European Molecular Biology Laboratory, Grenoble Outstation, 71 Avenue des Martyrs, CS 90181, 38042 Grenoble Cedex 9, France.
Nucleic Acids Res. 2018 Jan 25;46(2):956-971. doi: 10.1093/nar/gkx1210.
Influenza polymerase uses short capped primers snatched from nascent Pol II transcripts to initiate transcription of viral mRNAs. Here we describe crystal structures of influenza A and B polymerase bound to a capped primer in a configuration consistent with transcription initiation ('priming state') and show by functional assays that conserved residues from both the PB2 midlink and cap-binding domains are important for positioning the capped RNA. In particular, mutation of PB2 Arg264, which interacts with the triphosphate linkage in the cap, significantly and specifically decreases cap-dependent transcription. We also compare the configuration of the midlink and cap-binding domains in the priming state with their very different relative arrangement (called the 'apo' state) in structures where the potent cap-binding inhibitor VX-787, or a close analogue, is bound. In the 'apo' state the inhibitor makes additional interactions to the midlink domain that increases its affinity beyond that to the cap-binding domain alone. The comparison suggests that the mechanism of resistance of certain mutations that allow virus to escape from VX-787, notably PB2 N510T, can only be rationalized if VX-787 has a dual mode of action, direct inhibition of capped RNA binding as well as stabilization of the transcriptionally inactive 'apo' state.
流感聚合酶利用从新生 Pol II 转录本中抢夺的短加帽引物来启动病毒 mRNA 的转录。在这里，我们描述了与加帽引物结合的甲型和乙型流感聚合酶的晶体结构，其构象与转录起始一致（“引发状态”），并通过功能测定表明，来自 PB2 中链和帽结合结构域的保守残基对于定位加帽 RNA 很重要。特别是，与帽结合的 PB2 Arg264 的突变，与帽结合的三磷酸酯连接相互作用，显著且特异性地降低了帽依赖性转录。我们还将引发状态下的中链和帽结合结构域的构象与它们在结合强有效的帽结合抑制剂 VX-787 或其类似物时非常不同的相对排列（称为“apo”状态）进行了比较。在“apo”状态下，抑制剂与中链结构域发生额外的相互作用，使其亲和力超过单独与帽结合结构域的亲和力。这种比较表明，某些突变允许病毒逃避 VX-787 的机制，特别是 PB2 N510T，只有在 VX-787 具有双重作用模式的情况下才能得到合理的解释，即直接抑制加帽 RNA 结合以及稳定转录非活性的“apo”状态。
