Male-biased Cyp17a2 orchestrates antiviral sexual dimorphism in fish via STING stabilization and viral protein degradation
- Key Laboratory of Breeding Biotechnology and Sustainable Aquaculture, Institute of Hydrobiology, Chinese Academy of Sciences, China
- University of Chinese Academy of Sciences, China
- College of Fisheries and Life Science, Dalian Ocean University, China
- Key Laboratory of Mariculture, Ministry of Education, Ocean University of China, China
- Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, China
- Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, China
Figures
Figure 1
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 hr (n=3 per group). (C) Microscopy of H&E-stained liver and spleen sections from male and female zebrafish treated with SVCV for 48 hr. (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 hr. (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 hr. (G) The viral titer of heart in male and female zebrafish (n=40 per group) treated with SVCV for 48 hr.
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Figure 1—source data 1
PDF file containing original western blots for Figure 1F indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig1-data1-v1.pdf
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Figure 1—source data 2
Original files for western blot analysis displayed in Figure 1F.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig1-data2-v1.zip
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Figure 1—source data 3
Original data for graphs analysis in Figure 1A, E and G.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig1-data3-v1.xlsx
Figure 2 with 2 supplements
Male-biased cyp17a2 regulates RLR-mediated antiviral immunity.
(A) The differently expressed gene number of mRNA variations presented as a volcano plot in head-kidney of male and female zebrafish. (B) Heatmap view of mRNA variations of eight sex-regulated genes in the head-kidney of male and female zebrafish. Values are represented as log2(FPKM+1). (C) Heatmap view 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). Expression values [log2(fold change)] were transformed to Z-scores across rows for each gene to highlight patterns of relative expression. (D) 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). (E) 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). (F) 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 hr. (G) 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 hr.
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Figure 2—source data 1
PDF file containing original western blots for Figure 2D indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig2-data1-v1.pdf
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Figure 2—source data 2
Original files for western blot analysis displayed in Figure 2D.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig2-data2-v1.zip
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Figure 2—source data 3
Original data for graphs analysis in Figure 2B, C and E–G.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig2-data3-v1.xlsx
Figure 2—figure supplement 1
The male-biased expression of Cyp17a2 governs RLR antiviral signaling.
(A) qPCR analysis of cyp17a2 mRNA in the head-kidney of male and female zebrafish (n=50 per group). (B) qPCR analysis of cyp17a2 mRNA in the ZF4 or EPC cells infected with SVCV for the indicated times. (C) IB analysis of Cyp17a2 in ZF4 and EPC cells infected with SVCV for the indicated times. (B and C) Representative experiments are shown (n=3). (D) Microscopy of H&E-stained liver and spleen sections from cyp17a2+/+ and cyp17a2-/- male zebrafish treated with SVCV for 48 h. (E) IF analysis of SVCV-N protein in the liver and spleen of cyp17a2+/+ and cyp17a2-/- male zebrafish treated with SVCV for 48 hr. (F) The viral titer of heart in cyp17a2+/+ and cyp17a2-/- male zebrafish (n=15 per group) treated with SVCV for 48 hr. (G) 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 hr. (H) The differently expressed gene number of mRNA variations in the liver of cyp17a2+/+ and cyp17a2-/- male zebrafish infected with SVCV. (I) KEGG pathway enrichment map showing the top 10 signaling pathways of total. (J) 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.
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Figure 2—figure supplement 1—source data 1
PDF file containing original western blots for Figure 2—figure supplement 1C and G indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig2-figsupp1-data1-v1.pdf
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Figure 2—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 2—figure supplement 1C and G.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig2-figsupp1-data2-v1.zip
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Figure 2—figure supplement 1—source data 3
Original data for graphs analysis in Figure 2—figure supplement 1A, B and F.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig2-figsupp1-data3-v1.xlsx
Figure 2—figure supplement 2
Heatmap view of mRNA variations of RLR-mediated ISG sets in the liver of cyp17a2+/+ and cyp17a2-/- male zebrafish infected with SVCV.
Expression values are log2(FPKM+1) transformed and presented as row Z-scores.
Figure 3 with 1 supplement
Cyp17a2 upregulates IFN expression and inhibits viral replication.
