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

本项目主要研究红三叶草坏死花叶dianthovirus （RCNMV）组装序列（OAS）的起源及其他指导病毒粒子形成的包装信号。RCNMV的基因组由两个正义单链rna组成，总长度小于5.5 kb。目前尚不清楚这两种基因组rna是一起包装成病毒粒子还是分别包装。先前的研究表明，这两种基因组RNA通过碱基配对相互作用，指导多顺反子RNA-1合成亚基因组RNA。（亚基因组RNA编码衣壳蛋白。）在RNA-2上这个反式激活元件的下游，存在着另一个似乎指导病毒粒子组装的元件。基于这一观察以及其他观察，我们提出了一个关于RCNMV包的可测试假设。假设RCNMV组装的唯一同源起源位于RNA-2上。通过部分表征的反式激活剂（TA）相互作用与RNA-2相互作用，捕获RNA-1进行包装。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分子的多个拷贝结合成一个正在形成的病毒粒子，那些没有与RNA-1配对的RNA-2仍然可以被打包。这个假设有许多可以检验的约束条件。1)该假说排除了仅RNA-1病毒粒子的形成。II)它允许仅RNA-2病毒粒子的形成。III)它指出RNA-1上不存在OAS。IV)如果TA相互作用被破坏，将不会形成具有生物活性的病毒粒子制备或含有RNA-1的病毒粒子。V)它表明，OAS和反式活化元件虽然对包装都是必不可少的，但在物理上可能是不耦合的。解决这些问题的实验方法概述如下：1)鉴定和表征装配序列的RCNMV起源和rna上的最小/最大封装信号2.2)从装配过程中解耦sgRNA/CP表达的产生。这项研究将首次允许确定将多个基因组成分包装到病毒粒子中的二十面体病毒的OAS。这项研究将RNA-RNA相互作用的作用从转录激活扩展到病毒粒子组装，并进一步提出顺式或反式RNA-RNA相互作用的其他重要调控和结构作用。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，也可能为类似的动物病毒提供见解。这一机制的阐明可能导致基于病毒粒子组装特异性破坏的病毒疾病分子控制策略的发展。最后，植物病毒只有在形成全身性感染时才会引起疾病。对于大多数植物病毒来说，病毒粒子的形成与这一过程有关，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粒子形成。

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.