Journal: Journal of Virology

Article Title: Palmitoylation enhances the stability of porcine epidemic diarrhea virus spike protein by antagonizing its degradation via chaperone-mediated autophagy to facilitate viral proliferation

doi: 10.1128/jvi.00347-25

Figure Lengend Snippet: Palmitoylation of PEDV S protein enhances its stability. ( A ) Vero cells were transfected with the plasmid encoding PEDV S-Flag or S-ΔCRR. The cells were treated with or without 20 µM 2-BP for 36 h. The mRNA abundance of S protein was quantified by RT-qPCR. Statistical analysis was carried out using Student’s t -test. ns, not significant ( P > 0.05). ( B ) Vero cells were transfected with the plasmid encoding PEDV S-Flag and were then treated with 2-BP (5 µM, 10 µM, and 20 µM) or DMSO for 36 h. The S protein levels were measured by IB. ( C ) Vero cells were transfected with the plasmid encoding PEDV S-Flag, S-ΔCRR, or Flag-tagged empty vector for 36 h. The S protein levels were measured by IB. ( D ) Vero cells were transfected with si- ZDHHC5 or si-NC for 12 h and were then transfected with the plasmid encoding PEDV S-Flag or Flag-tagged empty vector for 24 h. The S protein levels were measured by IB. ( E ) Vero cells were co-transfected with the plasmids encoding S-Flag and ZDHHC5-Myc (0.5 µg, 1 µg, and 2 µg) for 36 h. The S protein levels were measured by IB. ( F ) Vero cells were transfected with the plasmid encoding PEDV S-Flag or S-ΔCRR. The S-Flag-overexpressed cells were treated with or without 20 µM 2-BP for 24 h. Then, the cells were stimulated with CHX (1 µg/mL) and lysed at the indicated time points (0 h, 2 h, 4 h, 6 h, and 8 h). The samples were analyzed by IB. ( G ) Vero cells were transfected with the plasmid encoding PEDV S-Flag or Flag-tagged empty vector. The cells were treated with DMSO or 20 µM 2-BP, and then co-incubated with DMSO, 3-MA (2 mM), carfilzomib (20 nM), NH 4 Cl (0.5 mM), or CQ (40 µM) for 36 h. The cells were lysed and analyzed by IB. ( H ) Vero cells were transfected with the plasmid encoding PEDV S-Flag, S-ΔCRR, or Flag-tagged empty vector. The cells were treated with DMSO, 3-MA (2 mM), carfilzomib (20 nM), NH 4 Cl (0.5 mM), or CQ (40 µM) for 36 h. The cells were lysed and analyzed by IB. ( I ) Vero cells were transfected with the plasmid encoding PEDV S-Flag, S-ΔCRR, or Flag-tagged empty vector. At 6 h post-transfection, the S-Flag-overexpressed groups were treated with or without 20 µM 2-BP, and all the cells were treated with NH 4 Cl (0.5 mM) for 24 h. The lysosomes were isolated from these cells and analyzed by IB. The mean gray values of S protein were quantified using ImageJ software.

Article Snippet: TransIntro PL transfection reagent (Cat. No. FT301-01) was purchased from TransGen Biotech.

Techniques: Transfection, Plasmid Preparation, Quantitative RT-PCR, Incubation, Isolation, Software

Journal: Journal of Virology

Article Title: Palmitoylation enhances the stability of porcine epidemic diarrhea virus spike protein by antagonizing its degradation via chaperone-mediated autophagy to facilitate viral proliferation

doi: 10.1128/jvi.00347-25

Figure Lengend Snippet: The KFERQ-like motif (QVDRL) in PEDV S protein is recognized and bound by HSC70. ( A ) Vero cells were transfected with the plasmid encoding PEDV S-ΔCRR or Flag-tagged empty vector for 36 h. The proteins were immunoprecipitated in WCL using anti-Flag magnetic beads, and then the associated proteins were separated by 7.5% SDS-PAGE and stained with silver. The black arrow indicated the expressed S-ΔCRR, and the red arrow indicated a different immunoprecipitated protein band in the S-ΔCRR-expressed cells. Lane M, protein marker. ( B ) The silver-stained protein band indicated by the red arrow was subjected to LC-MS/MS. ( C ) HEK-293T cells were co-transfected with the plasmids encoding PEDV S-Flag and HSC70-Myc, with Flag-tagged or Myc-tagged empty vector as control for 36 h. The supernatant of WCL was immunoprecipitated using anti-Flag magnetic beads or anti-Myc magnetic beads. The precipitated proteins were analyzed by IB. ( D ) Vero cells were co-transfected with the plasmids encoding PEDV S-Flag and HSC70-Myc for 24 h. The cells were visualized with the specific primary and secondary antibodies. Cell nuclei were stained with DAPI. Images were taken at a 630× magnification and representative of a single slice of a stack from three independent experiments. Representative images are shown. Scale bars, 10 µm. The co-localization was assessed by determination of the Pearson’s correlation coefficient using the JaCoP plugin in ImageJ software. The mean value ± SEM is representative of three individual enlarged pictures. ( E ) Analyses of KFERQ-like motifs in PEDV S proteins. The KFERQ-like motif typically consists of constant glutamine (Q) flanked on one side of the pentapeptide sequence, followed by one or two positively charged amino residues (lysine, [K] or arginine, [R]), one or two hydrophobic amino residues (phenylalanine, [F]; isoleucine, [I]; leucine, [L]; or valine, [V]), and one negatively charged amino residue (aspartate, [D] and glutamic acid, [E]) . ( F ) HEK-293T cells were co-transfected with the plasmids encoding HSC70-Myc and PEDV S-Flag, S Q1082A , S Q1117A , S Q1082A/Q1117A , or Flag-tagged empty vector. At 36 h post-transfection, the supernatant of WCL was immunoprecipitated using anti-Flag magnetic beads. The precipitated proteins were analyzed by IB.

