Studies on RNA Recombination In Vivo and In Vitro

9728277 Simon TCV RdRp在合成全长或接近全长的相关RNA sat-RNA D的正链后，转换为使用第二相关RNA sat-RNA C或TCV基因组RNA的负链，转录仅重新启动稳定发夹的3'端。该系统已被重建用于体外研究，通过创建嵌合RNA，该嵌合RNA由3'端的sat-RNA D正链通过短环连接到sat-RNA C负链的重组热点组成，该重组热点包括sat-RNA C重组热点发夹（基序1-发夹）。使用部分纯化的TCV RdRp，使用sat-RNA C-衍生区作为模板，体外重组被检测为来自sat-RNA D序列3'末端的分子内延伸产物。基序1-发夹和一个短的链内碱基配对区的基序1-发夹的上游是重要的引物延伸在体外和体内。Sat-RNA D和TCV基因组RNA在体内的相互作用显然不涉及TCV基序3 ′-发夹重组热点的碱基配对区。此外，亚基因组（sg）RNA启动子的转录起始位点是体内重组的热点。这项研究将：（1）建立TCV基序3-发夹区的体外引物延伸系统，和（2）类似于为sat-RNA C基序1-发夹建立的系统的sgRNA启动子区。使用诱变策略，将确定发夹和围绕基序发夹或sgRNA启动子发夹的单链区域的作用。此外，将确定在TCV基序3-发夹和sgRNA启动子的上游和下游添加能够与sat-RNA D正链形成异源双链体的序列的体内作用。(3)新生链的3'端的RdRp加工机制将使用在3'端标记的体外引物延伸构建体来确定。通过检查引物延伸反应的小标记产物，确定加工是否涉及核酸内切或核酸外切活性以及去除的3'末端序列的长度。 RNA重组是RNA病毒基因组进化的主要过程之一。此外，对重组重要的序列和结构的分析提供了对一种表征不佳的酶的重要见解，这种酶由所有RNA病毒编码，通过RNA中间体复制，称为RNA依赖性RNA聚合酶（RdRp）。芜菁皱缩病毒（TCV）系统是研究RNA重组的非常强大的系统，因为重组可以使用整个生物体（植物）、细胞培养（原生质体）和体外系统来研究。此外，TCV系统中的重组发生在非编码的且比病毒基因组RNA小得多的亚病毒（卫星）RNA（如卫星RNA C和D）之间，或卫星RNA和基因组RNA之间。这项研究将确定在重组事件中重要的RNA分子的序列和结构。此外，将分析RdRp使用类似于重组的过程修复病毒RNA末端截短的能力。

9728277 Simon The TCV RdRp, after synthesizing full-length or near full-length plus-strands of the associated RNA sat-RNA D, switches to using minus-strands of either a second associated RNA, sat-RNA C, or the TCV genomic RNA with transcription reinitiating just 3' of stable hairpins. This system has been reconstructed for in vitro studies by creating a chimeric RNA consisting of the recombination hot-spot from sat-RNA D plus-strands at the 3' end connected by a short loop to sat-RNA C minus-strands including the sat-RNA C recombination hot-spot hairpin (motif1-hairpin). Using partially purified TCV RdRp, recombination in vitro is detected as an intramolecular extension product from the 3' end of the sat-RNA D sequence using the sat-RNA C-derived region as template. The motif1-hairpin and a short intrastrand base-paired region just upstream of the motif1-hairpin are important for primer extension both in vitro and in vivo. Recombination between sat-RNA D and TCV genomic RNA in vivo does not apparently involve a base-paired region 3' of the TCV motif3-hairpin recombination hot-spot. In addition, transcription start sites for subgenomic (sg) RNA promoters are hot-spots for recombination in vivo. This research will: ( 1 ) establish an in vitro primer extension system for the TCV motif3-hairpin region and (2) sgRNA promoter region analogous to the system established for the sat-RNA C motif1-hairpin. Using a mutagenesis strategy, the roles of the hairpins and single-stranded regions surrounding the motif'3-hairpin or sgRNA promoter hairpin will be determined. In addition, the in vivo effects of adding sequences capable of forming heteroduplexes with sat-RNA D plus-strands upstream and downstream of the TCV motif3-hairpin and sgRNA promoter will be established. (3) The mechanism of RdRp processing of the 3' end of the nascent strand will be determined using the in vitro primer extension constructs labeled at the 3' end. By examining small labeled products of the primer-extension reaction, a determ ination of whether processing involves endonucleolytic or exonucleolytic activity and also the length of the 3' end sequence that is removed will be made. RNA recombination is one of the major processes involved in the evolution of RNA virus genomes. In addition, analyses of sequences and structures that are important for recombination have provided major insights into a poorly characterized enzyme, the enzyme encoded by all RNA viruses that replicate through RNA intermediates, called RNA-dependent RNA polymerase (RdRp). The turnip crinkle virus (TCV) system is an unusually powerful system for studying RNA recombination since recombination can be studied using the whole organism (plant), cell culture (protoplast) and in vitro systems. In addition, recombination in the TCV system is between subviral (satellite) RNAs (such as sat-RNAs C and D) that are non-coding and are substantially smaller than the viral genomic RNA, or between sat-RNAs and the genomic RNA. This research will establish the sequences and structures on the RNA molecules that are important in the recombination event. In addition, the ability of the RdRp to repair truncations at the ends of virus RNAs using a process similar to recombination will be analyzed.