IPEC-J2 autophagy induced by TLR4 and NSP6 interaction facilitate porcine epidemic diarrhea virus replication

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We attempted to reveal how PEDV and the functional domain of the viral protein NSP6 mediate autophagy during infection and how PEDV exploits the cellular autophagic machinery for replication. We report the receptor that is necessary for PEDV-induced autophagy as determined using RNA sequencing. In addition, we identified the functional sites and signaling pathways of PEDV NSP6-induced autophagy, providing a theoretical basis for understanding the pathogenesis of PEDV and controlling viral infection. FIG 1 PEDV infection in IPEC-J2 cells induced autophagy. (a-b) Western blot was used to detect changes in the expression of LC3-II, p62, and PEDV-N proteins in PEDV-infected IPEC-J2 cells at 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h and 48 h post-infection.(c) Quantitative analysis of LC3-II and β-actin. (d) Quantitative analysis of P62a and β-actin. (e) Quantitative analysis of PEDV-N and β-actin. (f) PEDV-N gene copy number analysis. FIG 2 Autophagosome observed using TEM. (a) Cell control group and magnified autophagosome structure in the control group. (b) PEDV-infected cells and magnified autophagosome structure in PEDV-infected group. FIG 3 The role of autophagy in PEDV replication. (a) Expression levels of LC3-II and p62 in IPEC-J2 cells pretreated with different concentrations of chloroquine. (b) Expression level of LC3-II in IPEC-J2 cells treated with different concentrations of insulin. (c) Expression level of LC3-II in IPEC-J2 cells treated with different concentrations of rapamycin. The control group was normal cells. (d) Autophagic flow was detected using GFP-mCherry-LC3 dual fluorescence labeling. FIG 4 Statistics, volcano plot and pattern clustering heat maps of differentially expressed mRNAs. FIG 5 PEDV N gene expression in TLR4-/-, TFRC-/-, and GABRG3-/- IPEC-J2 cells. *(P < 0.05). FIG 6 The effects of TLR4-/-, TFRC-/-, and GABRG3-/- on autophagy as assessed using western blotting. FIG 7 Analysis of AKT-mTOR phosphorylation using western blotting. FIG 8 Analysis of LC3-II and p62 protein levels by western blotting. FIG 9 Autophagic flow was detected using GFP-mCherry-LC3 dual fluorescence labeling. FIG 10 Analysis of LC3-II and p62 expression in IPEC-J2 cells using western blotting. FIG 11 Autophagic flow was detected using GFP-mCherry-LC3 dual fluorescence labeling. FIG 12 Analysis of AKT-mTOR signaling pathway activation using western blotting. FIG 13 Detection of TLR4 and NSP61-2C co-localization using indirect immunofluorescent assay FIG 14. Molecular mechanism model diagram of autophagy induced by PEDV. Figure S1. GO enrichment analysis of the differentially expressed mRNAs. Figure S2. KEGG pathway analysis of the differentially expressed mRNAs.

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FIG 1 PEDV infection in IPEC-J2 cells induced autophagy. (a-b) Western blot was used to detect changes in the expression of LC3-II, p62, and PEDV-N proteins in PEDV-infected IPEC-J2 cells at 6 h, 12 h, 18 h, 24 h, 30 h, 36 h, 42 h and 48 h post-infection.(c) Quantitative analysis of LC3-II and β-actin. (d) Quantitative analysis of P62a and β-actin. (e) Quantitative analysis of PEDV-N and β-actin. (f) PEDV-N gene copy number analysis. FIG 2 Autophagosome observed using TEM. (a) Cell control group and magnified autophagosome structure in the control group. (b) PEDV-infected cells and magnified autophagosome structure in PEDV-infected group. FIG 3 The role of autophagy in PEDV replication. (a) Expression levels of LC3-II and p62 in IPEC-J2 cells pretreated with different concentrations of chloroquine. (b) Expression level of LC3-II in IPEC-J2 cells treated with different concentrations of insulin. (c) Expression level of LC3-II in IPEC-J2 cells treated with different concentrations of rapamycin. The control group was normal cells. (d) Autophagic flow was detected using GFP-mCherry-LC3 dual fluorescence labeling. FIG 4 Statistics, volcano plot and pattern clustering heat maps of differentially expressed mRNAs. FIG 5 PEDV N gene expression in TLR4-/-, TFRC-/-, and GABRG3-/- IPEC-J2 cells. *(P < 0.05). FIG 6 The effects of TLR4-/-, TFRC-/-, and GABRG3-/- on autophagy as assessed using western blotting. (a) The changes in protein expression of LC3-II, PEDV-N, and p62 in PEDV-J2-infected IPEC-J2 cells treated with siRNA as detected using western blotting. (b) Quantitative analysis of LC3-II and β-actin. (c) Quantitative analysis of p62 and β-actin. (d) Quantitative analysis of PEDV-N and β-actin. FIG 7 Analysis of AKT-mTOR phosphorylation using western blotting. FIG 8 Analysis of LC3-II and p62 protein levels by western blotting. FIG 9 Autophagic flow was detected using GFP-mCherry-LC3 dual fluorescence labeling. FIG 10 Analysis of LC3-II and p62 expression in IPEC-J2 cells using western blotting. FIG 11 Autophagic flow was detected using GFP-mCherry-LC3 dual fluorescence labeling. FIG 12 Analysis of AKT-mTOR signaling pathway activation using western blotting. FIG 13 Detection of TLR4 and NSP61-2C co-localization using indirect immunofluorescent assay FIG 14. Molecular mechanism model diagram of autophagy induced by PEDV.

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