Viral Genomic RNA-RNA Interactions Programming Virion Content and Assembly

本项目将充分表征RNA-RNA和RNA-衣壳蛋白（CP）相互作用在红三叶草坏死花叶病毒（RCNMV）装配起源（OAS）中的作用。RCNMV基因组被分成两个正义单链RNA。这两个基因组RNA彼此碱基配对，作为病毒生命周期中的关键分子开关。 RNA-2的34个核苷酸的茎环结构称为反式激活因子（TA）碱基对，其与RNA-1反式激活因子结合位点（TABS）形成稳定的反式假结。到目前为止，亚基因组RNA合成，CP表达，RNA-2复制和病毒体组装已被分配到单独的RNA-2 TA或与RNA-1 TABS复合。 计划中的研究将充分确定TA和TA/TABS相互作用在病毒体内容和组装中的作用。RNA-2 TA是指导病毒体组装所必需的，因此被认为是OAS。 这个项目要回答的主要问题是，TA本身是否具有OAS活性，或者它是否需要与另一种RNA复合？在该项目中待检验的主要假设是RNA-2 TA是否必须与RNA-1特异性地在TABS处碱基配对，以形成用于感染性病毒体组装的功能性OAS。实验方法将涉及使用体内组装测定结合遗传学研究和结构和生化方法，涉及组装过程中分离的组分，即病毒RNA和CP亚基。病毒学中一个长期存在的问题是，具有多个基因组片段的病毒如何将基因组包装成病毒粒子？ 在这个项目的背景下所发现的将适用于其他多组分二十面体RNA植物和动物病毒。这种RCNMV开关在结构上和机制上与参与翻译（猿猴逆转录病毒1型）和病毒体组装（人类免疫缺陷病毒1型）的其他病毒RNA元件相似。 RCNMV在单一元件中具有与许多健康相关的RNA病毒在形式和功能上保守的这些特征，证明了对这种植物病原体的研究所提供的独特机会。 这一机制的阐明可能会导致发展的病毒疾病的分子控制策略的基础上，特定的破坏病毒体组装。最后，植物病毒只有在形成系统感染时才会引起疾病。对于大多数植物病毒，病毒体的形成与此过程有关，但尚未确定。RCNMV OAS的阐明将允许直接测试长距离运输和全身感染所需的病毒体形成。 高中生、本科生和研究生、技术人员、博士后和访问科学家将在拟议的赠款期间接受培训并在实验室进行研究。所有这些人都接触到高度创新的研究，这种经验为他们提供了宝贵的学习经验。

This project will fully characterize the role of RNA-RNA and RNA-capsid protein (CP) interactions in the Red clover necrotic mosaic virus (RCNMV) origin of assembly (OAS). The RCNMV genome is split between two positive-sense single-stranded RNAs. The two genomic RNAs base-pair with each other to serve as a key molecular switch in the virus life cycle. The RNA-2 34-nucleotide stem-loop structure termed the trans-activator (TA) base-pairs with the RNA-1 trans-activator binding site (TABS) forming a stable trans-pseudoknot. So far, subgenomic RNA synthesis, CP expression, RNA-2 replication and virion assembly have been assigned to the RNA-2 TA alone or complexed with the RNA-1 TABS. The planned research will fully determine the role of the TA and the TA/TABS interaction in virion content and assembly. The RNA-2 TA is necessary and sufficient to direct virion assembly and is therefore deemed to be the OAS. The main question to be answered in this project is, does the TA itself possess OAS activity or does it need to be complexed with another RNA? The main hypothesis to be tested in this project is whether the RNA-2 TA must base pair with RNA-1 specifically at the TABS to form a functional OAS for the assembly of infectious virions. The experimental approach will involve the use of an in vivo assembly assay coupled with genetic studies and structural and biochemical approaches involving the isolated components of the assembly process, namely viral RNAs and CP subunits.A long standing question in virology is how do viruses with multiple genomic segments package the genome into virions? What is discovered in the context of this project will be applicable to other multicomponent icosahedral RNA plant and animal viruses. This RCNMV switch structurally and mechanistically parallels other viral RNA elements involved in both translation (Simian retrovirus type-1) and virion assembly (Human immunodeficiency virus-1). That RCNMV possesses these features conserved in both form and function with a number of health related RNA viruses in a single element attests to the unique opportunity offered by research on this plant pathogen. The elucidation of this mechanism may lead to the development of molecular control strategies for virus diseases based on the specific disruption of virion assembly. Finally, plant viruses only cause a disease when they form a systemic infection. For most plant viruses, virion formation has been implicated in this process, but not definitively established. The elucidation of the RCNMV OAS will allow for a direct test of the need for virion formation for long distance transport and systemic infection. High school, undergraduate and graduate students, technicians, post-docs and visiting scientists will be trained and conduct research in the laboratory during the proposed grant period. All of these people are exposed to highly innovative research and this experience provides them with an invaluable learning experience.