(A and E) Luciferase activity of IFNφ1pro in EPC cells transfected with indicated plasmids for 24 hr, and then untreated or transfected with poly I:C (0.5 μg) or infected with SVCV (MOI = 1) for 24 hr before luciferase assays. (B) qPCR analysis of cyp17a2 in EPC cells transfected with indicated plasmids for 24 hr, and then untreated or transfected with poly I:C (2 μg) or infected with SVCV (MOI = 1) for 24 hr. (C and D) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 hr. (A–E) Representative experiments are shown (n=3). (F) Heatmap view of mRNA variations of SVCV-activated ISG sets in the Cyp17a2-overexpressing cells or cyp17a2 knockdown cells and infected with SVCV for 24 hr. Expression values are log2(FPKM+1) transformed and presented as row Z-scores. (G) qPCR analysis of ifn in EPC cells transfected with indicated plasmids for 24 hr, and then untreated or infected with SVCV (MOI = 1) for 24 hr. (H) Plaque assay of virus titers in EPC cells or GICB cells transfected with indicated plasmids for 24 hr, followed by SVCV or CyHV-2 challenge for 24 hr or 48 hr. (I and J) qPCR and IB analysis of SVCV genes in EPC cells transfected with indicated plasmids for 24 hr, followed by SVCV challenge for 24 hr. (K) IF analysis of SVCV proteins in EPC cells transfected with indicated plasmids for 24 hr, followed by SVCV challenge for 24 hr. The fluorescence intensity (arbitrary unit, a.u.) was recorded by the LAS X software, and the data were expressed as mean ± SD, n=5. (G–K) Representative experiments are shown (n=3).
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Figure 3—source data 1
PDF file containing original western blots for Figure 3C, D and J indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig3-data1-v1.pdf
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Figure 3—source data 2
Original files for western blot analysis displayed in Figure 3C, D and J.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig3-data2-v1.zip
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Figure 3—source data 3
Original data for graphs analysis in Figure 3A, B, E, G–I and K.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig3-data3-v1.xlsx
Figure 3—figure supplement 1
Cyp17a2 enhances IFN production to inhibit viral replication.
(A–D) Luciferase activity of IFNφ3pro and ISRE in EPC cells transfected with indicated plasmids for 24 hr, and then untreated or transfected with poly I:C (0.5 μg) or infected with SVCV (MOI = 1) for 24 hr before luciferase assays. (E) qPCR analysis of vig1 in EPC cells transfected with indicated plasmids for 24 hr, and then untreated or infected with SVCV (MOI = 1) for 24 hr. (F) Plaque assay of virus titers in EPC cells or GICB cells transfected with indicated plasmids for 24 hr, followed by SVCV or CyHV-2 challenge for 24 hr or 48 hr. (G and H) qPCR and IB analysis of SVCV genes in EPC cells transfected with indicated plasmids for 24 hr, followed by SVCV challenge for 24 hr. (I) IF analysis of SVCV proteins in EPC cells transfected with indicated plasmids for 24 hr, followed by SVCV challenge for 24 hr. All experiments were repeated for at least three times with similar results.
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Figure 3—figure supplement 1—source data 1
PDF file containing original western blots for Figure 3—figure supplement 1H indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig3-figsupp1-data1-v1.pdf
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Figure 3—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 3—figure supplement 1H.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig3-figsupp1-data2-v1.zip
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Figure 3—figure supplement 1—source data 3
Original data for graphs analysis in Figure 3—figure supplement 1A-G and I.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig3-figsupp1-data3-v1.xlsx
Figure 4 with 1 supplement
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 hr. Representative experiments are shown (n=3). (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 hr. 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 hr, followed by untreated or infected with SVCV (MOI = 1) for 24 hr. (I) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 hr. (J) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 hr, followed by untreated or infected with SVCV (MOI = 1) for 24 hr. (K) IB analysis of proteins in EPC cells transfected with indicated plasmids for 18 hr, then treated with CHX for 4 hr and 8 hr. (F–K) Representative experiments are shown (n=3).
