Direct observation of backtracking by influenza A and B polymerases upon consecutive incorporation of the nucleoside analog T1106

The antiviral pseudo-base T705 and its de-fluoro analog T1106 mimic adenine or guanine and can be compet-itively incorporated into nascent RNA by viral RNA-dependent RNA polymerases. Although dispersed, single pseudo-base incorporation is mutagenic, consecutive incorporation causes polymerase stalling and chain termination. Using a template encoding single and then consecutive T1106 incorporation four nucleotides later, we obtained a cryogenic electron microscopy structure of stalled influenza A/H7N9 polymerase. This shows that the entire product-template duplex backtracks by 5 nt, bringing the singly incorporated T1106 to the +1 position, where it forms an unexpected T1106:U wobble base pair. Similar structures show that influ-enza B polymerase also backtracks after consecutive T1106 incorporation, regardless of whether prior single incorporation has occurred. These results give insight into the unusual mechanism of chain termination by pyr-azinecarboxamide base analogs. Consecutive incorporation destabilizes the proximal end of the product -template duplex, promoting irreversible backtracking to a more energetically favorable overall configuration.

Automated summary

The antiviral pseudo-base T705 and its de-fluoro analog T1106 can compete with adenine or guanine in viral RNA-dependent RNA polymerases. Single incorporation of these analogs is mutagenic, while consecutive incorporation causes polymerase stalling and chain termination. Cryogenic electron microscopy shows that the product-template duplex backtracks after consecutive T1106 incorporation, which sheds light on the mechanism of chain termination by pyrazinecarbox-amide base analogs.