USP22 controls type III interferon signaling and SARS-CoV-2 infection through activation of STING

Abstract

Pattern recognition receptors (PRRs) and interferons (IFNs) serve as essential antiviral defense against SARS-CoV-2, the causative agent of the COVID-19 pandemic. Type III IFNs (IFN-λ) exhibit cell-type specific and long-lasting functions in auto-inflammation, tumorigenesis, and antiviral defense. Here, we identify the deubiquitinating enzyme USP22 as central regulator of basal IFN-λ secretion and SARS-CoV-2 infections in human intestinal epithelial cells (hIECs). USP22-deficient hIECs strongly upregulate genes involved in IFN signaling and viral defense, including numerous IFN-stimulated genes (ISGs), with increased secretion of IFN-λ and enhanced STAT1 signaling, even in the absence of exogenous IFNs or viral infection. Interestingly, USP22 controls basal and 2'3'-cGAMP-induced STING activation and loss of STING reversed STAT activation and ISG and IFN-λ expression. Intriguingly, USP22-deficient hIECs are protected against SARS-CoV-2 infection, viral replication, and the formation of de novo infectious particles, in a STING-dependent manner. These findings reveal USP22 as central host regulator of STING and type III IFN signaling, with important implications for SARS-CoV-2 infection and antiviral defense.

Fig. 1. Profiling USP22-mediated gene expression in HT-29 hIECs.

A. Volcano plot showing the differential gene expression patterns of two independent single-cell HT-29 USP22 CRISPR/Cas9 KO clones (#16 and #62) compared to CRISPR/Cas9 control (NHT) HT-29 cells. Color code represents the log2 fold change compared to NHT. B Heatmap of the top-50 differentially regulated genes between HT-29 USP22 KO single clones #16 and #62 and the NHT control. Color coding represents the row-wise scaled (Z-score) RNA intensities. Genes are sorted according to their log2 fold change, compared to NHT. C Basal mRNA expression levels of the indicated genes were determined in control and two independent USP22 KO HT-29 single clones using qRT-PCR. Gene expression was normalized against 28S mRNA and is presented as x-fold mRNA expression compared to NHT. Mean and SD of three independent experiments in triplicate are shown. *P < 0.05; **P < 0.01, ***P < 0.001.

Fig. 2. Loss of USP22 specifically enriches for genes involved in IFN signaling and response to viral infection.

A Bar plot showing the top-20 regulated GO terms in two independent single-cell HT-29 USP22 CRISPR/Cas9 KO clones (#16 and #62) compared to control (NHT) HT-29 cells. Color code represents the number of annotated genes within each gene set. B Heatmap of differentially expressed genes contributing to the GO terms response to type I IFN (left) and type II signaling (right). Color code represents the log2 fold change compared to NHT. Note that due to lack of annotation and overlapping ISGs between type I/II and type III IFNs, response to type III IFN as GO term was not included. C Basal mRNA expression levels of GO- enriched genes related to IFN signaling in control (NHT) and two independent USP22 KO HT-29 single clones using qRT-PCR. Gene expression was normalized against 28S mRNA and is presented as x-fold mRNA expression compared to NHT. Mean and SD of three independent experiments in triplicate are shown. *P < 0.05; **P < 0.01, ***P < 0.001, n.s. not significant. D Western blot analysis of basal MX1, IRF9, ISG56, ISG20, and USP22 expression levels in control and USP22 KO HT-29 cells (clone USP22 KO #62). GAPDH served as loading control. Representative blots of at least two different independent experiments are shown.

Fig. 3. USP22 negatively regulates STAT1 signaling and IFN-λ1 expression.