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Figure 4—source data 1
PDF file containing original western blots for Figure 4A-C, E and I–K indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig4-data1-v1.pdf
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Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4A–C, E and I–K.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig4-data2-v1.zip
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Figure 4—source data 3
Original data for graphs analysis in Figure 4F, G and H.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig4-data3-v1.xlsx
Figure 4—figure supplement 1
STING stability is enhanced by Cyp17a2.
(A) Colocalization analyses of Figure 4G were performed by calculating Pearson correlation coefficient. (B) Fluorescent analysis of proteins in EPC cells transfected with indicated plasmids for 24 hr. The fluorescence intensity (arbitrary unit, a.u.) was recorded by the LAS X software, and the data were expressed as mean ± SD, n=5. (C) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 hr, followed by untreated or transfected with poly I:C (2 μg) for 24 hr. (D and E) IB analysis of proteins in EPC cells transfected with indicated plasmids for 18 hr, then treated with CHX for 4 hr and 8 hr. (B-E) Representative experiments are shown (n=3).
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Figure 4—figure supplement 1—source data 1
PDF file containing original western blots for Figure 4—figure supplement 1C–E indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig4-figsupp1-data1-v1.pdf
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Figure 4—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 4—figure supplement 1C–E.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig4-figsupp1-data2-v1.zip
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Figure 4—figure supplement 1—source data 3
Original data for graphs analysis in Figure 4—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig4-figsupp1-data3-v1.xlsx
Figure 5 with 1 supplement
Cyp17a2 potentiates STING-induced IFN production and antiviral responses.
(A and B) Luciferase activity of IFNφ1pro in EPC cells transfected with indicated plasmids for 24 hr before luciferase assays. (C and D) qPCR analysis of ifn in EPC cells transfected with indicated plasmids for 24 hr. (E and H) Detection of virus titers in EPC cells transfected with indicated plasmids for 24 hr and then infected with SVCV (MOI = 1) for 24 hr. (F and J) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 hr and then untreated or infected with SVCV (MOI = 1) for 24 hr. (G and I) qPCR analysis of SVCV genes in EPC cells transfected with indicated plasmids for 24 hr, followed by SVCV challenge for 24 hr. All experiments were repeated at least three times with similar results.
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Figure 5—source data 1
PDF file containing original western blots for Figure 5F and J indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig5-data1-v1.pdf
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Figure 5—source data 2
Original files for western blot analysis displayed in Figure 5F and J.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig5-data2-v1.zip
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Figure 5—source data 3
Original data for graphs analysis in Figure 5A–E and G–I.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig5-data3-v1.xlsx
Figure 5—figure supplement 1
Cyp17a2 enhances STING-mediated IFN activation and antiviral defense.
(A and B) Luciferase activity of ISRE in EPC cells transfected with indicated plasmids for 24 hr before luciferase assays. (C and D) qPCR analysis of vig1 in EPC cells transfected with indicated plasmids for 24 hr. (E and H) Plaque assay in EPC cells transfected with indicated plasmids for 24 hr and then infected with SVCV (MOI = 1) for 24 hr. (F and I) IF analysis of N protein and P protein in EPC cells transfected with indicated plasmids for 24 hr, followed by SVCV challenge for 24 hr. The fluorescence intensity (arbitrary unit, a.u.) was recorded by the LAS X software, and the data were expressed as mean ± SD, n=5. (G) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 hr. All experiments were repeated for at least three times with similar results.
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Figure 5—figure supplement 1—source data 1
PDF file containing original western blots for Figure 5—figure supplement 1G indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig5-figsupp1-data1-v1.pdf
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Figure 5—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 5—figure supplement 1G.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig5-figsupp1-data2-v1.zip
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Figure 5—figure supplement 1—source data 3
Original data for graphs analysis in Figure 5—figure supplement 1A–D, F and I.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig5-figsupp1-data3-v1.xlsx
Figure 6 with 1 supplement
Cyp17a2 stabilizes STING by orchestrating btr32-catalyzed K33-linked polyubiquitination.
