High-resolution structure of a replication-initiation like configuration of influenza polymerase active site visualises the essential role of a conserved dibasic motif in the PA subunit

Influenza polymerase, comprising subunits PA, PB1 and PB2, transcribes the negative-sense genomic viral RNA (vRNA) into mRNA or replicates it first into complementary RNA (cRNA) and then back to vRNA. Here we investigate the mechanism of de novo (unprimed) initiation of vRNA to cRNA replication. We present a high-resolution structure of A/little-yellow-shouldered-bat/H17N10 polymerase with the 3’ end of the template in the RNA synthesis active site, both in the apo-state and after soaking with GTP and CTP. The priming GTP and incoming CTP are observed to base-pair to template nucleotides C2 and G3 at the -1 and +1 positions respectively, thus representing a replication initiation-like state. This configuration is stabilised by partial stacking of the tip of the priming loop on the GTP:C2 base-pair and the interaction of PB1/H649 and dibasic motif residues PA/R658 and K659 with the triphosphate of the priming GTP. The dibasic motif is universally conserved in orthomyxovirus PA subunits. Trans-complementation assays in cells using mutants of PA/K659 show that the dibasic motif is specifically essential for replication. These results shed light on the mechanism of replication initiation even though vRNA to cRNA replication is expected to be terminally initiated, with priming ATP and incoming GTP base-pairing to template nucleotides U1 and C2 at the -1 and +1 positions respectively, implying a different position of the template.