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，这是复制的基本过程 致病性和毒力，（ ）链 RNA 病毒，包括许多 人类、动物和植物病原体。使用强大的模型真核病毒 系统，例如萝卜皱纹病毒系统，对于解决许多问题是必要的 RNA病毒复制中的重要问题，结果已经得到 已证明适用于人类病毒。 TCV 与小、 非编码寄生 RNA，如 satC，含有已识别的顺式作用元件 通过 TCV RdRp。已确定的是： 3 端胡萝卜病毒共有序列 序列（CCS）； 3PE也含有CCS；基序 1-发夹 (M1 H) 是 重组热点，转录增强子，还包含CCS；和 ( )-链合成所需的 5 PE。组成相似的序列 元素以及3PE和5PE作为独立启动子的能力 在体外，表明它们都吸引 RdRp。为了解决函数和 元素之间的关系，具体目标 1 将检验以下假设： satC (-)链的体内和体外结构不同 RdRp 3 端的可及性，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。我们还将转换体外结构 使用突变进入更活跃的体内结构以稳定体内 结构和RNA伴侣。在具体目标 2 中，对 M1 H 的分析 将进行 satC 和 TCV 的类似基序 3 发夹。我们将测试 功能替换序列可以增强任一功能的假设 satC 的复制或 TCV 的全身运动。我们还将测试为什么最 常见的 Ml H 替换基序 CAACCCC，也在相关的 5PE 中发现 碎碎念病毒褪绿斑点 (CCFV)，抑制某些细胞的复制 序列上下文。将测试环出和扫描模型的 RdRp 找到 M1H 结合后的 3 端启动子。在具体目标 3 中，函数 将进行 satC、TCV 和 CCFV 的 5PE 分析，解决 5 PE的序列特异性并确定该元素是否 在体外可作为 TCV RdRp 的独立启动子，发挥 增强剂或体内结构作用。

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.