(A) IB analysis of proteins in EPC cells transfected with indicated plasmids for 18 hr, then treated with CHX and DMSO, MG132, or CQ for 4 hr and 8 hr. (B and C) STING ubiquitination assays in EPC cells transfected with indicated plasmids for 24 hr. (D–G, K) IB analysis of WCLs and proteins immunoprecipitated with anti-Flag Ab-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 hr. (H and I) Confocal microscopy of btr32 and ER or STING or Cyp17a2 in EPC cells transfected with indicated plasmids for 24 hr. The coefficient of colocalization was determined by qualitative analysis of the fluorescence intensity of the selected area in Merge. (A–I, K) Representative experiments are shown (n=3). (J) Colocalization analyses of (I) were performed by calculating Pearson correlation coefficient. (L and M) Luciferase activity of IFNφ1pro and qPCR analysis of vig1 in EPC cells transfected with indicated plasmids for 24 hr. (N) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 hr. (L–N) Representative experiments are shown (n=3).
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Figure 6—source data 1
PDF file containing original western blots for Figure 6A–G, K and N indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig6-data1-v1.pdf
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Figure 6—source data 2
Original files for western blot analysis displayed in Figure 6A–G, K and N.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig6-data2-v1.zip
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Figure 6—source data 3
Original data for graphs analysis in Figure 6H–J, L and M.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig6-data3-v1.xlsx
Figure 6—figure supplement 1
Cyp17a2 promotes STING stabilization by facilitating btr32-catalyzed K33-linked polyubiquitination.
(A) STING ubiquitination assays in EPC cells transfected with indicated plasmids for 24 hr. (B) IB analysis of WCLs and proteins immunoprecipitated with anti-Flag Ab-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 hr. (C and D, G) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 hr. (E and F) Luciferase activity of IFNφ1pro and qPCR analysis of vig1 in EPC cells transfected with indicated plasmids for 24 hr. All experiments were repeated for at least three times with similar results.
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Figure 6—figure supplement 1—source data 1
PDF file containing original western blots for Figure 6—figure supplement 1A–D and G indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig6-figsupp1-data1-v1.pdf
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Figure 6—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 6—figure supplement 1A–D and G.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig6-figsupp1-data2-v1.zip
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Figure 6—figure supplement 1—source data 3
Original data for graphs analysis in Figure 6—figure supplement 1E and F.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig6-figsupp1-data3-v1.xlsx
Figure 7 with 1 supplement
Cyp17a2-mediated ubiquitination of STING is dependent on the K203.
(A and B, G and H, and M) STING ubiquitination assays in EPC cells transfected with indicated plasmids for 24 hr. Representative experiments are shown (n=3). (C) Mass spectrometry analysis of a peptide derived from ubiquitinated STING-Myc. (D) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 hr. (E and F) Luciferase activity of IFNφ1pro and qPCR analysis of ifn in EPC cells transfected with indicated plasmids for 24 hr. (D–F) Representative experiments are shown (n=3). (I) Schematic representation of full-length btr32 and its mutants. (J–L) IB of WCLs and proteins immunoprecipitated with anti-Flag/HA Ab-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 h. Representative experiments are shown (n=3).
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Figure 7—source data 1
PDF file containing original western blots for Figure 7A, B, D, G, H and J–M indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig7-data1-v1.pdf
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Figure 7—source data 2
Original files for western blot analysis displayed in Figure 7A, B, D, G, H and J–M.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig7-data2-v1.zip
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Figure 7—source data 3
Original data for graphs analysis in Figure 7E and F.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig7-data3-v1.xlsx
Figure 7—figure supplement 1
The K203 residue is essential for the ubiquitination of STING mediated by Cyp17a2.
(A–E) STING ubiquitination assays in EPC cells transfected with indicated plasmids for 24 hr. All experiments were repeated for at least three times with similar results.
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Figure 7—figure supplement 1—source data 1
PDF file containing original western blots for Figure 7—figure supplement 1A–E indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig7-figsupp1-data1-v1.pdf
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Figure 7—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 7—figure supplement 1A–E.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig7-figsupp1-data2-v1.zip
Figure 8
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 hr. (D) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 hr. (E) Fluorescent analysis of proteins in EPC cells transfected with indicated plasmids for 24 hr. (F) Confocal microscopy of P and Cyp17a2 in EPC cells transfected with indicated plasmids for 24 hr. 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 hr, followed by treatments of MG132 (10 μM), 3-MA (2 mM), Baf-A1 (100 nM), and CQ (50 μM) for 6 hr, respectively. (H–K) P protein ubiquitination assays in EPC cells transfected with indicated plasmids for 18 hr, followed by MG132 treatments for 6 hr. All experiments were repeated at least three times with similar results.
