Unité de Virologie et Immunologie moléculaires, INRAE, Université Paris-Saclay, Jouy-en-Josas, France.
Institut de biologie structurale, CNRS, Université de Grenoble, Grenoble, France.
PLoS Pathog. 2024 Jun 14;20(6):e1011642. doi: 10.1371/journal.ppat.1011642. eCollection 2024 Jun.
Influenza viruses transcribe and replicate their genome in the nucleus of the infected cells, two functions that are supported by the viral RNA-dependent RNA-polymerase (FluPol). FluPol displays structural flexibility related to distinct functional states, from an inactive form to conformations competent for replication and transcription. FluPol machinery is constituted by a structurally-invariant core comprising the PB1 subunit stabilized with PA and PB2 domains, whereas the PA endonuclease and PB2 C-domains can pack in different configurations around the core. To get insights into the functioning of FluPol, we selected single-domain nanobodies (VHHs) specific of the influenza A FluPol core. When expressed intracellularly, some of them exhibited inhibitory activity on type A FluPol, but not on the type B one. The most potent VHH (VHH16) binds PA and the PA-PB1 dimer with an affinity below the nanomolar range. Ectopic intracellular expression of VHH16 in virus permissive cells blocks multiplication of different influenza A subtypes, even when induced at late times post-infection. VHH16 was found to interfere with the transport of the PA-PB1 dimer to the nucleus, without affecting its handling by the importin β RanBP5 and subsequent steps in FluPol assembly. Using FluPol mutants selected after passaging in VHH16-expressing cells, we identified the VHH16 binding site at the interface formed by PA residues with the N-terminus of PB1, overlapping or close to binding sites of two host proteins, ANP32A and RNA-polymerase II RPB1 subunit which are critical for virus replication and transcription, respectively. These data suggest that the VHH16 neutralization is likely due to several activities, altering the import of the PA-PB1 dimer into the nucleus as well as inhibiting specifically virus transcription and replication. Thus, the VHH16 binding site represents a new Achilles' heel for FluPol and as such, a potential target for antiviral development.
流感病毒在感染细胞的细胞核中转录和复制其基因组，这两个功能由病毒 RNA 依赖性 RNA 聚合酶（FluPol）支持。FluPol 显示出与不同功能状态相关的结构灵活性，从无活性形式到具有复制和转录能力的构象。FluPol 机制由一个结构不变的核心组成，该核心包含 PB1 亚基，该亚基与 PA 和 PB2 结构域稳定，而 PA 内切酶和 PB2 C 结构域可以围绕核心以不同的构象包装。为了深入了解 FluPol 的功能，我们选择了针对流感 A 型 FluPol 核心的单域纳米抗体（VHH）。当在细胞内表达时，其中一些对 A 型 FluPol 具有抑制活性，但对 B 型 FluPol 没有。最有效的 VHH（VHH16）以低于纳摩尔范围的亲和力结合 PA 和 PA-PB1 二聚体。在病毒允许的细胞中外源表达 VHH16 可阻止不同流感 A 亚型的增殖，即使在感染后晚期诱导时也是如此。发现 VHH16 干扰 PA-PB1 二聚体向细胞核的转运，而不影响其被 importin β RanBP5 处理以及 FluPol 组装的后续步骤。使用在表达 VHH16 的细胞中传代后选择的 FluPol 突变体，我们确定了 VHH16 结合位于 PA 残基与 PB1 N 端形成的界面上的结合位点，该结合位点与两个宿主蛋白的结合位点重叠或接近，这两个宿主蛋白是病毒复制和转录所必需的，分别为 ANP32A 和 RNA 聚合酶 II RPB1 亚基。这些数据表明，VHH16 的中和可能是由于多种活性引起的，这些活性改变了 PA-PB1 二聚体进入细胞核的导入，并且特异性抑制病毒转录和复制。因此，VHH16 结合位点代表 FluPol 的新阿喀琉斯之踵，因此是抗病毒药物开发的潜在靶标。