Article Snippet: TransIntro PL transfection reagent (Cat. No. FT301-01) was purchased from TransGen Biotech.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Magnetic Beads, SDS Page, Staining, Marker, Liquid Chromatography with Mass Spectroscopy, Control, Software, Sequencing, Residue

Journal: Journal of Virology

Article Title: Palmitoylation enhances the stability of porcine epidemic diarrhea virus spike protein by antagonizing its degradation via chaperone-mediated autophagy to facilitate viral proliferation

doi: 10.1128/jvi.00347-25

Figure Lengend Snippet: Palmitoylation of PEDV S protein prevents its QVDRL motif from being recognized by HSC70. ( A ) HEK-293T cells were transfected with the plasmids encoding HSC70-Myc and PEDV S-Flag, S-ΔCRR, or Flag-tagged empty vector. At 6 h post-transfection, the S-Flag-overexpressed cells were treated with or without 40 µM 2-BP. After 36 h post-transfection, the supernatant of WCL was immunoprecipitated using anti-Flag magnetic beads, and the precipitated proteins were analyzed by IB. ( B ) Vero cells were transfected with the plasmid encoding PEDV S-Flag or S-ΔCRR. At 6 h post-transfection, the S-Flag-overexpressed groups were treated with or without 20 µM 2-BP. At 24 h post-transfection, S-Flag, S-ΔCRR, or HSC70 was visualized with the specific primary and secondary antibodies. Cell nuclei were stained with DAPI. Images were taken at a 630× magnification and representative of a single slice of a stack from three independent experiments. Representative images are shown. Scale bars, 10 µm. The co-localization was assessed by determination of the Pearson’s correlation coefficient using the JaCoP plugin in ImageJ software. The mean value ± SEM is representative of three individual enlarged pictures. Statistical analysis was carried out using Student’s t -test. ** P < 0.01. ( C ) HEK-293T cells were co-transfected with the plasmids encoding HSC70-Myc and PEDV S-Flag, S-ΔCRR, S Q1082A , S-ΔCRR Q1082A , or Flag-tagged empty vector. At 6 h post-transfection, the S-Flag-overexpressed and the S Q1082A -overexpressed cells were treated with or without 40 µM 2-BP. Subsequent assays were performed as described for panel A. (D through F) Vero cells were transfected with si- HSC70 or si-NC. At 12 h post-transfection, the cells were transfected with the plasmid encoding PEDV S-Flag, S-ΔCRR, or Flag-tagged empty vector, and treated with or without 20 µM 2-BP for another 24 h. The cells were lysed and analyzed by IB. The mean gray values of S protein and HSC70-Myc were quantified using ImageJ software.

Article Snippet: TransIntro PL transfection reagent (Cat. No. FT301-01) was purchased from TransGen Biotech.

Techniques: Transfection, Plasmid Preparation, Immunoprecipitation, Magnetic Beads, Staining, Software

Journal: Journal of Virology

Article Title: Palmitoylation enhances the stability of porcine epidemic diarrhea virus spike protein by antagonizing its degradation via chaperone-mediated autophagy to facilitate viral proliferation

doi: 10.1128/jvi.00347-25

Figure Lengend Snippet: Palmitoylation of PEDV S protein prevents its degradation via CMA. ( A ) Vero cells were transfected with the plasmid encoding PEDV S-Flag or S-ΔCRR. At 6 h post-transfection, the S-Flag-overexpressed cells were treated with or without 20 µM 2-BP and then treated with NH 4 Cl (0.5 mM). At 24 h post-transfection, S-Flag, S-ΔCRR, or LAMP2A was visualized with the specific primary and secondary antibodies. Cell nuclei were stained with DAPI. Images were taken at a 630× magnification and representative of a single slice of a stack from three independent experiments. Representative images are shown. Scale bars, 10 µm. The co-localization was assessed by determination of the Manders’ overlap coefficient using the JaCoP plugin in ImageJ software. The mean value ± SEM is representative of three individual enlarged pictures. Statistical analysis was carried out using Student’s t -test. ** P < 0.01; *** P < 0.001. (B through D) Vero cells were transfected with si- LAMP2A or si-NC. At 12 h post-transfection, the cells were transfected with the plasmid encoding PEDV S-Flag, S-ΔCRR, or Flag-tagged empty vector and treated with or without 20 µM 2-BP for another 24 h. The cells were lysed and analyzed by IB. The mean gray values of S protein were quantified using ImageJ software.

Article Snippet: TransIntro PL transfection reagent (Cat. No. FT301-01) was purchased from TransGen Biotech.

Techniques: Transfection, Plasmid Preparation, Staining, Software