Elements Required for Replication of a Model Viral RNA

模型病毒 RNA 复制所需的元素

• 批准号: 6469408
• 负责人: Anne Elizabeth Simon
• 金额: $ 22.2万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2002
• 资助国家: 美国
• 起止时间: 2002-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6469408
• 关键词: Arabidopsis; RNA virus; gene mutation; molecular chaperones; nucleic acid sequence; virus RNA; virus genetics; virus replication

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.

申请人提供的描述）：这项研究的长期目标是 确定复制的分子机制，这是一个基本的过程 致病性干旱毒力，在（+） - 链RNA病毒中，包括许多 人，动物和植物病原体。使用强大的模型真核病毒 诸如萝卜皱纹病毒系统之类的系统对于解决许多系统是必要的 RNA病毒复制中的重要问题，结果已经是 被证明适用于人类病毒。 TCV与小有关 非编码寄生虫RNA（如SATC）含有顺式作用元件已识别 由TCV RDRP。已经确定的是：3端carmovirus共识 序列（CCS）; 3PE还包含CCS；主序（M1 H）是一个 重组热点，转录增强子，还包含CCS；和 （+） - 链合成所需的5个PE。构成的类似序列 元素和3PE和5PE充当独立启动子的能力 在体外，建议它们都吸引了RDRP。解决该功能和 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在 RDRP的3端可访问性，并促进体外结构 内部底漆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 使用突变稳定体内的更活跃的体内结构 结构和RNA伴侣。在特定的目标2中，对M1 H的分析 将进行TCV的SATC和Theanalogous Motif-3发夹。我们将测试 功能替代序列可以增强的假设 SATC或TCV系统运动的复制。我们还将测试为什么最多 常见的Ml H替换基序CAACCCC，也发现了相关的5PE 病毒氨基氨基绿化斑点（CCFV），抑制了某些人的复制 序列上下文。将测试循环和扫描模型的RDRP 在M1H结合后找到3端启动子。在特定目标3中，功能性 将对SATC，TCV和CCFV的5PE分析，以解决 5 PE的序列特定性质，并确定该元素是否（哪个元素）是否 可以在体外充当TCV RDRP的独立启动子，播放 增强子或体内的结构作用。