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

点击翻译按钮获取中文摘要

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.