European Molecular Biology Laboratory, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France.
Molecular Modeling & Drug Discovery Lab, Istituto Italiano di Tecnologia, Via Morego 30, 16163 Genova, Italy.
Cell Rep. 2023 Jan 31;42(1):111901. doi: 10.1016/j.celrep.2022.111901. Epub 2023 Jan 2.
The antiviral pseudo-base T705 and its de-fluoro analog T1106 mimic adenine or guanine and can be competitively 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 influenza 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 pyrazinecarboxamide base analogs. Consecutive incorporation destabilizes the proximal end of the product-template duplex, promoting irreversible backtracking to a more energetically favorable overall configuration.
抗病毒假碱基 T705 及其去氟类似物 T1106 模拟腺嘌呤或鸟嘌呤，可以被病毒 RNA 依赖性 RNA 聚合酶竞争性地掺入新生 RNA 中。尽管分散，但单个假碱基的掺入具有诱变作用，连续掺入会导致聚合酶停滞和链终止。使用编码单个 T1106 掺入的模板，然后在 4 个核苷酸后连续掺入 T1106，我们获得了停滞的流感 A/H7N9 聚合酶的低温电子显微镜结构。这表明整个产物-模板双链向后移动 5 个核苷酸，将单个掺入的 T1106 带到+1 位置，在那里它形成一个意想不到的 T1106:U 摆动碱基对。类似的结构表明，流感 B 聚合酶在连续掺入 T1106 后也会向后移动，无论之前是否发生了单个掺入。这些结果深入了解了嘧啶酰胺碱基类似物导致链终止的不寻常机制。连续掺入会使产物-模板双链的近端不稳定，从而促进不可逆的回溯到更有利的整体构象。