A. Basal mRNA expression levels of total IFNA (panIFNA) and IFNB1 in control (NHT) and the CRISPR/Cas9-generated USP22 KO HT-29 single clone (USP22 KO #62). Gene expression was normalized against 28 S mRNA and is presented as x-fold mRNA expression compared to NHT. Mean and SD of three independent experiments in triplicate are shown. *P < 0.05; **P < 0.01. B Western blot analysis of basal phosphorylated and total levels of STAT1 and USP22 in control and USP22 KO HT-29 cells (USP22 KO #62). GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. C FACS-based analysis of the indicated basal secretion patterns of the viral defense cytokine panel in supernatants of control and USP22 KO HT-29 cells (USP22 KO #62). Data are presented as absolute levels of cytokines (in pg/ml). Samples below lower detection limit were set to zero, values above upper detection limit were set to detection limit. Mean and SD of three independent experiments in triplicate are shown. *P < 0.05; n.s. not significant. D Basal mRNA expression levels of IFNL1 in control and USP22 KO HT-29 single clone (USP22 KO #62). Gene expression was normalized against 28 S mRNA and is presented as x-fold mRNA expression compared to NHT. Mean and SD of three independent experiments in triplicate are shown. *P < 0.05. E Western blot analysis of phosphorylated and total levels of STAT1 as well as USP22 in control and USP22 KO HT-29 cells (USP22 KO #62) after 24 h incubation with 1 µg/ml IFNAR2 blocking antibody. As positive control, cells were pre-treated for 1 h with IFNAR2 blocking antibody, then treated 1 h with 0.05 ng/ml IFN-β. Vinculin served as loading control. Representative blots of at least two different independent experiments are shown.

Fig. 4. USP22 regulates type III IFN signaling via STING.

A. Western blot analysis of basal RIG-I, MDA5, TLR3, and USP22 expression levels in control (NHT) and the CRISPR/Cas9-generated USP22 KO HT-29 single clone (USP22 KO #62). GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. B Western blot analysis of basal RIG-I, phosphorylated and total STAT1, ISG56 and USP22 expression levels in control, USP22 KO HT-29 cells (USP22 KO #62) as well as two NHT-control and one USP22-DDX58 dKO HT-29 single clones. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. C Idem as (B), one MDA5/IFIH1 KO single clone instead of RIG-I/DDX58. D Idem as (B), three TLR3 KO single clones instead of RIG-I/DDX58. E Western blot analysis of phosphorylated and total STAT1, RIG-I, STING, and USP22 expression levels in control and USP22 KO HT-29 cells (USP22 KO #62) subjected to transfection with transfection reagent (control) alone or in the presence of ISD and poly(I:C) (2 µg/well) for 24 h. Vinculin served as loading control. Representative blots of at least two different independent experiments are shown. F mRNA expression levels of OAS3 (left) and IRF9 (right) in control and USP22 KO HT-29 cells (USP22 KO #62) subjected to transfection with ISD and poly(I: C) (each 2 µg/well) for 24 h. Gene expression was normalized against 28 S mRNA and is presented as x-fold mRNA expression compared to NHT. Mean and SD of three independent experiments in triplicate are shown. *P < 0.05; **P < 0.01; n.s. not significant. G Western blot analysis of phosphorylated and total STAT1, STING, and USP22 expression levels in control NHT and USP22 KO HT-29 cells (USP22 KO #62) as well as in the indicated NHT- and USP22-STING dKO cells. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. H Basal mRNA expression levels of the indicated genes in control NHT and USP22 KO HT-29 cells (USP22 KO #62) as well as in the indicated NHT and USP22 STING dKO cells. Gene expression was normalized against 28 S mRNA and is presented as x-fold mRNA expression compared to NHT. Mean and SD of three independent experiments in triplicate are shown. **P < 0.01; ***P < 0.001. I Basal mRNA expression levels of IFNA, IFNB, and IFNL1 in control NHT and USP22 KO HT-29 cells (USP22 KO #62) as well as in the indicated NHT- and USP22-STING dKO cells. Gene expression was normalized against 28 S mRNA and is presented as x-fold mRNA expression compared to NHT. Mean and SD of three independent experiments in triplicate are shown. **P < 0.01; ***P < 0.001.

Fig. 5. USP22 negatively regulates STING activation and ubiquitination.

