SARS-CoV-2 Nsp1 suppresses the canonical NF-κB pathway by promoting ubiquitin-dependent degradation of TAK1 kinase
Fig 5
SARS-CoV-2 Nsp1 promotes TAK1 K48-ubiquitin degradation.
a: The ubiquitination of TAK1 increased in N-Caco-2 cells infected with GFP-expressing ΔN SARS-CoV-2 RDPs at an MOI of 0.01. The cells were transfected with plasmids expressing HA-Ub and FLAG-tagged TAK1 (TAK1-FLAG). Twenty-four hours later, the cells were infected at an MOI of 0.01, and samples were collected at different time points. Ubiquitinated TAK1 was immunoprecipitated (IP) using anti-FLAG magnetic beads, and the ubiquitination level was analyzed with an anti-ubiquitin antibody. TAK1 was detected by anti-FLAG. The relative signals quantified by ImageJ are shown in the right panels of a-c and e-f. b: HEK293T cells were transfected with plasmids expressing HA-Ub and TAK1-FLAG 24 h prior to transfection with different quantities of plasmids expressing cMYC-tagged Nsp1 (Nsp1-cMYC; 0, 0.5, 1, 2 or 4 μg). Nsp1 was detected by an anti-cMYC antibody in b-d. c: HEK293T cells were transfected with plasmids containing TAK1-FLAG and wild-type (WT) or mutant HA-Ub in which the corresponding ubiquitin-accepting lysine (K) residue was replaced with arginine (R), and 24 h later, the cells were transfected again with the cMYC-tagged Nsp1 (Nsp1-cMYC) or GFP control (Ctrl, GFP-cMYC) plasmid. Green arrowhead, GFP-cMYC; red arrowhead, Nsp1-cMYC in c, e and f. d: HEK293T cells were transfected with the Nsp1-cMYC plasmid 24 hr prior to treatment with different concentrations of MG132 (0, 2, 5 or 10 μM) for 12 hr. The relative signals between TAK1 (by anti-TAK1) and β-ACTIN (by anti-β-ACTIN) were quantified by ImageJ and are shown in the right panel. e: HEK293T cells were treated with 10 μM MG132 or DMSO (Ctrl) prior to transfection with the TAK1-FLAG and HA-Ub plasmids, and 24 h later, they were transfected again with the Nsp1-cMYC or control GFP-cMYC (Ctrl) plasmid. f: Polyubiquitination levels of TAK1 mutants in which the lysine (K) residue at position 34, 72, 209 or 589 was mutated to alanine (A). HEK293T cells were first transfected with plasmids expressing HA-Ub and wild-type (WT) or mutant TAK1-FLAG; 24 h later, the cells were transfected again with Nsp1-cMYC or GFP-cMYC (Ctrl) plasmids. The results in a, c, d, e, and f are representative of two independent experiments.