Polymerase trapping as the mechanism of H5 highly pathogenic avian influenza virus genesis

Highly pathogenic avian influenza viruses (HPAIVs) derive from H5 and H7 low pathogenic avian influenza viruses (LPAIVs). Although insertion of a furin-cleavable multibasic cleavage site (MBCS) in the hemagglutinin gene was identified decades ago as the genetic basis for the LPAIV-to-HPAIV transition, the mechanisms underlying the occurrence of insertion are unknown. Here, we show that transient H5 RNA structures, predicted to trap the influenza virus polymerase on purine-rich sequences, drive nucleotide insertions, providing empirical evidence of RNA structure involvement in MBCS acquisition. Introduction of H5-like sequences and structures into an H6 hemagglutinin resulted in MBCS-yielding insertions. Our results show that nucleotide insertions that underlie H5 HPAIV emergence result from an RNA structure-driven diversity-generating mechanism, which could also occur in other RNA viruses.