Replication of the RNA segments of a bipartite viral genome is coordinated by a transactivating subgenomic RNA

The insect nodavirus Flock house virus (FHV) has a small genome divided between two segments of positive-sense RNA, RNA1 and RNA2. RNA1 encodes the RNA-dependent RNA polymerase (RdRp) catalytic subunit and templates the synthesis of a subgenomic RNA (RNA3) that encodes two small nonstructural proteins. Replication of RNA2, which encodes a precursor to the viral capsid proteins, suppresses RNA3 synthesis. Here we report that RNA1 mutants deficient in RNA3 synthesis failed to support RNA2 replication. This effect was not caused by alterations in the RdRp catalytic subunit nor by a lack of the proteins encoded by RNA3. Furthermore, RNA3 supplied in trans from an exogenous source restored RNA2 replication. These data indicate that RNA3 transactivates the replication of RNA2, a novel property for a viral RNA. We propose that the RNA3 dependence of RNA2 replication serves to coordinate replication of the FHV genome segments.