Internal initiation is responsible for synthesis of Wuhan nodavirus subgenomic RNA

Nodaviruses are small nonenveloped spherical viruses with a bipartite genome of RNAs. In nodaviruses, subgenomic RNA3 (sgRNA3) plays a critical role in viral replication and survival, as it coordinates the replication of two viral genomic RNAs (RNA1 and RNA2) and encodes protein B2, which is a potent RNA-silencing inhibitor. Despite its importance, the molecular mechanism of nodaviral sgRNA3 synthesis is still poorly understood. Here, we propose that sgRNA3 of Wuhan nodavirus (WhNV) is internally initiated from a promoter on the negative template of genomic RNA1. Serial deletion and mutation analyses further revealed that the core promoter of WhNV sgRNA3 is between nucleotide positions -22 and +6 of its transcription start site. Besides, a stem-loop structure of WhNV sgRNA3 was computationally predicted upstream of sgRNA3's transcription start site. Both the secondary structure and the primary sequence were determined to be required for promoter activity. Furthermore, our results show that the synthesis of WhNV sgRNA3 is counterregulated by the replication of WhNV genomic RNA2, which encodes a viral capsid precursor protein. And this sgRNA3 synthesis is also able to trans-activate the replication of RNA2. Altogether, findings in this study indicate that there is a newly discovered internal initiation model for the synthesis of nodaviral sgRNA.