Backtracking of influenza polymerase upon consecutive incorporation of nucleoside analogue T1106 directly observed by high resolution cryo-electron microscopy

The broad-spectrum antiviral pseudobase T705, a fluorinated pyrazinecarboxamide, is incorporated via its triphosphate form into nascent viral RNA by viral RNA-dependent RNA polymerases. Since it mimics guanine or adenine it can act as a mutagen, whereas consecutive incorporation leads to chain termination. Here we examine the structural basis for incorporation and stalling for the case of influenza polymerase, using T1106-TP, the nucleotide form of T1105, the de-fluoro analogue of T705. We used a specially designed template that allows single T1106-MP incorporation at a defined site followed by consecutive T1106-MP incorporation and stalling four nucleotides later, as demonstrated by biochemical analysis. A high-resolution cryoEM structure of influenza A/H7N9 polymerase, stalled after transcribing this template, revealed that the entire product-template duplex has backtracked by five nucleotides. Consequently, the singly incorporated T1106-MP resides at the +1 position and forms an unexpected wobble base-pair with a U in the template. The relative stability of the canonical and wobble T1106:U base-pairs in different contexts is investigated by molecular dynamics simulations. Using a different template and influenza B polymerase we also observe stalling after double incorporation of T1106-MP and structural analysis showed again that backtracking occurs, this time by four nucleotides. These results show that, at least in early elongation, consecutive T1106-MP incorporation into the product destabilises the proximal end of the product-template duplex, promoting irreversible backtracking until a more favourable overall configuration is achieved. These results give new insight into the unusual mechanism of chain termination by pyrazinecarboxamide base analogues.