Fig. 7: TRAF6 interacts with the ATPase domains of PCV2 ORF1 to modulate its ubiquitination and degradation. | Communications Biology

Fig. 7: TRAF6 interacts with the ATPase domains of PCV2 ORF1 to modulate its ubiquitination and degradation.

From: Retinol binding protein 4 restricts PCV2 replication via selective autophagy degradation of viral ORF1 protein

Fig. 7

a Schematic of PCV2 ORF1 domains and mutants. ED (amino acids 1–118) indicates the N-terminal endonuclease ___domain, OD (amino acids 119–157) indicates the oligomerization ___domain, and AD (amino acids 158–314) is the C-terminal ATPase ___domain. The ORF1-∆OD mutant was generated by deletion of the OD ___domain. b Immunoblotting analysis of TRAF6 and PCV2 ORF1 in immunoprecipitated samples or whole-cell lysates of 3D4/21 cells transfected with GFP-pTRAF6 and empty vector, full-length PCV2 ORF1 Myc-ORF1-FL (FL), or truncated PCV2 ORF1 Myc-ORF1-ED (ED), Myc-ORF1-AD (AD), or Myc-ORF1-∆OD (∆OD). Anti-Myc immunoprecipitates were analyzed by immunoblotting with anti-GFP or anti-Myc antibody as indicated. Levels of the transfected proteins were analyzed by immunoblotting with anti-GFP or anti-Myc antibody. c Colocalization of TRAF6 and full-length PCV2 ORF1 or truncated ORF1. 3D4/21 cells were transfected with GFP-pTRAF6 and full-length or truncated PCV2 ORF1 plasmids for 24 h before confocal microscopy. Scale bar, 10 μm. d Immunoblotting analysis of SQSTM1 and PCV2 ORF1 in immunoprecipitated samples or whole-cell lysates of 3D4/21 cells transfected with empty vector, full-length or truncated PCV2 ORF1. Anti-Myc immunoprecipitates were analyzed by immunoblotting with an anti-SQSTM1 antibody or anti-Myc antibody as indicated. Levels of the transfected proteins were analyzed by immunoblotting with an anti-Myc or anti-SQSTM1 antibody. e Immunoprecipitation analysis of 3D4/21 cells expressing His-Ub with full-length or truncated PCV2 ORF1. Anti-Myc immunoprecipitates were analyzed by immunoblotting with an anti-His or anti-Myc antibody. Levels of the transfected proteins were analyzed by immunoblotting with anti-Myc antibody as indicated. f Immunoprecipitation analysis of 3D4/21 cells expressing His-Ub, GFP-pTRAF6 together with wild-type (WT) PCV2 ORF1 or ORF1 mutants (K4, K5, K15, K30, K31, K66, K67, K72, K74, K85, K87, K94, and K99 only; or Lys-to-Arg mutants of all lysines in ORF1 ED ___domain, AKR) as indicated. Anti-Myc immunoprecipitates were analyzed by immunoblotting with an anti-His or anti-Myc antibody. Levels of the transfected proteins were analyzed by immunoblotting with anti-Myc or anti-GFP antibody as indicated. g Immunoprecipitation analysis of 3D4/21 cells expressing His-Ub-K63, GFP-pTRAF6 together with WT PCV2 ORF1-ED or ORF1-ED mutants (K15, K66, K67, K94, K99 only or AKR) as indicated. Anti-Myc immunoprecipitates were analyzed by immunoblotting with an anti-His or anti-Myc antibody. Levels of the transfected proteins were analyzed by immunoblotting with anti-Myc or anti-GFP antibody as indicated. Data are representative of three (b–d) or two (e–g) independent experiments.

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