Fig. 4
HSP90α maintains the stability of ORF9b. a Co-Immunoprecipitation was performed to test the interaction of SARS-CoV-2 ORF9b and ElonginB/C in HEK293T cells. b HEK293T cells overexpressing the ORF9b protein were transfected with ElonginB or ElonginC expressing plasmid as indicated. The ORF9b protein level was detected at 24 h after transfection. c HEK293T cells stably expressing the indicated siRNA were transfected with plasmids containing Flag-ORF9b, treated with CHX and collected at indicated time for Western blot. d In vivo ubiquitination assay of SARS-CoV-2 ORF9b when Elongin B or Elongin C was knocked down in HEK293T cells. e Co-Immunoprecipitation was performed to test the interaction of ORF9b and HSP90 in HEK293T cells. f and g HEK293T cells overexpressing Flag-ORF9b-WT (f) or Flag-ORF9b-K67R (g) were treated with increasing concentrations of GA or 17-AAG for 24 h and the ORF9b protein level was detected. h HEK293T cells expressing ORF9b were treated with DMSO, GA or 17-AAG for 12 h and then infected with SeV (100 HAU/ml). Total RNA was extracted to detect the relative levels of IFNb, CCL5, and CXCL10. i HEK293T cells overexpressing Flag-ORF9b were treated with 1.0 μM GA or 17-AAG, together treated with MG132 or not for 24 h. Cells were collected to detect the protein level of ORF9b. j In vivo ubiquitination assay of SARS-CoV-2 ORF9b in HEK293T cells when treated with HSP90 inhibitors (GA or 17-AAG) or not. k Half-life analyses of SARS-CoV-2 ORF9b when treated with GA or 17-AAG. l Co-Immunoprecipitation was performed to test the interaction of SARS-CoV-2 ORF9b and HSP90α/β in HEK293T cells. m In vivo ubiquitination assay of SARS-CoV-2 ORF9b when overexpressing HSP90α or HSP90β in HEK293T cells. n The ORF9b protein level was detected in HEK293T cells transfected with indicated plasmids. o The HEK293T, Calu3 and HCT116 cells expressing ORF9b were transfected with indicated siRNA for 36 h and collected to detect the HSP90α, HSP90β, and ORF9b protein levels. 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 < 0.05; **P < 0.01; ***P < 0.001
