Department of Microbiology and Molecular Medicine, Medical Faculty, University of Geneva, Geneva, Switzerland.
Bioimaging Core Facility, Medical Faculty, University of Geneva, Geneva, Switzerland.
J Virol. 2024 May 14;98(5):e0041124. doi: 10.1128/jvi.00411-24. Epub 2024 Apr 3.
Influenza A virus infection activates the NLRP3 inflammasome, a multiprotein signaling complex responsible for the proteolytic activation and release of the proinflammatory cytokine IL-1β from monocytes and macrophages. Some influenza A virus (IAV) strains encode a short 90-amino acid peptide (PB1-F2) on an alternative open reading frame of segment 2, with immunomodulatory activity. We recently demonstrated that contemporary IAV PB1-F2 inhibits the activation of NLRP3, potentially by NEK7-dependent activation. PB1-F2 binds to NLRP3 with its C-terminal 50 amino acids, but the exact binding motif was unknown. On the NLRP3 side, the interface is formed through the leucine-rich-repeat (LRR) domain, potentially in conjunction with the pyrin domain. Here, we took advantage of PB1-F2 sequences from IAV strains with either weak or strong NLRP3 interaction. Sequence comparison and structure prediction using Alphafold2 identified a short four amino acid sequence motif (TQGS) in PB1-F2 that defines NLRP3-LRR binding. Conversion of this motif to that of the non-binding PB1-F2 suffices to lose inhibition of NLRP3 dependent IL-1β release. The TQGS motif further alters the subcellular localization of PB1-F2 and its colocalization with NLRP3 LRR and pyrin domain. Structural predictions suggest the establishment of additional hydrogen bonds between the C-terminus of PB1-F2 and the LRR domain of NLRP3, with two hydrogen bonds connecting to threonine and glutamine of the TQGS motif. Phylogenetic data show that the identified NLRP3 interaction motif in PB1-F2 is widely conserved among recent IAV-infecting humans. Our data explain at a molecular level the specificity of NLRP3 inhibition by influenza A virus.
Influenza A virus infection is accompanied by a strong inflammatory response and high fever. The human immune system facilitates the swift clearance of the virus with this response. An essential signal protein in the proinflammatory host response is IL-1b. It is released from inflammatory macrophages, and its production and secretion depend on the function of NLRP3. We had previously shown that influenza A virus blocks NLRP3 activation by the expression of a viral inhibitor, PB1-F2. Here, we demonstrate how this short peptide binds to NLRP3 and provide evidence that a four amino acid stretch in PB1-F2 is necessary and sufficient to mediate this binding. Our data identify a new virus-host interface required to block one signaling path of the innate host response against influenza A virus.
甲型流感病毒感染会激活 NLRP3 炎性小体，这是一种负责从单核细胞和巨噬细胞中蛋白水解激活和释放促炎细胞因子 IL-1β的多蛋白信号复合物。一些甲型流感病毒（IAV）株在第 2 节的替代开放阅读框上编码一个短的 90 个氨基酸肽（PB1-F2），具有免疫调节活性。我们最近证明，当代 IAV PB1-F2 通过 NEK7 依赖性激活来抑制 NLRP3 的激活。PB1-F2 通过其 C 末端的 50 个氨基酸与 NLRP3 结合，但确切的结合基序尚不清楚。在 NLRP3 方面，该接口通过富含亮氨酸重复（LRR）结构域形成，可能与吡喃结构域结合。在这里，我们利用具有弱或强 NLRP3 相互作用的 IAV 株的 PB1-F2 序列。使用 Alphafold2 进行序列比较和结构预测，确定了 PB1-F2 中一个短的四氨基酸序列基序（TQGS），该基序定义了 NLRP3-LRR 结合。将该基序转换为非结合的 PB1-F2 足以丧失对 NLRP3 依赖性 IL-1β释放的抑制作用。TQGS 基序进一步改变了 PB1-F2 的亚细胞定位及其与 NLRP3 LRR 和吡喃结构域的共定位。结构预测表明，在 PB1-F2 的 C 末端和 NLRP3 的 LRR 结构域之间建立了另外两个氢键，其中两个氢键连接到 TQGS 基序中的苏氨酸和谷氨酰胺。系统发育数据显示，在最近感染人类的 IAV 中，PB1-F2 中的鉴定出的 NLRP3 相互作用基序广泛保守。我们的数据从分子水平解释了流感病毒对 NLRP3 抑制的特异性。
甲型流感病毒感染伴随着强烈的炎症反应和高热。人体免疫系统通过这种反应迅速清除病毒。在炎症反应中，IL-1b 是宿主产生的一种重要信号蛋白。它从炎症性巨噬细胞中释放出来，其产生和分泌依赖于 NLRP3 的功能。我们之前已经表明，流感病毒通过表达一种病毒抑制剂 PB1-F2 来阻断 NLRP3 的激活。在这里，我们展示了这种短肽如何与 NLRP3 结合，并提供了证据表明 PB1-F2 中的四个氨基酸片段足以介导这种结合。我们的数据确定了一个新的病毒-宿主界面，该界面对于阻断宿主对流感病毒的先天免疫反应的一条信号通路是必需的。
