Male fish show stronger viral resistance than females

(A) Survival (Kaplan-Meier Curve) of male and female zebrafish (n = 15 per group) at various days after i.p. injected with SVCV (5 × 108 TCID50/ml, 5 μL/individual). (B) Sex-specific morphological alterations in zebrafish given i.p. injection of SVCV for 48 h (n = 3 per group). (C) Microscopy of H&E-stained liver and spleen sections from male and female zebrafish treated with SVCV for 48 h. (D) IF analysis of SVCV-N protein in liver and spleen of male and female zebrafish treated with SVCV for 48 h. (E) qPCR analysis of svcv-n mRNA in the liver and spleen of male and female zebrafish (n = 26 per group) given i.p. injection of SVCV for 48 h. (F) IB analysis of SVCV proteins in the liver and spleen sections of male and female zebrafish (n = 7 per group) treated with SVCV for 48 h. (G) The viral titer of heart in male and female zebrafish (n = 40 per group) treated with SVCV for 48 h.

Male-biased cyp17a2 regulates RLR-mediated antiviral immunity

(A) Schematic representation of zebrafish tissue dissection and RNA extraction for transcriptome sequencing. The head-kidney from male and female zebrafish. Total RNAs were extracted and used for transcriptome sequencing and analysis. (B) The differently expressed gene number of mRNA variations in head-kidney of male and female zebrafish. (C) Heatmap view of mRNA variations of sex-regulated genes in the head-kidney of male and female zebrafish. (D) qPCR analysis of cyp17a2 mRNA in the head-kidney of male and female zebrafish (n = 50 per group). (E) qPCR analysis of cyp17a2 mRNA in the brain, heart, head-kidney, gill, liver, spleen, body-kidney, gonad, gut, muscle, and skin of male and female zebrafish (n = 5 per group). (F) IB analysis of Cyp17a2 in the heart, head-kidney, gill, liver, spleen, body-kidney, gonad, and skin of male and female zebrafish (n = 5 per group). (G) qPCR analysis of cyp17a2 mRNA in the ZF4 or EPC cells infected with SVCV for the indicated times. (H) IB analysis of Cyp17a2 in ZF4 and EPC cells infected with SVCV for the indicated times. (I) Survival (Kaplan-Meier Curve) of cyp17a2+/+ and cyp17a2−/− male zebrafish (n = 16 per group) at various days after i.p. injected with SVCV (5 × 108 TCID50/ml, 5 μl/individual). (J) Microscopy of H&E-stained liver and spleen sections from cyp17a2+/+ and cyp17a2−/− male zebrafish treated with SVCV for 48 h. (K) IF analysis of SVCV-N protein in the liver and spleen of cyp17a2+/+ and cyp17a2−/− male zebrafish treated with SVCV for 48 h. (L) The viral titer of heart in cyp17a2+/+ and cyp17a2−/− male zebrafish (n = 15 per group) treated with SVCV for 48 h. (M) qPCR analysis of svcv-n mRNA in the liver and spleen of cyp17a2+/+ and cyp17a2−/− male zebrafish (n = 20 per group) given i.p. injection of SVCV for 48 h. (N) IB analysis of SVCV proteins in the liver and spleen sections of cyp17a2+/+ and cyp17a2−/− male zebrafish (n = 3 per group) treated with SVCV for 48 h. (O) KEGG pathway enrichment map showing the top 10 signaling pathways of total. (P) GSEA of differentially expressed genes in the liver of cyp17a2+/+ and cyp17a2−/−male zebrafish infected with SVCV and enrichment of RLR signaling pathway. The p-values were corrected for the False Discovery Rate (FDR) method, and modules with p-values less than 0.05 were selected as significant. (Q) qPCR analysis of ifnφ1 mRNA in the liver and spleen of cyp17a2+/+ and cyp17a2−/− male zebrafish (n = 20 per group) given i.p. injection of SVCV for 48 h.

