TRIM8 inhibits porcine epidemic diarrhoea virus replication by targeting and ubiquitinately degrading the nucleocapsid protein

Figures

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  Figure 1. PEDV infection induces the expression of TRIM8 in IPEC-J2 cells. Relative mRNA A and protein B expression levels of TRIM8 at different time points (0, 12, 24, 48 h). hpi: hours post-infection. *P < 0.05, ** P < 0.01.

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  Figure 2. TRIM8 inhibits PEDV infection in host cells. Relative expression of PEDV M gene A and virus titres B in PEDV-infected TRIM8 knockout and wild-type cells. Relative expression of PEDV M gene C and virus titres D in PEDV-infected TRIM8 overexpression and wild-type cells. WT: wild-type cells; TRIM8-KO: TRIM8 knockout cells; TRIM8-OE: TRIM8 overexpression cells. *P < 0.05, **P < 0.01.

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  Figure 3. TRIM8 colocalises and interacts with PEDV N protein. A Co-IP of HEK293T cells expressing TRIM8 and HA-tagged PEDV N proteins. B Fluorescence co-localization of FLAG-tagged TRIM8 and HA-tagged N proteins in HEK293T cells. Staining was performed using DAPI, GFP, and RFP, respectively. Scale bars, 10 μm. C Prediction of the interaction domain between the PEDV N protein and TRIM8 by PyMOL software. Green and cyan indicates TRIM8 and PEDV N protein, respectively. D Key binding sites predicted for the interaction domain of PEDV N and TRIM8 proteins. Ribbon representations are coloured according to the proteins: TRIM8 (green) and PEDV N (cyan). E Co-IP of FLAG-tagged TRIM8 and its truncated mutants with HA-tagged PEDV N proteins in HEK293T cells. WT: full length of TRIM8; RB: RING and B box domains of TRIM8; Box: B box domain of TRIM8; CC: coiled coil domain of TRIM8.

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  Figure 4. TRIM8 catalyses PEDV N protein degradation via K48-linked ubiquitination. A Co-IP and IB analysis of HEK293T cells expressing HA-tagged PEDV N protein and increasing amounts of TRIM8. B IB analysis of cells expressing HA-tagged PEDVN with or without TRIM8 under CHX treatment for different time points (0, 9, 12, 15 h). CHX: Cycloheximide. C IB analysis of cells expressing HA-tagged PEDV N under the treatment of proteasome inhibitor MG132 for 12 h. D IB analysis of cells expressing HA-tagged PEDV N under the treatment of autophagy-lysosome inhibitor Leupeptin for 12 h. E Co-IP and IB analysis of MG132-treated cells expressing MYC-tagged PEDV N, with or without TRIM8, HA-tagged wild-type ubiquitin, mutant ubiquitin K48 and K63. WCL: whole cell lysates.

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  Figure 5. Changes in the transcriptomes of PEDV-infected TRIM8 overexpression and control cells. A Volcano plot of differentially expressed genes between PEDV-infected and uninfected cells. B Volcano plot of differentially expressed genes between PEDV-infected TRIM8 overexpression and wild-type cells. C-D GO annotation and KEGG enrichment analysis for differentially expressed genes are shown in A. E–F GO annotation and KEGG enrichment analysis for differentially expressed genes shown in B. G Intersected genes between different groups. Group A represents differentially expressed genes shown in A; Group B represents differentially expressed genes shown in B. H Expression profiles of the intersected genes shown in G.