Decoding the molecular cues regulating the transition of Influenza virus polymerase from transcriptase to replicase.

Abstract

Influenza virus polymerase (FluPol) dynamically transitions between distinct conformational states to coordinate viral gene transcription and genome replication. While vRNP-associated monomeric FluPol supports transcription, ANP32-dependent asymmetric and ANP32-independent symmetric polymerase dimers mediate genome and antigenome (vRNA/cRNA) replication. How FluPol coordinates these distinct functional states to maintain the transcription-replication balance remains poorly understood. Here, we identify and characterize interdomain interactions within PB2 that govern the stability of the replication-competent conformation of FluPol. Specifically, a highly conserved salt bridge between PB2 Mid-E282 and NLS-K752 stabilizes the replicase conformation, while the charge state of residue 627 (E or K) dictates ANP32 engagement, together coordinating polymerase dimerization and replication activity. Strikingly, bat influenza A viruses H17N10 and H18N11 harbor unconventional serine substitutions at both positions, compromising polymerase dimerization, ANP32 interaction, and vRNA/cRNA replication. Remarkably, introducing these bat-signature substitutions (E282S/K627S) into human H1N1 polymerase results in phosphorylation at both positions in human cells. These findings suggest that phosphorylation can partially compensate for the loss of negative charge at these residues, revealing a phosphomimetic adaptation mechanism that modulates polymerase conformational dynamics and function. Collectively, our study reveals how a conserved interdomain interaction within PB2 regulates conformational transitions and transcription-replication balance of FluPol, which might have been driven by phosphomimetic adaptations during the course of a diverse range of influenza virus evolution.