The differently expressed gene number of mRNA variations in liver of cyp17a2+/+ and cyp17a2−/− male zebrafish infected with SVCV.

Heatmap view of mRNA variations of RLR-mediated ISGs sets in the liver of cyp17a2+/+ and cyp17a2−/− male zebrafish infected with SVCV.

Cyp17a2 upregulates IFN expression and inhibits viral replication

(A-C and G) Luciferase activity of IFNφ1pro, IFNφ3pro, and ISRE in EPC cells transfected with indicated plasmids for 24 h, and then untreated or transfected with poly I:C (0.5 μg) or infected with SVCV (MOI = 1) for 24 h before luciferase assays. (D) qPCR analysis of cyp17a2 in EPC cells transfected with indicated plasmids for 24 h, and then untreated or transfected with poly I:C (2 μg) or infected with SVCV (MOI = 1) for 24 h. (E and F) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 h. (H) Heatmap view of mRNA variations of SVCV-activated ISG sets in the Cyp17a2-overexpressing cells or cyp17a2 knockdown cells and infected with SVCV. (I) qPCR analysis of ifn and vig1 in EPC cells transfected with indicated plasmids for 24 h, and then untreated or infected with SVCV (MOI = 1) for 24 h. (J and K) Plaque assay of virus titers in EPC cells or GICB cells transfected with indicated plasmids for 24 h, followed by SVCV or CyHV-2 challenge for 24 h or 48 h. (L and M) qPCR and IB analysis of SVCV genes in EPC cells transfected with indicated plasmids for 24 h, followed by SVCV challenge for 24 h. (N and O) IF analysis of SVCV proteins in EPC cells transfected with indicated plasmids for 24 h, followed by SVCV challenge for 24 h.

Cyp17a2 physically interacts with STING and stabilizes its expression

(A-C and E) IB analysis of WCLs and proteins immunoprecipitated with anti-Flag or HA Ab-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 h. (D) Schematic representation of full-length STING and its mutants. (F and G) Confocal microscopy of Cyp17a2 and ER or STING and its mutants in EPC cells transfected with indicated plasmids for 24 h. The coefficient of colocalization was determined by qualitative analysis of the fluorescence intensity of the selected area in Merge. (H) qPCR analysis of sting in EPC cells transfected with indicated plasmids for 24 h, followed by untreated or infected with SVCV (MOI = 1) for 24 h. (I) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 h. (J) Fluorescent analysis of proteins in EPC cells transfected with indicated plasmids for 24 h. The fluorescence intensity (arbitrary unit, a.u.) was recorded by the LAS X software, and the data were expressed as mean ± SD, n = 5. (K) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 h, followed by untreated or infected with transfected with poly I:C (2 μg) or SVCV (MOI = 1) for 24 h. (L-N) IB analysis of proteins in EPC cells transfected with indicated plasmids for 18 h, then treated with CHX for 4 h and 8 h.

Cyp17a2 potentiates STING-induced IFN production and antiviral responses

(A-D) Luciferase activity of IFNφ1pro and ISRE in EPC cells transfected with indicated plasmids for 24 h before luciferase assays. (E and F) qPCR analysis of ifn and vig1 in EPC cells transfected with indicated plasmids for 24 h. (G, H, and M) Plaque assay of virus titers in EPC cells transfected with indicated plasmids for 24 h and then infected with SVCV (MOI = 1) for 24 h. (I and N) qPCR analysis of SVCV genes in EPC cells transfected with indicated plasmids for 24 h, followed by SVCV challenge for 24 h. (J and Q) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 h and then untreated or infected with SVCV (MOI = 1) for 24 h. (K, O, and P) IF analysis of N protein and P protein in EPC cells transfected with indicated plasmids for 24 h, followed by SVCV challenge for 24 h. The fluorescence intensity (arbitrary unit, a.u.) was recorded by the LAS X software, and the data were expressed as mean ± SD, n = 5. (L) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 h.

