Cheng Yan, Li Yuhang, Bi Xiaoqing, Chen Jinxia, Zhao Bingqian, Bai Jishan, Ye Yinbo, Dai Qi, Zou Linke, Chen Jing, Feng Xiuli, Zhou Bin
MOE Joint International Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agricultural University, Nanjing, China.
Key Laboratory of Animal Bacteriology, Ministry of Agriculture and Rural Affairs, Nanjing Agricultural University, Nanjing, China.
Autophagy. 2025 Jul 9:1-21. doi: 10.1080/15548627.2025.2523734.
Classical swine fever virus (CSFV) infection induces complete mitophagy, which is essential for the clearance of damaged mitochondria. The endosomal sorting complex required for transport (ESCRT) machinery plays a vital role in mediating phagophore closure and autophagosome-lysosome fusion during starvation-induced autophagy. Nevertheless, its involvement in CSFV-induced mitophagy and the underlying mechanisms remain insufficiently understood. Here, we found that the ESCRT-III subunits including CHMP1A, CHMP1B, and CHMP4B, along with the AAA-ATPase VPS4, were actively recruited to autophagosomes during CSFV-induced mitophagy. Consistent with this, depletion of CHMP1A, CHMP1B, CHMP4B or VPS4A disrupted mitophagic flux, impairing both PINK1-PRKN-dependent and -independent pathways. Further investigations revealed that CSFV transiently recruited these subunits to nascent autophagosomes for phagophore sealing during mitophagy. Remarkably, multiple CSFV nonstructural proteins (NSPs) including NS3, NS4B, NS5A and NS5B interacted with these ESCRT key subunits and colocalized on mitophagosomes. Taken together, our study identifies CHMP1A, CHMP1B, CHMP4B, and VPS4A as pivotal regulators of phagophore closure in CSFV-induced mitophagy, unveiling novel mechanisms by which the virus manipulates host cellular pathways and highlighting potential therapeutic targets for infection control.Abbreviation: ATF4: activating transcription factor 4; ATG5: autophagy related 5; BafA1: bafilomycin A; BFP: blue fluorescent protein; BNIP3L/NIX: BCL2 interacting protein 3like; BSA: bovine serum albumin; CALCOCO2/NDP52: calcium binding andcoiled-coil domain 2; CCCP: carbonyl cyanide 3-chlorophenylhydrazone; CHMP: charged multivesicular body protein; COX4: cytochrome c oxidase subunit 4; CSFV: classical swine fever virus; DAPI: 4',6-diamidino-2-phenylindole; DN: dominant-negative; ER: endoplasmic reticulum; ESCRT: endosomal sorting complex required for transport; FUNDC1: FUN14 domain containing 1; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; hpt: hours post-transfection; HSPD1/HSP60: heat shock protein family D (Hsp60) member 1; IB: immunoblotting; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFF: mitochondrial fission factor; MFN2: mitofusin 2; MITO: mitochondria; MOI: multiplicity of infection; mtDNA: mitochondrial DNA; OPTN: optineurin; PBS: phosphate-buffered saline; PINK1: PTEN induced kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; RAPA: rapamycin; RFP: redfluorescent protein; RT-qPCR: reverse transcription-quantitativereal-time polymerase chain reaction; RT-PCR: real-time polymerasechain reaction; SD: standard deviation; siCtrl: negative control siRNA; siRNA: small interfering RNA; SQSTM1/p62: sequestosome 1; TOMM20: translocase of outer mitochondrial membrane 20; VDAC1: voltage dependent anion channel 1; VPS4A: vacuolar protein sorting 4 homolog A; WCL: whole-cell lysate; WT: wild-type.
经典猪瘟病毒（CSFV）感染会诱导完全的线粒体自噬，这对于清除受损线粒体至关重要。运输所需的内体分选复合体（ESCRT）机制在饥饿诱导的自噬过程中，对介导吞噬泡封闭和自噬体-溶酶体融合起着至关重要的作用。然而，其在CSFV诱导的线粒体自噬中的参与情况及潜在机制仍未得到充分了解。在此，我们发现包括CHMP1A、CHMP1B和CHMP4B在内的ESCRT-III亚基，以及AAA-ATP酶VPS4，在CSFV诱导的线粒体自噬过程中被积极招募到自噬体上。与此一致的是，CHMP1A、CHMP1B、CHMP4B或VPS4A的缺失会破坏线粒体自噬通量，损害PINK1-PRKN依赖性和非依赖性途径。进一步的研究表明，CSFV在自噬过程中会短暂地将这些亚基招募到新生自噬体上进行吞噬泡密封。值得注意的是，包括NS3、NS4B、NS5A和NS5B在内的多种CSFV非结构蛋白（NSPs）与这些ESCRT关键亚基相互作用，并共定位于线粒体自噬体上。综上所述，我们的研究确定CHMP1A、CHMP1B、CHMP4B和VPS4A是CSFV诱导的线粒体自噬中吞噬泡封闭的关键调节因子，揭示了病毒操纵宿主细胞途径的新机制，并突出了感染控制的潜在治疗靶点。缩写：ATF4：激活转录因子4；ATG5：自噬相关5；BafA1：巴弗洛霉素A；BFP：蓝色荧光蛋白；BNIP3L/NIX：BCL2相互作用蛋白3样；BSA：牛血清白蛋白；CALCOCO2/NDP52：钙结合和卷曲螺旋结构域2；CCCP：羰基氰化物3-氯苯基腙；CHMP：带电荷的多囊泡体蛋白；COX4：细胞色素c氧化酶亚基4；CSFV：经典猪瘟病毒；DAPI：4',6-二脒基-2-苯基吲哚；DN：显性负性；ER：内质网；ESCRT：运输所需的内体分选复合体；FUNDC1：含FUN14结构域1；GAPDH：甘油醛-3-磷酸脱氢酶；GFP：绿色荧光蛋白；hpt：转染后小时数；HSPD1/HSP60：热休克蛋白家族D（Hsp60）成员1；IB：免疫印迹；MAP1LC3/LC3：微管相关蛋白1轻链3；MFF：线粒体裂变因子；MFN2：线粒体融合蛋白2；MITO：线粒体；MOI：感染复数；mtDNA：线粒体DNA；OPTN：视黄醛结合蛋白；PBS：磷酸盐缓冲盐水；PINK1：PTEN诱导激酶1；PRKN：帕金RBR E3泛素蛋白连接酶；RAPA：雷帕霉素；RFP：红色荧光蛋白；RT-qPCR：逆转录定量实时聚合酶链反应；RT-PCR：实时聚合酶链反应；SD：标准差；siCtrl：阴性对照小干扰RNA；siRNA：小干扰RNA；SQSTM1/p62：聚集体蛋白1；TOMM20：线粒体外膜转位酶20；VDAC1：电压依赖性阴离子通道1；VPS4A：液泡蛋白分选4同源物A；WCL：全细胞裂解物；WT：野生型
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