Classical swine fever virus hijacks ESCRT-III and VPS4A to promote phagophore closure for accelerating mitophagy

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 A₁; 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.