Fig 1: H7N9 PB1 facilitates RNF5 to ubiquitinate MAVS.(A) RNF5 catalyzes K27-linked ubiquitination of MAVS. HEK293 cells were transfected with Flag-MAVS, HA-ubiquitin, or its mutants (KO, K-only) together with a control and Myc-RNF5 plasmids for 20 h. The cells were then treated with 3-MA for 6 h before use in ubiquitination assays with the indicated antibodies. The intensities of the indicated protein bands were determined by using image J, were normalized to Flag-MAVS, and are shown as the fold-change of HA/Flag. (B) RNF5 deficiency inhibits SZ19-triggered K27-linked ubiquitination of MAVS. RNF5-/- and control cells were infected with SZ19 virus (MOI = 0.1) for 24 h. The cells were then treated with 3-MA for 6 h before use in ubiquitination assays with the indicated antibodies. The intensities of the indicated protein bands were determined by using image J, were normalized to MAVS, and are shown as the fold-change of the indicated Ub/MAVS. (C) K27R ubiquitin inhibits PB1-mediated K27-linked ubiquitination of MAVS. HEK293 cells were transfected with Myc-MAVS, Flag-K27O, and HA-K27R (which K27 lysine residues was mutated to arginine) together with a control and GFP-PB1 plasmids for 20 h. The cells were then treated with 3-MA for 6 h before use in ubiquitination assays with the indicated antibodies. The intensities of the indicated protein bands were determined by using image J, were normalized to Myc-MAVS, and are shown as the fold-change of Flag/Myc. (D) MAVS bearing the K362 and 461R mutations is resistant to PB1-mediated degradation. HEK293 cells were transfected with Myc-MAVS and the indicated mutants in the presence or absence of Flag-PB1 for 24 h before immunoblot analysis. (E) The effects of overexpression or knockout of RNF5 on SZ19-ΔF2 virus replication. HEK293 cells were transfected with control vector or RNF5 plasmid for 24 h. The cells were then infected with SZ19-ΔF2 virus (MOI = 0.01) for the indicated times. The supernatants were harvested for virus titration (EID50/ml) (left). RNF5-/- or control cells were infected with SZ19-ΔF2 virus (MOI = 0.01) for the indicated times. The supernatants were harvested for virus titration (EID50/ml) (right). The data shown represent three independent experiments; bars represent the mean ± SD of the three independent experiments (n = 3). [P< 0.05(*), P < 0.01(**), P < 0.001(***), P < 0.0001(****)].
Fig 2: H7N9 PB1 facilitates RNF5 to degrade MAVS through the autophagosomal pathway.(A) Overexpression of PB1 interacts with RNF5. HEK293 cells were transfected with the indicated plasmids for 24 h before co-immunoprecipitation and immunoblotting analysis. (B) PB1 interacts with RNF5 directly. Purified GST-RNF5 was used to pull down purified His-PB1. The immunoprecipitants were analyzed by immunoblotting. (C) Endogenous PB1 interacts with RNF5 after SZ19 virus infection. A549 cells were left uninfected or were infected with SZ19 virus (MOI = 0.1) for 24 h before coimmunoprecipitation and immunoblot analysis. (D) PB1 co-localizes with RNF5 in the cytoplasm. HeLa cells transfected with Flag-PB1 along with the GFP-RNF5 or MARCH8 plasmids. Twenty hours later, the cells were fixed with 4% paraformaldehyde and stained with anti-Flag before confocal microscopy. The nuclei were stained by DAPI. The fluorescence intensity profile of DAPI (blue), GFP-RNF5/MARCH8 (green), and Flag-PB1 (red) was measured along the line drawn by Image J. (E) MAVS interacts with RNF5 directly. Purified GST-RNF5 was used to pull down purified His-MAVS. The immunoprecipitants were analyzed by immunoblotting. (F) RNF5-deficiency inhibits PB1-mediated degradation of MAVS. RNF5 knockdown or knockout cells were transfected with the indicated plasmids for 24 h before immunoblotting analysis. (G) PB1 enhances RNF5-mediated autophagic degradation of MAVS. HEK293 cells were transfected with the indicated plasmids for 20 h. The cells then were treated with 3-MA (10 mM), CQ (50 μM), MG132 (10 μM), or ZVAD (20 μM) for another 6 h before immunoblot analysis. The data shown represent three independent experiments.
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