Elements Required for Replication of a Model Viral RNA

申请人提供的描述）：本研究的长期目标是 确定复制的分子机制，这是 致病性和毒力，在（+）链RNA病毒，包括许多 人类、动物和植物病原体。使用强大的模式真核病毒 系统，如芜菁皱缩病毒系统，是必要的，以解决许多 RNA病毒复制中的重要问题，结果已经被 显示其适用于人类病毒。TCV与小， 非编码寄生RNA如satC，含有识别的顺式作用元件， 由TCV RdRp。已经确定的是：一个3-末端的卡莫病毒共识 序列（CCS）; 3 PE也含有CCS;基序1-发夹（M1 H）是一个 重组热点、转录增强子，并且还含有CCS;并且 （+）-链合成需要5个PE。组成这些序列的相似序列 元件以及3 PE和5 PE作为独立启动子的能力 表明它们都能吸引RdRp。为了解决功能和 具体目标1将检验以下假设： satC（-）链的体内和体外结构不同， 3端对RdRp的可及性，体外结构促进 内部引物延伸和体内结构促进全长 互补链合成我们还将把体外结构 使用突变来稳定体内结构， 结构和RNA分子伴侣。在具体目标2中，分析了 SatC和TCV的类似模体-3发夹结构的研究。我们将测试 假设功能性替换序列可以增强 SatC的复制或TCV的全身运动。我们还将测试为什么大多数 常见的M1 H置换基序CAACCCC，也发现于相关的 碎米荠褪绿斑点病毒（CCFV），抑制某些 序列上下文。将测试环出和扫描模型，以了解RdRp 发现M1 H结合后的3端启动子。在具体目标3中， 将对satC、TCV和CCFV的5 PE进行分析， 序列特异性性质的5 PE，并确定是否元素， 在体外可作为TCV RdRp的独立启动子， 增强子或体内结构作用。

DESCRIPTION provided by applicant): The long-term goal of this research is to determine the molecular mechanisms of replication, a process fundamental to pathogenicity arid virulence, in (+)-strand RNA viruses that include many human, animal and plant pathogens. The use of powerful model eukaryotic virus systems, such as the turnip crinkle virus system, is necessary to address many important questions in RNA virus replication, and results have already been shown to be applicable to human viruses. TCV is associated with small, non-coding parasitic RNAs like satC that contain cis-acting elements recognized by the TCV RdRp. Already identified are: a 3-terminal carmovirus consensus sequence (CCS); 3PE also containing a CCS; the motif1-hairpin (M1 H) that is a recombination hot-spot, enhancer of transcription, and also contains a CCS; and 5 PE required for (+)-strand synthesis. The similar sequences that comprise the elements and the ability of the 3PE and 5PE to serve as independent promoters in vitro, suggests that they all attract the RdRp. To address the function and relationship among the elements, Specific Aim 1 will test the hypothesis that the in vivo and in vitro structures of satC (-)strands differ in the accessibility of the 3 end to the RdRp, with the in vitro structure promoting internal primer extension and the in vivo structure promoting full-length complementary strand synthesis. We will also convert the in vitro structure into the more active in vivo structure using mutations to stabilize the in vivo structure and RNA chaperones. In Specific Aim 2, an analysis of the M1 H of satC and theanalogous motif-3 hairpin of TCV will be conducted. We will test the hypothesis that functional replacement sequences can enhance either replication of satC or systemic movement of TCV. We will also test why the most common Ml H replacement motif CAACCCC, also found in the 5PE of the related virus Cardamine chlorotic fleck (CCFV), inhibits replication in certain sequence contexts. Loop-out and scanning models will be tested for how the RdRp finds the 3 end promoter after M1H binding. In Specific Aim 3, a functional analysis of the 5PE of satC, TCV and CCFV will be conducted, addressing the sequence specific nature of the 5 PE and determining whether the element, which can serve as an independent promoter for the TCV RdRp in vitro, plays an enhancer or a structural role in vivo.