A predicted 3'-terminal structure serves to stabilize Nodamura virus RNA2 in vitro

The bipartite positive-strand Nodamura virus (NoV) genome replicates to high levels in a wide range of host cells, providing a model for genome replication of other positive-strand RNA viruses. NoV RNA1 and RNA2 encode the viral RNA-dependent RNA polymerase (RdRp) and capsid protein precursor, respectively. The 3’ end of nodaviral genomic and subgenomic RNAs are blocked to modification by poly(A) polymerase and T4 RNA ligase enzymes. We and others have used RNA secondary structure prediction software to predict that the extreme 3’ termini of nodaviral RNA2 segments may form a structural element to protect the ends from degradation. For NoV RNA2, the 3’-terminal 10-nt form a small stem-loop. We hypothesize that this stable terminal structure protects the 3’-terminus from reactivity and degradation. To test this hypothesis, we deleted these nts from a full-length RNA2 cDNA clone under transcriptional control of a T7 promoter. We synthesized in vitro transcripts of wildtype (WT) and the delta-terminus mutant and analyzed them by Northern blot hybridization with a radioactively labeled RNA2-specific probe. Our preliminary results show marked degradation of the mutant but not WT RNA2 under these conditions. Additional experiments are underway to test the ability of the mutant to be replicated by the viral RdRp in transfected mammalian cells and to test the reactivity of the mutant to enzymatic modification using an in vitro polyadenylation assay. We conclude from our preliminary data that base pairing of the extreme 3’-terminus of NoV RNA2 serves to protect it from degradation.