Fig. 5
TOM70 serves as a substrate receptor of CUL5-based E3 ligase for ORF9b. a HEK293T cells expressing siNC or siTOM70 were transfected with plasmids containing ORF9b and treated with DMSO, GA or 17-AAG for 24 h. The protein levels of ORF9b were detected by Western blot. b Half-life analyses of SARS-CoV-2 ORF9b when knocking down TOM70 or not in HEK293T cells. c In vivo ubiquitination assay of SARS-CoV-2 ORF9b when overexpressing TOM70 or not in HEK293T cells. d In vivo ubiquitination assay of SARS-CoV-2 ORF9b when knocking down TOM70 or not in HEK293T cells. e–g Co-Immunoprecipitation was performed to test the interaction among TOM70, CUL5 and HSP90α in HEK293T cells. h The interactions between SARS-CoV-2 ORF9b and CUL5, or ORF9b and HSP90α were tested in HEK293T cells when siTOM70 was transfected or not. i–k HEK293T cells expressing indicated siRNAs were transfected with plasmids expressing Flag-tagged proteins. The whole cell lysates and precipitated proteins were analyzed by immunoblotting with indicated antibodies. l A structural model of the ORF9b–TOM70–CUL5–HSP90α complex is presented in two orthogonal views. The complex includes two copies of HSP90α (light green and blue-white) and one copy each of ORF9b (red), CUL5 (magenta), and TOM70 (cyan). The model was generated using AlphaFold v2.3.2. 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
