Fig. 1
K48-linked polyubiquitin chain at the K67 site mediates the ubiquitinated degradation of SARS-CoV-2 ORF9b. a Half-life analyses of N and ORF9b proteins of SARS-CoV and SARS-CoV-2 in HEK293T cells. The cells were transfected with plasmids expressing the aforementioned viral proteins for 30 h and then split into 12-well plates. Following treatment with CHX, cells were collected as indicated time points for Western blot analysis. b The protein levels of ORF9b in HEK293T cells with treatment of DMSO, MG132, BTM, CQ, and NH4Cl. c Half-life analyses of SARS-CoV-2 ORF9b in the primary human airway epithelial (HAE) cells with treatment of CHX + MG132 or CHX + DMSO. d In vivo ORF9b ubiquitination assay in HEK293T cells. The HA-Ub WT, K48R, and K63R mutant plasmids were individually co-transfected with the Flag-ORF9b plasmid into HEK293T cells, followed by culturing and co-immunoprecipitation. Western blot was performed to detect ubiquitinated chains. e Half-life analyses of SARS-CoV-2 ORF9b-WT and SARS-CoV-2 ORF9b-3R mutant in HEK293T cells. f Flag-ORF9b was expressed in HEK293T and purified by anti-Flag beads, and then analyzed by mass spectrometry. One peptide containing lysine residues was identified. K67 was shown in red. g The protein levels of SARS-CoV-2 ORF9b-WT and indicated mutants expressed in HEK293T cells were detected after treatment with MG132 or DMSO. h In vivo ubiquitination assay of SARS-CoV-2 ORF9b-WT and SARS-CoV-2 ORF9b-K67R mutant in HEK293T cells. i and j Half-life analyses of indicated SARS-CoV-2 ORF9b mutants in HEK293T cells. Quantification was shown as mean±s.d. n = 3 independent experiments. Student’s t-test (unpaired, two-tailed) was used to compare two independent groups, and a two-way ANOVA test was performed for comparisons of multiple groups. **P < 0.01; ***P < 0.001; n.s. not significant
