Fig. 6: RBP4 activates TRAF6 to initiate K63-linked polyubiquitination of PCV2 ORF1. | Communications Biology

Fig. 6: RBP4 activates TRAF6 to initiate K63-linked polyubiquitination of PCV2 ORF1.

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

Fig. 6

a Immunoprecipitation analysis of WT and RBP4-KO 3D4/21 cells transfected with PCV2 ORF1 and His-Ubiquitin (His-Ub) or His-Ub at K6 (His-Ub-K6), K11 (His-Ub-K11), K27 (His-Ub-K27), K29 (His-Ub-K29), K33 (His-Ub-K33), K48 (His-Ub-K48) or K63 (His-Ub-K63) only for 24 h. b Immunoprecipitation analysis of RBP4-KO 3D4/21 cells expressing PCV2 ORF1 and His-Ub, His-Ub-K48, His-Ub-K63, His-Ub-K48R, or His-Ub-K63R as indicated. Cells were left untreated or stimulated with rpRBP4 protein for 6 h before harvest. c Immunoprecipitation analysis of 3D4/21 cells expressing GFP-TRAF6 together with empty vector (EV), PCV2 ORF1, PCV3 ORF1, or PCV4 ORF1 as indicated. Anti-GFP immunoprecipitates were analyzed by immunoblotting with anti-Myc antibody. Levels of the transfected proteins were analyzed by immunoblotting with anti-Myc and anti-GFP antibodies. d Colocalization of exogenous TRAF6 and PCV2 ORF1 in 3D4/21 cells. Cells were transfected with GFP-TRAF6 and PCV2 ORF1 for 24 h before confocal microscopy. Scale bar, 10 μm. e GST pull-down analysis of the interaction between His-ORF1 and GST, GST-SQSTM1/p62, GST-LC3, or GST-TRAF6 as indicated. Recombinant proteins were pulled down by GST magnetic beads and were analyzed by immunoblotting with anti-His or anti-GST antibodies (upper). Recombinant proteins in the assay were examined by SDS–PAGE and coomassie blue staining (lower). f Schematic drawings of the TRAF6-binding motif Pro-X-Glu-X-X-Ar/Ac. The presence of the putative Pro-X-Glu-X-X-Ar/Ac motifs in ORF1 of PCV2 and other representative circoviruses (PCV3, KX778720.1(Genebank number); PCV4, MK986820.1; PCV1, U49186.1; HuCV2, ON226770.2; GoCV, MT831941.1; DuCV, MN078101.1; and CaCV, JQ821392.1) were analyzed and consistent amino acids are indicated in color. g Immunoprecipitation analysis of the association of PCV2 ORF1 and TRAF6 in 3D4/21 cells transfected with GFP-TRAF6 and wild-type PCV2 ORF1 (WT), or PCV2 ORF1 mutants (P309T, E311A, or P309T/E311A). 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. h Immunoprecipitation analysis of 3D4/21 cells expressing PCV2 ORF1 and His-Ub, His-Ub-K6, His-Ub-K11, His-Ub-K27, His-Ub-K29, His-Ub-K33, His-Ub-K48 or His-Ub-K63 together with or without GFP-TRAF6 as indicated. i Immunoblotting analysis of the indicated proteins in immunoprecipitated samples and whole-cell lysates of RBP4-KO 3D4/21 cells transfected with TRFA6 siRNA or control siRNA (100 nM). Twenty-four hours after transfection, cells were further transfected with PCV2 ORF1 and His-Ub (left) or His-Ub-K63 (right) for 24 h. Cells were then stimulated with rpRBP4 (30 μg/mL) for 6 h before analysis. Data are representative of three (c–g) or two (a, b, h, i) independent experiments.

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