Fig. 6

HSP90 inhibitors restrict the replication of SARS-CoV-2 by promoting ORF9b degradation. a The plasmids containing Strep-tagged SARS-CoV-2 viral genes were transfected into HEK293T cells and treated with indicated concentrations of GA or 17-AAG for 24 h. The cells were lysed to detect the viral protein levels and quantification of different viral proteins was normalized to β-actin. b and c Calu3 cells treated with indicated increasing concentrations of GA (b-up), 17-AAG (b-down) or DMSO were inoculated with SARS-CoV-2 at MOI = 1 for 24 h. Quantification of relative virus amount was measured by immunofluorescence (c). d–g Calu3 cells infected with SARS-CoV-2 at a MOI = 1 were treated with indicated concentrations of GA (d and e) or 17-AAG (f and g) for 24 h. Relative mRNA (left) and protein levels (right) were detected by qRT-PCR and Western blot as indicated. h A schematic diagram illustrates the regulation of the TOM70–CUL5–HSP90α complex on ORF9b. The interaction model of the complex was based on a predicted model generated using AlphaFold, as shown in Fig. 5l. Different components are represented by distinct colors: ORF9b is depicted in sky blue, TOM70 in yellow, CUL5 in yellow-green, HSP90α monomer 1 in cyan, HSP90α monomer 2 in aquamarine, and the nonfunctional HSP90α dimer in gray. This diagram demonstrates how, upon SARS-CoV-2 entry, host cells counteract viral immune evasion by targeting ORF9b for ubiquitination and subsequent degradation. In the absence of HSP90α, TOM70 and CUL5 mediate ORF9b degradation via the ubiquitin-proteasome pathway (left). Conversely, in the presence of HSP90α, ORF9b is shielded from degradation (middle). However, HSP90 inhibitors such as 17-AAG/GA deactivate HSP90αα, leading to the degradation of ORF9b (right). i–k A dataset was obtained from GEO with accession number GSE171524 to analyze the difference in RNA levels of HSP90AA1 between lung epithelial cells of COVID-19 patients and healthy control. Group origins of cells (i) and RNA levels of HSP90AA1 in single cells (j) were shown with a UMAP plot. Violin plot (k) was performed to show the difference in HSP90AA1 RNA levels between lung epithelial cells of COVID-19 patients and healthy control. 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