A Western blot analysis of STING, phosphorylated and total TBK1, phosphorylated and total IRF3 and USP22 expression levels in control (NHT) and CRISPR/Cas9-generated USP22 KO HT-29 single clone (USP22 KO #62) subjected to 2’3’-cGAMP (10 µg/ml) for the indicated time points. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. B mRNA expression levels of IFNA, IFNB, and IFNL1 in control and USP22 KO HT-29 cells (USP22 KO #62) subjected to 2’3’-cGAMP (10 µg/ml) for 3 h. Gene expression was normalized against 28 S mRNA and is presented as x-fold mRNA expression compared to NHT. Mean and SD of three independent experiments in triplicate are shown. *P < 0.05; **P < 0.01. C Western blot analysis of STING, phosphorylated and total STAT1 and USP22 expression levels in control and USP22 KO HT-29 cells (USP22 KO #62) subjected to the JAK/STAT inhibitor ruxolitinib (5 µM) for the indicated time points. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. D Western blot analysis of STING and USP22 expression levels in control and USP22 KO HT-29 cells (USP22 KO #62) subjected to CHX (100 µg/ml) for the indicated time points. Vinculin served as loading control. Representative blots of at least two different independent experiments are shown. E Western blot analysis of TUBE-enriched ubiquitin-modified STING from control and USP22 KO HT-29 cells (USP22 KO #62) subjected to 2’3’-cGAMP (10 µg/ml) for 24 h. GAPDH served as loading control and Ponceau S staining confirms equal loading of GST-TUBE beads. Representative blots of at least two different independent experiments are shown.

Fig. 6. Loss of USP22 protects against SARS-CoV-2 infection, replication and de novo infectious virus production in a STING-dependent manner.

A Western blot analysis of phosphorylated and total STAT1, STING, and USP22 expression levels in WT, control (NHT), and two CRISPR/Cas9-generated USP22 KO Caco-2 single clones (USP22 KO #1 and #6). GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. B Basal mRNA expression levels of IRF9 and OAS3 in WT, control, and USP22 KO Caco-2 cells (USP22 KO #1 and #6). Gene expression was normalized against 28 S mRNA and is presented as x-fold mRNA expression compared to NHT. Mean and SD of three independent experiments in triplicate are shown. *P < 0.05; **P < 0.01, n.s. not significant. C Western blot analysis of phosphorylated and total STAT1 and total STING expression levels after 24 h of treatment with 1 µM STING agonist diABZI in control and USP22 KO Caco-2 cells (USP22 KO #6). GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. D Quantification of immunofluorescence-stained SARS-CoV-2-infected cells, normalized against non-infected cells. WT, control, and USP22 KO Caco-2 cells (USP22 KO #1 and #6) were stained with anti-dsRNA (J2) at 24 hpi. Mean and SD of three independent experiments in triplicate are shown. ***P < 0.001. E Quantification of relative SARS-CoV-2 genome expression of SARS-CoV-2-infected WT, control and USP22 KO Caco-2 cells (USP22 KO #1 and #6) at 6 hpi (left) and 24 hpi (right). Data are normalized against non-infected cells. Mean and SD of three independent experiments in triplicate are shown. ***P < 0.001. F Quantification of relative SARS-CoV-2 genome expression of SARS-CoV-2-infected control-NHT, control-USP22 KO #1 and #6, STING KO-NHT and STING-USP22 dKO (USP22 KO #1 and #6) Caco-2 cells at 24 hpi. Mean and SD of three independent experiments in triplicate are shown. **P < 0.005. G Western blot analysis of total and phosphorylated STAT1, total STING and USP22 expression levels in control-NHT, control-USP22 KO #1 and #6, STING KO-NHT and STING-USP22 dKO (USP22 KO #1 and #6) Caco-2 cells. GAPDH served as loading control. Representative blots of at least two different independent experiments are shown. H Basal mRNA expression levels of IFNL1, IFNB1 and panIFNA, and ISGs IRF9 and OAS3 as well as USP22 in control and USP22 KO Caco-2 cells (USP22 KO #6) and STING KO-NHT and STING-USP22 dKO (USP22 KO #6) Caco-2 cells. Gene expression was normalized against 28 S mRNA and is presented as x-fold mRNA expression compared to NHT. Mean and SD of three independent experiments in triplicate are shown. *P < 0.05.