The role of RNA secondary structure in replication of Nodamura virus RNA2

Our laboratory studies genome replication of positive-strand RNA viruses, using a reverse-genetic system in yeast cells. Nodamura virus (NoV) provides an excellent model system for the study of RNA replication due to its genetic simplicity, its robust yield of replication products, and its ability to replicate in a wide variety of host cells. NoV contains a bipartite positive strand RNA genome: RNA1 encodes the viral RNA replicase, while RNA2 encodes the capsid protein. The role of RNA secondary structure in the genome replication of other RNA viruses has been well established. For NoV, sequences at the 3’ end of RNA2 are critical for RNA replication and we hypothesized that the secondary structure adopted by these RNA sequences is essential for RNA replication. Three different software programs were used to generate predicted secondary structures of the 3’-terminal end of NoV RNA2; all three consistently predicted the presence of a conserved stem-loop structure within this region. We tested whether the predicted structure has biological relevance in the viral life cycle and whether its formation could be verified experimentally. The structure was deleted from a cDNA clone of RNA2 using site directed mutagenesis. Yeast cells were transformed with wildtype or mutant RNA2 together with RNA1 as a source of RdRp and RNA replication products were detected by Northern blot hybridization. Our results showed that deletion of the predicted stem-loop structure resulted in a severe defect in RNA replication. We also used nuclease mapping to confirm that NoV RNA2 forms this structure in solution. These data suggest that the 3’-terminal region plays a significant role in RNA2 replication. Its exact role is, as yet, unknown