Dissecting the mechanism of viral RNA recombination

描述(申请人提供)：RNA重组通过促进新的致病或耐药病毒和毒株的出现，极大地促进了RNA病毒的进化。RNA重组的另一个重要功能是修复受损或突变的病毒基因组，有助于病毒适应。RNA重组也可以发生在病毒和宿主序列之间，导致具有新特性的重组病毒的出现。大量的人类病毒，包括微冠状病毒、黄状病毒、罗塔病毒、冠状病毒、Calici病毒、动脉病毒、天文病毒、甲型病毒、丁型肝炎病毒和甲型流感病毒都存在RNA重组。尽管它的重要性，但对RNA重组的机制的理解是不完整的。 PI已经开发了基于纯化的Tombus和CarmoVirus RNA依赖的RNA聚合酶(RdRp)的高效和易操作的体外RNA重组系统，这与许多重要的人类病毒的RdRp相似。PI还开发了一种在整个植物和单细胞(原生质体)中有效的体内RNA重组系统，这使得Tombus病毒特别适合于研究RNA重组。本工作检验的中心假设是，在重组RNA中存在特定的RNA重组信号，这些信号可以被病毒RdRp识别。在初步数据的鼓励下，PI将剖析(目标1)由(I)复制增强剂支持的RNA重组机制；(Ii)富含AU的序列；以及(Iii)使用Tombus和CarmoVirus RdRp系统的高度结构的序列；他将在单细胞和整个生物体中测试(目标2)促进重组的序列，以评估它们在RNA重组中的效率。这些元素在病毒间重组中的可能意义将通过测试CarmoVirus复制增强剂支持Tombus病毒系统中的RNA重组的能力来检验。 本文的研究将有助于更好地理解RNA重组的机制，以及在RNA重组过程中复制元件与病毒RdRp之间的相互作用。实现所提出的目标将为识别普遍的重组信号提供关键信息，并将有助于开发预测重组和病毒进化的计算机软件。重组的知识提供了限制病毒感染和更安全的疫苗的手段，有望带来数不清的健康益处。

DESCRIPTION (provided by applicant): RNA recombination contributes greatly to RNA virus evolution via promoting emergence of new pathogenic or drug-resistant viruses and strains. Another important function of RNA recombination is to repair damaged or mutated viral genomes, contributing to virus fitness. RNA recombination can also occur between viral and host sequences, leading to the emergence of recombinant viruses having novel traits. RNA recombination has been demonstrated to occur for a large number of human viruses, including Picorna-, Flavi-, Rota-, Corona-, Calici-, Arteri-, Astro-, Alphaviruses, hepatitis D virus and influenza A virus. In spite of its significance, understanding of the mechanism of RNA recombination is incomplete. The PI has developed efficient and tractable in vitro RNA recombination systems based on purified Tombus- and Carmovirus RNA-dependent RNA polymerases (RdRp), which are similar to the RdRp of a number of significant human viruses. The PI has also developed an efficient in vivo RNA recombination system in whole plants and single cells (protoplasts), which make Tombusviruses exceptionally suitable for studying RNA recombination. The central hypothesis tested in this work is that there are specific RNA recombination signals in the recombining RNAs and these signals can be recognized by the viral RdRp. Encouraged by preliminary data, the PI will dissect (Aim 1) the mechanism of RNA recombination supported by (i) replication enhancers; (ii) AU-rich sequences; and (iii) highly structured sequences using the Tombus- and Carmovirus RdRp systems; and he will test (Aim 2) recombination-promoting sequences in single cells and in whole organisms in order to estimate their efficiencies in RNA recombination. The possible significance of these elements in interviral recombination will be examined by testing the ability of a Carmovirus replication enhancer to support RNA recombination in the Tombusvirus system. The research described here will lead to better understanding of the mechanism of RNA recombination and the interaction between replication elements and the viral RdRp during RNA recombination. Accomplishing the proposed goals will provide crucial information towards identification of universal recombination signals and will help developing computer software to predict recombination and virus evolution. Knowledge of recombination promises untold health benefits by providing means to limit virus infections and safer vaccines.