ABSTRACT

Influenza A virus (IAV) infection induces mitophagy, which is essential for the clearance of damaged mitochondria. Dysfunctional mitochondria can be selectively targeted by PINK1, which recruits PRKN/PARK2 and leads to subsequent mitochondrial sequestration within autophagosomes. The IAV PB1-F2 protein translocates to mitochondria, accelerates the mitochondrial fragmentation and impairs the innate immunity. However, whether PB1-F2 mediates IAV–induced mitophagy and the relation between mitophagy and PB1-F2-attenuated innate immunity remain obscure. Here, we showed that PB1-F2 translocated to mitochondria by interacting and colocalizing with TUFM (Tu translation elongation factor, mitochondrial). Further studies revealed that PB1-F2 induced complete mitophagy, which required the interactions of PB1-F2 with both TUFM and MAP1LC3B/LC3B that mediated the autophagosome formation. PB1-F2-induced mitophagy was critical for the MAVS (mitochondrial antiviral signaling protein) degradation and led to its suppression of the type I IFN production. Importantly, the C-terminal LIR motif of PB1-F2 protein was demonstrated to be essential for its mitophagy induction and attenuated innate immunity. In conclusion, PB1-F2-induced mitophagy strongly correlates with impaired cellular innate immunity, revealing it is a potential therapeutic target.

Abbreviations: BCL2L13: BCL2 like 13; BECN1: beclin 1; BNIP3L/Nix: BCL2 interacting protein 3 like; CQ: chloroquine; DDX58: DExD/H-box helicase 58; eGFP: enhanced green fluorescent protein; hpi: hours post infection; IAV: influenza A virus; IFN: interferon; IP: immunoprecipitation; LIR: LC3-interacting region; MAP1LC3B/LC3B: microtubule associated protein 1 light chain 3 beta; MAVS: mitochondrial antiviral signaling protein; MMP: mitochondrial membrane potential; MOI, multiplicity of infection; mRFP: monomeric red fluorescent protein; NBR1: NBR1 autophagy cargo receptor; NC: negative control; NLRP3: NLR family pyrin domain containing 3; PINK1: PTEN induced kinase 1; PRKN/PARK2: parkin RBR E3 ubiquitin protein ligase; RLR: RIG-I-like-receptor; ROS: reactive oxygen species; SEV: sendai virus; SQSTM1/p62: sequestosome 1; TAX1BP1: Tax1 binding protein 1; TM: transmembrane; TOMM20/40: translocase of outer mitochondrial membrane 20/40; TUFM: Tu translation elongation factor, mitochondrial.

摘要

甲型流感病毒 (IAV) 感染诱导线粒体自噬，这对于清除受损线粒体至关重要。PINK1 可以选择性地靶向功能失调的线粒体，它会招募 PRKN/PARK2 并导致随后的自噬体内的线粒体隔离。IAV PB1-F2 蛋白易位到线粒体，加速线粒体断裂并损害先天免疫。然而，PB1-F2 是否介导 IAV 诱导的线粒体自噬以及线粒体自噬与 PB1-F2 减弱的先天免疫之间的关系仍不清楚。在这里，我们表明 PB1-F2 通过与 TUFM（Tu 翻译延伸因子，线粒体）相互作用和共定位而易位到线粒体。进一步的研究表明，PB1-F2 诱导了完全的线粒体自噬，这需要 PB1-F2 与介导自噬体形成的 TUFM 和 MAP1LC3B/LC3B 相互作用。PB1-F2 诱导的线粒体自噬对于 MAVS（线粒体抗病毒信号蛋白）降解至关重要，并导致其抑制 I 型干扰素的产生。重要的是，PB1-F2 蛋白的 C 端 LIR 基序被证明对其线粒体自噬诱导和减弱先天免疫至关重要。总之，PB1-F2 诱导的线粒体自噬与细胞先天免疫受损密切相关，表明它是一个潜在的治疗靶点。PB1-F2 蛋白的 C 端 LIR 基序被证明对其线粒体自噬诱导和先天免疫减弱至关重要。总之，PB1-F2 诱导的线粒体自噬与细胞先天免疫受损密切相关，表明它是一个潜在的治疗靶点。PB1-F2 蛋白的 C 端 LIR 基序被证明对其线粒体自噬诱导和先天免疫减弱至关重要。总之，PB1-F2 诱导的线粒体自噬与细胞先天免疫受损密切相关，表明它是一个潜在的治疗靶点。

缩写：BCL2L13：BCL2 像 13；BECN1: beclin 1; BNIP3L/Nix：BCL2 相互作用蛋白 3 样；CQ：氯喹；DDX58：DExD/H-box 解旋酶 58；eGFP：增强型绿色荧光蛋白；hpi：感染后的小时数；IAV：甲型流感病毒；干扰素：干扰素；IP：免疫沉淀；LIR：LC3 相互作用区；MAP1LC3B/LC3B：微管相关蛋白 1 轻链 3 β；MAVS：线粒体抗病毒信号蛋白；MMP：线粒体膜电位；MOI，感染复数；mRFP：单体红色荧光蛋白；NBR1：NBR1 自噬货物受体；NC：阴性对照；NLRP3：NLR家族pyrin结构域，含3个；PINK1：PTEN 诱导激酶 1；PRKN/PARK2：parkin RBR E3泛素蛋白连接酶；RLR：RIG-I-like-receptor；ROS：活性氧；SEV：仙台病毒；SQSTM1/p62：sequestosome 1；TAX1BP1：Tax1 结合蛋白 1；TM值：跨膜；TOMM20/40：线粒体外膜20/40的转位酶；TUFM：Tu 翻译延伸因子，线粒体。