Mutagenesis studies suggest a mechanism for influenza polymerase stalling during polyadenylation.

Influenza polymerase (FluPol) carries out both viral transcription and replication using the same viral genome segment as a template to yield distinct end products. However, it remains largely unclear how FluPol synthesizes transcripts containing poly (A) tails during transcription termination, while producing fully complementary products during replication termination. In this study, through structural analysis combined with cell-based and biochemical assays, we identified that the PB1 Leu675/Asn676 and PB2 Arg38 residues of FluPol are critical for transcription termination and polyadenylation. During transcription termination, these three residues adopt the PB1 Leu675/Asn676down and PB2 Arg38out conformations, with their side chains positioned against the G12 and G14 residues of the RNA template at the 5' end. These steric hindrances block template translocation and facilitate FluPol 'stuttering' at U17, which is required for viral messenger RNA polyadenylation. Importantly, both structural analysis and mutational studies suggest that this specific conformation of these residues is unique to the transcription termination state. Overall, our findings provide novel insights into the mechanisms by which FluPol generates distinct 3' end products during transcription and replication termination.