A Novel Viral RNA-RNA Interaction Coupling Gene Expression and Assembly

NSF MCB-0077964摘要Lommel该项目涉及红三叶草坏死花叶石竹病毒（RCNMV）组装序列（OAS）和其他指导病毒体形成的包装信号的来源的表征。RCNMV的基因组由两个总长度小于5.5kb的正义单链RNA组成。目前尚不清楚这两种基因组RNA是否一起包装到病毒体中，或者是否各自单独包装。先前已经表明，两个基因组RNA通过碱基配对相互作用，以指导从多顺反子RNA-1合成亚基因组RNA。(The亚基因组RNA编码衣壳蛋白。 RNA-2上的反式激活元件下游存在另一个元件，其似乎指导病毒体组装。基于这一观察以及其他，一个可测试的假设如何RCNMV包可以提出。假设RCNMV装配的唯一同源起源位于RNA-2上。RNA-1通过与RNA-2相互作用而被捕获用于包装，所述相互作用经由部分表征的反式激活因子（TA）相互作用。那些在包装过程中与RNA-2碱基配对的RNA-1形成RNA-1和RNA-2颗粒。如果RNA-2分子的多个拷贝合并成一个正在形成的病毒体，那么那些没有与RNA-1配对的RNA-2仍然可以被包装。这一假设有一些可以检验的限制。I）该假设排除了仅RNA-1病毒体的形成。II）其允许仅RNA-2病毒体的形成。III）它表明RNA-1上不存在OAS。IV）如果TA相互作用被破坏，则不会形成生物活性病毒体制剂或含有RNA-1的病毒体。V）这表明OAS和反式激活元件虽然对于包装是相互必需的，但可以物理地解偶联。解决这些问题所采取的实验方法概述于以下总结的目标中：1）鉴定和表征RNA-2上的组装序列和最小/最大包装信号的RCNMV来源。2）将sgRNA/CP表达的产生与组装过程解偶联。这项研究将允许，第一次，确定OAS的二十面体病毒，包装成一个病毒粒子一个以上的基因组成分。这项研究将扩大的作用RNA-RNA相互作用的转录激活以外的病毒粒子组装，并进一步提出额外的重要的调节和结构的作用，无论是顺式或反式RNA-RNA相互作用。在这个项目的背景下发现的东西肯定会适用于其他多组分二十面体RNA植物病毒，并可能提供类似的动物病毒的见解。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途运输和全身感染所需的病毒体形成。

NSF MCB-0077964 ABSTRACTLommelThe project involves the characterization of the origin of assembly sequence (OAS) and other packaging signals directing virion formation of red clover necrotic mosaic dianthovirus (RCNMV). The genome of RCNMV is composed of two positive sense single-stranded RNAs totaling less that 5.5 kb. It is not known whether both genomic RNAs are packaged into virions together or whether each is packaged separately. It has been previously shown that the two genomic RNAs interact by base-pairing to direct the synthesis of a subgenomic RNA from the polycistronic RNA-1. (The subgenomic RNA encodes the capsid protein.) Down-stream from this trans-activating element on RNA-2 resides another element that appears to direct virion assembly. Based on this observation as well as others, a testable hypothesis for how RCNMV packages can be proposed. The hypothesis is that the only cognate origin of assembly for RCNMV resides on RNA-2. RNA-1 is captured for packaging by interacting with RNA-2 via the partially characterized trans-activator (TA) interaction. Those RNA-1s that are base-paired with RNA-2 during packaging form an RNA-1 and RNA-2 particle. Those RNA-2's that are not paired with an RNA-1 can still be packaged, if multiple copies of RNA-2 molecules coalesce into a forming virion. This hypothesis has a number of constraints that can be tested. I) The hypothesis precludes the formation of RNA-1 only virions. II) It allows for the formation of RNA-2 only virions. III) It states that no OAS is present on RNA-1. IV) No biologically active virion preparation or RNA-1 containing virions will form if the TA interaction is disrupted. V) It suggests that the OAS and trans-activating element, while mutually essential for packaging, may be physically uncoupled. The experimental approach to be taken to address these questions is outlined in the objectives summarized below:1) Identify and characterize the RCNMV origin of assembly sequence and minimum/maximum packaging signal on RNA-2.2) Uncouple the generation of sgRNA/CP expression from the assembly process. This research will allow, for the first time, the determination of the OAS for an icosahedral virus that packages more than one genomic component into a virion. This study will extend the role of RNA-RNA interactions beyond transcriptional activation to virion assembly and further suggest additional important regulatory and structural roles for either cis or trans RNA-RNA interactions. What is discovered in the context of this project will certainly be applicable to other multicomponent icosahedral RNA plant viruses and likely provide insights into similar animal viruses as well. 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.