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Figure 8—source data 1
PDF file containing original western blots for Figure 8A–D and G–K indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig8-data1-v1.pdf
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Figure 8—source data 2
Original files for western blot analysis displayed in Figure 8A–D and G–K.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig8-data2-v1.zip
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Figure 8—source data 3
Original data for graphs analysis in Figure 8E and F.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig8-data3-v1.xlsx
Figure 9 with 1 supplement
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, O) IB analysis of WCLs and proteins immunoprecipitated with anti-HA/Myc Ab-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 hr. Representative experiments are shown (n=3). (D) Confocal microscopy of USP8 and Cyp17a2 or P protein in EPC cells transfected with indicated plasmids for 24 hr. The coefficient of colocalization was determined by qualitative analysis of the fluorescence intensity of the selected area in Merge. (F and G, K) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 hr. (H and I, L and M, P) P protein ubiquitination assays in EPC cells transfected with indicated plasmids for 18 hr, followed by MG132 treatments for 6 hr. (F-I, K-M, and P) Representative experiments are shown (n=3). (J) Mass spectrometry analysis of a peptide derived from ubiquitinated P-Myc. (N) Schematic representation of full-length USP8 and its mutants.
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Figure 9—source data 1
PDF file containing original western blots for Figure 9B, C, E–I, K–M, O and P indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig9-data1-v1.pdf
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Figure 9—source data 2
Original files for western blot analysis displayed in Figure 9B, C, E–I, K–M, O and P.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig9-data2-v1.zip
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Figure 9—source data 3
Original data for graphs analysis in Figure 9D.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig9-data3-v1.xlsx
Figure 9—figure supplement 1
Cyp17a2 mediates K33-linked polyubiquitination of the P protein at K12.
(A and C) IB analysis of WCLs and proteins immunoprecipitated with anti-Flag/Myc Ab-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 hr. Representative experiments are shown (n=3). (B) Colocalization analyses of Figure 9D were performed by calculating Pearson correlation coefficient. (D) IB analysis of proteins in EPC cells transfected with indicated plasmids for 24 hr. (E–I) P protein ubiquitination assays in EPC cells transfected with indicated plasmids for 18 hr, followed by MG132 treatments for 6 hr. (D–I) Representative experiments are shown (n=3).
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Figure 9—figure supplement 1—source data 1
PDF file containing original western blots for Figure 9—figure supplement 1A and C–I indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig9-figsupp1-data1-v1.pdf
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Figure 9—figure supplement 1—source data 2
Original files for western blot analysis displayed in Figure 9—figure supplement 1A and C–I.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig9-figsupp1-data2-v1.zip
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Figure 9—figure supplement 1—source data 3
Original data for graphs analysis in Figure 9—figure supplement 1B.
- https://cdn.elifesciences.org/articles/108048/elife-108048-fig9-figsupp1-data3-v1.xlsx
Figure 10
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 btr32, 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.
Additional files
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Supplementary file 1
List of primer sequences used in this study.
- https://cdn.elifesciences.org/articles/108048/elife-108048-supp1-v1.xlsx
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Supplementary file 2
List of all identified genes in FHK-vs-MHK in transcriptomic analysis.
- https://cdn.elifesciences.org/articles/108048/elife-108048-supp2-v1.xlsx
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Supplementary file 3
Heatmap view of mRNA variations of SVCV-activated ISG sets in the Cyp17a2-overexpressing cells or cyp17a2 knockdown cells.
- https://cdn.elifesciences.org/articles/108048/elife-108048-supp3-v1.xlsx
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MDAR checklist
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Male-biased Cyp17a2 orchestrates antiviral sexual dimorphism in fish via STING stabilization and viral protein degradation
eLife 14:RP108048.
https://doi.org/10.7554/eLife.108048.4
