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.
