Sequences and Structures Involved in Replication and Recombination of TCV RNAs

9419303西蒙芜菁皱缩病毒(TCV)基因组和亚病毒RNA将用于研究涉及复制和重组的序列和结构。TCV的优点包括：从克隆的基因组和大量亚病毒RNA体外产生的转录本在植物和分离的植物细胞(原生质体)中具有生物活性；含有依赖于模板的RNA依赖的RNA聚合酶(RdRp)活性的植物提取物可以从(+)-链和(-)-链TCV亚病毒RNA模板转录RNA；大量嵌合RNA已被鉴定为基因组和亚病毒RNA重组的产物。与TCV相关的RNA重组体在3‘交叉点有三个基序之一(与其他病毒重组体不同)，其中一个是病毒在植物和原生质体中正常积累所必需的，这表明重组与病毒复制之间存在联系。初步结果引出了许多耐人寻味的问题：TCV基因组RNA中特定区域的复制和重组之间有什么联系？稳定的TCV RNA发夹的序列或结构对重组/复制重要吗？RdRp转录所需的序列/结构是什么？与交叉位点有共同之处吗？使用(-)链卫星模板体外合成大于全长的产物和重组之间是否有联系？在删除模板的不同部分后，相同反应中其大小以不寻常方式变化的小于全长的主要产物的身份是什么？为了开始回答这些和其他相关的问题，将进行以下实验：(1)将通过遗传操作和结构确定来研究TCV基因组RNA中参与复制和重组的区域。(2)TCV卫星RNA C体外转录(+)和(-)链所需的顺式作用序列/结构将通过进一步删除末端和内部序列、结构测定和推测的结构元件的修饰来确定。(3)将使用模板测试重组与体外合成全长产物之间的联系，其中该区域的结构保持不变，但已知影响重组的特定序列已被改变。(4)对小于全长的150个碱基的产物进行克隆，并对其合成机理进行研究。研究TCV RdRp在转录启动或交叉事件后重新启动转录过程中识别的序列/结构，将有助于更好地理解病毒RNA复制过程。萝卜皱缩病毒是一种二十面体病毒，具有单部分RNA基因组，侵染范围广泛的双子叶植物。4054碱基基因组RNA编码病毒的复制和封装功能。芜菁皱缩病毒基因组和亚病毒RNA将用于研究病毒RNA复制和重组所涉及的RNA序列和结构。作为一种模式系统，萝卜皱缩病毒具有技术优势，包括能够通过体外转录克隆的DNA来产生具有生物活性的病毒RNA，能够制备具有病毒RNA依赖的RNA聚合酶功能的植物细胞提取物，该酶负责病毒RNA的复制。这些实验将极大地加深我们对依赖于RNA的RNA聚合酶介导的几种植物RNA病毒复制和重组特性的分子机制的理解。***

9419303 Simon Turnip crinkle virus (TCV) genomic and subviral RNAs will be used to study sequences and structures involved replication and recombination. The advantages of TCV include: transcripts produced in vitro from cloned cDNA of genomic and numerous subviral RNAs are biologically active in plants and isolated plant cells (protoplasts); plant extracts containing template-dependent RNA-dependent RNA polymerase (RdRp) activity can transcribe RNA from both (+)- and (-)-strand TCV subviral RNA templates; numerous chimeric RNAs have been characterized that are the products of recombination between the genomic and subviral RNAs. RNA recombinants associated with TCV have one of three motifs at the 3' crossover point (unlike other virus recombinants), one of which is absolutely required for normal accumulation of the virus in plants and protoplasts, suggesting a link between recombination and virus replication. Preliminary results have led to numerous intriguing questions: What is the connection between replication and recombination at a particular region in the TCV genomic RNA? Is the sequence or structure of a stable TCV RNA hairpin important for recombination/replication? What are the sequences/structures required for transcription by the RdRp and is there a commonality with the crossover sites? Is there a connection between in vitro synthesis of a larger-than-full-length product using (-)-strand satellite template and recombination? What is the identity of the major smaller-than-full-length product in the same reactions, whose size varies in an unusual manner after deletion of various portions of the template? To begin answering these and other related questions, the following experiments will be performed: (1) The region involved in replication and recombination in the TCV genomic RNA will be studied by genetic manipulation and structural determination. (2) Cis-acting sequences/structures of TCV satellite RNA C that are required for transcription of (+)-and (-)-strands in vitro wil l be determined by further deletions of terminal and internal sequences, structural determinations, and modification of putative structural elements. (3) The connection between recombination and synthesis of larger-than-full-length product in vitro will be tested by using templates where the structure in the region is maintained but specific sequences, known to affect recombination, have been altered. (4) The 150 base smaller-than-full-length product will be cloned and the mechanism of its synthesis determined. Studying the sequences/structures that are recognized by the TCV RdRp during initiation of transcription or re-initiation of transcription following a crossover event, should lead to a better understanding of the process viral RNA replication. %%% Turnip crinkle virus is an icosahedral virus with and monopartite RNA genome that infects a broad range of dicot plants. The 4054 base genomic RNA encodes the viral replication and encapsidation functions. Turnip crinkle virus genomic and subviral RNAs will be used to study the RNA sequences and structures involved in viral RNA replication and recombination. As a model system, turnip crinkle virus has technical advantages including the ability to produce biologically active viral RNAs by in vitro transcription of cloned DNAs, the ability to prepare plant cell extracts that have functioning viral RNA-dependent RNA polymerase, the enzyme responsible for virus RNA replication. These experiments will greatly increase our understanding of the molecular mechanism of RNA-dependent RNA polymerase mediated replication and recombination characteristic of several plant RNA viruses. ***