Cyp17a2 stabilizes STING by orchestrating TRIM11-catalyzed K33-linked polyubiquitination

(A) IB analysis of proteins in EPC cells transfected with indicated plasmids for 18 h, then treated with CHX and DMSO, MG132, or CQ for 4 h and 8 h. (B-D) STING ubiquitination assays in EPC cells transfected with indicated plasmids for 24 h. (E-G, J) IB analysis of WCLs and proteins immunoprecipitated with anti-Flag Ab-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 h. (H and I) Confocal microscopy of TRIM11 and ER or STING or Cyp17a2 in EPC cells transfected with indicated plasmids for 24 h. The coefficient of colocalization was determined by qualitative analysis of the fluorescence intensity of the selected area in Merge. (K and L, O and P) Luciferase activity of IFNφ1pro and qPCR analysis of vig1 in EPC cells transfected with indicated plasmids for 24 h. (M and N, Q) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 h.

Cyp17a2-mediated ubiquitination of STING is dependent on the K203

(A-D, I-L, and P-Q) STING ubiquitination assays in EPC cells transfected with indicated plasmids for 24 h. (E) Mass spectrometry analysis of a peptide derived from ubiquitinated STING-Myc. (F) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 h. (G and H) Luciferase activity of IFNφ1pro and qPCR analysis of ifn1 in EPC cells transfected with indicated plasmids for 24 h. (M) Schematic representation of full-length TRIM11 and its mutants. (N and O) IB of WCLs and proteins immunoprecipitated with anti-Flag/HA Ab-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 h.

Cyp17a2 induces proteasomal degradation of the SVCV P protein by catalyzing K33-linked polyubiquitination

(A-C) IB analysis of WCLs and proteins immunoprecipitated with anti-Flag, anti-HA, or anti-Myc Ab-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 h. (D) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 h. (E) Fluorescent analysis of proteins in EPC cells transfected with indicated plasmids for 24 h. (F) Confocal microscopy of P and Cyp17a2 in EPC cells transfected with indicated plasmids for 24 h. The coefficient of colocalization was determined by qualitative analysis of the fluorescence intensity of the selected area in Merge. (G) IB analysis of proteins in EPC cells transfected with indicated plasmids for 18 h, followed by treatments of MG132 (10 μM), 3-MA (2 mM), Baf-A1 (100 nM), and CQ (50 μM) for 6 h, respectively. (H-K) P protein ubiquitination assays in EPC cells transfected with indicated plasmids for 18 h, followed by MG132 treatments for 6 h.

Cyp17a2 targets the K12 site of the P protein for K33-linked polyubiquitination

(A) List of proteins interacting with P protein detected by mass spectrometry. (B and C, E and F) IB analysis of WCLs and proteins immunoprecipitated with anti-HA, anti-Flag, or anti-Myc Ab-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 h. (D) Confocal microscopy of USP8 and Cyp17a2 or P protein in EPC cells transfected with indicated plasmids for 24 h. The coefficient of colocalization was determined by qualitative analysis of the fluorescence intensity of the selected area in Merge. (G-I and M) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 h. (J and K, N and O, R) P protein ubiquitination assays in EPC cells transfected with indicated plasmids for 18 h, followed by MG132 treatments for 6 h. (L) Mass spectrometry analysis of a peptide derived from ubiquitinated P-Myc. (P) Schematic representation of full-length USP8 and its mutants.

A mechanistic model illustrating the dual regulatory roles of Cyp17a2 in upregulating STING expression and degrading SVCV P protein

Upon virus infection, male-biased autosomal gene cyp17a2 enhances antiviral responses in males. Mechanistically, Cyp17a2, an ER-localized protein, stabilizes STING expression through recruitment of the E3 ubiquitin ligase TRIM11, promoting K33-linked polyubiquitination. Furthermore, Cyp17a2 facilitates proteasomal degradation of the SVCV P protein by engaging USP8 to reduce its K33-linked polyubiquitination, thereby amplifying antiviral IFN responses.