烟草丛顶病毒RNA依赖RNA聚合酶(RdRp)的-1型移码翻译机制研究

Viral RNA-dependent RNA polymerase (RdRp) is the core component of replicase complex, which is responsible for the replication of RNA virus genome. RdRp could be expressed by various type of alternative mechanism including polyprotein processing, translation reinitiation, and post-translational mechanism (leaky scanning, ribosomal frameshift and stop-codon readthrough) based on the location of RdRp ORF within the viral genome, in which -1 frameshift and readthrough are the principal mechanism used by the majority of RNA viruses. -1 frameshift and readthrough could ensure the expression of RdRp at the certain ratio via multi-level cis-elements, which is critical for the accumulation of RNA viruses. -1 frameshift had the similar characteristic with readthrough in the requirement for the local secondary structure and potential long distance RNA-RNA interaction, although they required conserved slippery sequence and preferred type of stop-codon in nucleotide level respectively. It is necessary to further study the distal elements involved in long distance RNA-RNA interaction and relationship between -1 frameshift and readthrough..In this project, we will reveal the detailed mechanism of -1 frameshift expressing RdRp in Tobacco bushy top virus (TBTV) and consequently seek the possible evolutionary correlation between -1 frameshift and readthrough using TBTV as template. Firstly, we will confirm that RdRp of TBTV is expressed by -1 frameshift. Then the multi-level cis-elements required by -1 frameshift will be identified. The conserved seven nucleotide slippery sequences of -1 frameshift will be mapped by mutagenesis assay. Further，the local secondary structural element located downstream slippery sequence will be resolved by mutagenesis assay and in vitro structure probing method such as in-line probing. Finally, the potential long-distance RNA-RNA interaction involved in frameshift will be analyzed by EMSA and mutagenesis. On the basis of adequately understanding -1 frameshift mechanism for RdRp, the mutual-replacement correlation between -1 frameshift and readthrough on the regulation of gene expression will be explored. Firstly, under the condition of keeping the local secondary structure and potential long-distance RNA-RNA interaction constant, TBTV will be changed to express possibly RdRp by readthrough mechanism through subtle mutation. Then, the local secondary structure and potential long-distance RNA-RNA interaction initially involved in frameshift were respectively destroyed to analyze the effect on readthrough of RdRp so that we can conclude whether there are mutual-replacement correlation between frameshift and readthrough on the expression of viral gene, even the evolution of RNA viruses. This project will provide the support of theory and data on managing disease caused by RNA viruses and studying the relationship between -1 frameshift and readthrough during the evolution of RNA viruses.

RNA病毒编码的RNA依赖RNA聚合酶（RdRp）作为复制酶系统核心组分对病毒基因组的复制至关重要，主要以-1型移码或通读机制表达。-1型移码机制的研究以移码滑动序列和临近RNA元件为主，对远端RNA元件的调控作用以及移码和通读在基因表达中的可置换性研究有待深入。课题利用突变分析烟草丛顶病毒（TBTV）RdRp -1型移码所需的七核苷酸滑动序列及其下游合适距离内的RNA元件；通过凝胶阻滞实验（EMSA）定位潜在的远端RNA元件；利用RNA结构体外分析技术In-line probing解析临近元件和远端元件的结构特征并定位其核心组件。通过突变构建以通读机制表达RdRp的TBTV衍生载体，分析其移码元件在通读调控中作用。在深入理解TBTV RdRp -1型移码机制的同时，探索性研究移码元件调控通读的潜力；为通过调控移码效率防治RNA病毒病以及研究移码和通读在病毒进化中的关系提供理论和数据支持。

RNA病毒编码的RNA依赖RNA聚合酶（RdRp）作为复制酶系统核心组分对病毒基因组的复制至关重要，主要以-1型移码或通读机制表达。-1型移码机制的研究以移码滑动序列和临近RNA元件为主，对远端RNA元件的调控作用以及移码和通读在基因表达中的可置换性研究有待深入。课题以烟草丛顶病毒（TBTV）为对象开展RdRp的-1型移码翻译机制调控研究，包括七核苷酸滑动序列及其下游合适距离内的RNA元件、远距离RNA互作元件、移码核心元件在通读机制中的调控作用。.通过突变及蛋白表达的检测，定位了TBTV-1型移码所需的七核苷酸滑动序列是946GGATTTT。利用SHAPE技术解析了TBTV中七核苷酸序列临近下游移码相关RNA元件的结构特征，该结构包含3个大小不等的茎环结构fSL1, fSL2 和fSL3，以及2个茎环间隔区fLR1和fLR2。其中fLR1和fLR2可以形成潜在的kissing-linker（KL）。这3个茎环均参与TBTV RdRp的-1位移码的调控，并且KL的存在对于-1位移码至关重要。利用凝胶阻滞实验（EMSA）确定了与移码区域发生远距离互作的RNA元件位于基因组RNA的3’末端的Pr区。然后利用In-line probing技术解析了Pr区的RNA特征，并且发现Pr区的噜噗（loop）可以与移码区的三处位点发生远距离RNA互作，这些互作处于动态调整状态，只需要任何两处远距离RNA-RNA互作存在即可满足-1型移码的调控。通过突变替换实验将TBTV的移码区元件替换到甜菜黑色焦枯病毒（BBSV）的通读元件区，分析移码元件对通读机制的调控能力，结果表明移码元件不能调控通读。.本研究阐述了TBTV中的-1型移码调控的新模型，其中三处远距离RNA-RNA互作的存在是区别于其他-1型移码调控的根本所在，而移码下游的KL结构则可以保证三处远距离RNA-RNA互作的同步发生。本研究丰富了正单链RNA病毒-1位移码翻译调控的研究内容，提供了新的调控模型；也为烟草丛顶病毒病的防治提供了防治靶标。