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，这是一个基本的过程 致病性和毒力，在()链RNA病毒，包括许多 人类、动物和植物的病原体。强大的真核细胞模型病毒的应用 系统，如芜菁皱纹病毒系统，是解决许多 RNA病毒复制中的重要问题和结果已经被 被证明适用于人类病毒。TCV与小型、 非编码寄生RNA，如SATC，含有识别的顺式作用元件 由TCV RdRp提供。已经确定的是：一种3-末端的卡摩病毒共识 序列(CCS)；3PE也包含CCS；基序1-发夹(M1 H)是 重组热点、转录增强子，还含有CCS； 5合成()-链需要PE。组成这些序列的相似序列 3PE和5PE作为独立推动者的要素和能力 在体外，表明它们都吸引了RdRp。要解决这一功能和 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不同于 RdRp的3端可及性，体外结构促进 内引物延伸与全长启动子的体内结构 互补链合成。我们还将把体外结构 进入更活跃的体内结构，使用突变来稳定体内 结构和RNA伴侣。在具体目标2中，分析了M1H 将进行SATC和TCV的类似Motif-3发夹。我们将测试 假设功能替换序列可以增强 SATC复制或TCV全身性运动。我们还将测试为什么大多数 常见的mLH替换基序CAACCCC，也在5PE中发现了相关的 豆蔻碱褪绿斑病毒(CCFV)，在一定程度上抑制复制 序列上下文。将测试环回和扫描模型以了解RdRp如何 在M1H结合后找到3端启动子。在具体目标3中，一个功能 将对SATC、TCV和CCFV的5PE进行分析，解决 5个PE的特定序列性质，并确定该元件是否 在体外可作为TCV RdRp的独立启动子，起到 增强剂或在体内的结构作用。

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.