Fish CDK2 recruits Dtx4 to degrade TBK1 through ubiquitination in the antiviral response
- 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
- State Key Laboratory of Freshwater Ecology and Biotechnology, Institute of Hydrobiology, China
- Key Laboratory of Aquaculture Disease Control, Ministry of Agriculture, China
- Laboratory for Marine Biology and Biotechnology, Qingdao Marine Science and Technology Center, China
Figures
Figure 1
In vivo and in vitro, CDK2 is upregulated during viral infection.
(A) Schematic representation of zebrafish tissue dissection and RNA extraction for transcriptome sequencing. The liver and spleen tissues from male and female zebrafish injected with phosphate-buffered saline (PBS) (5 μL/individual), spring viremia of carp virus (SVCV) (5×108 TCID50/mL, 5 μL/individual) for 48 hr. Total RNAs were extracted and used for transcriptome sequencing and analysis. (B) Heatmap view of mRNA variations of CDKs in the liver and spleen of zebrafish with or without SVCV infection. (C) qPCR analysis of cdk2 mRNA in the THP-1, GICB, ZF4, or epithelioma papulosum cyprini (EPC) cells infected with vesicular stomatitis virus (VSV), CyHV-2, or SVCV for the indicated times. Representative experiments are shown (n=3). (D) qPCR analysis of cdk2 mRNA in the liver and spleen of zebrafish (n=5 per group) given injected intraperitoneally (i.p.) with PBS or SVCV for 48 hr. (E) IB of proteins in the liver and spleen of zebrafish (n=3 per group) given i.p. injection of PBS or SVCV for 48 hr. (F) IB of proteins in ZF4 and EPC cells infected with SVCV for the indicated times. Representative experiments are shown (n=3).
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Figure 1—source data 1
PDF file containing original western blots for Figure 1E and F, indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig1-data1-v1.zip
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Figure 1—source data 2
Original files for western blot analysis displayed in Figure 1E and F.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig1-data2-v1.zip
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Figure 1—source data 3
Original data for graphs analysis in Figure 1B and C.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig1-data3-v1.xlsx
Figure 2
CDK2 deficiency protects fish from spring viremia of carp virus (SVCV) infection.
(A) Survival (Kaplan-Meier Curve) of cdk2+/+ and cdk2-/- zebrafish (n=15 per group) at various days after i.p. infected with SVCV (5×108 TCID50/mL, 5 μL/individual). (B) Microscopy of hematoxylin and eosin (H&E)-stained liver, spleen, and kidney sections from cdk2+/+ and cdk2-/- zebrafish treated with SVCV for 48 hr. (C) qPCR analysis of svcv-n mRNA in the liver, spleen, and kidney of cdk2+/+ and cdk2-/- zebrafish (n=6 per group) given i.p. injection of SVCV for 48 hr. (D) Immunoblotting (IB) of proteins in the liver, spleen, and kidney sections from cdk2+/+ and cdk2-/- zebrafish (n=3 per group) treated with SVCV for 48 hr. (E) CDK2 regulates antiviral response-relevant target genes, presented as a volcano plot of geneiss with differential expression after SVCV infection in the liver of cdk2+/+ and cdk2-/- zebrafish. (F) Gene set enrichment analysis (GSEA) of differentially expressed genes in the liver of cdk2+/+ and cdk2-/- zebrafish with SVCV infection and enrichment of interferon (IFN). FDR (q-value) was shown. (G) Heatmap view of mRNA variations of IFN-mediated IFN-stimulated gene (ISG) sets in the liver of cdk2+/+ and cdk2-/- zebrafish with SVCV infection. (H) qPCR analysis of ifnφ1 mRNA in the liver, spleen, and kidney of cdk2+/+ and cdk2-/- zebrafish (n=6 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/98357/elife-98357-fig2-data1-v1.zip
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Figure 2—source data 2
Original files for western blot analysis displayed in Figure 2D.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig2-data2-v1.zip
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Figure 2—source data 3
Original data for graphs analysis in Figure 2A, C, G and H.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig2-data3-v1.xlsx
Figure 3
CDK2 negatively regulates interferon (IFN) production and promotes viral replication.
(A and C) Luciferase activity of IFNφ1pro and ISRE in epithelioma papulosum cyprini (EPC) cells transfected with indicated plasmids for 24 hr, and then untreated or infected with spring viremia of carp virus (SVCV) (MOI = 1) or transfected with poly I:C (0.5 μg) for 24 hr before luciferase assays. (B) IB of proteins in EPC cells transfected with indicated plasmids for 24 hr. (D) qPCR analysis of ifn and vig1 in EPC cells transfected with indicated plasmids for 24 hr, and then untreated or infected with SVCV (MOI = 1) or transfected with poly I:C (2 μg) for 24 hr. (E and F) Plaque assay of virus titers in EPC, Ctenopharyngodon idellus kidney (CIK), and Gibel carp brain (GiCB) cells transfected with indicated plasmids for 24 hr, followed by SVCV, Grass carp reovirus (GCRV), and CyHV-2 challenge for 24–72 hr. (G and H) qPCR and immunoblotting (IB) analysis of SVCV genes in epithelioma papulosum cyprini (EPC) cells transfected with indicated plasmids for 24 hr, followed by SVCV challenge for 24 hr. (I) Interferon (IF) analysis of N 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. All experiments were repeated for at least three times with similar results.
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Figure 3—source data 1
PDF file containing original western blots for Figure 3B and H indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig3-data1-v1.zip
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Figure 3—source data 2
Original files for western blot analysis displayed in Figure 3B and H.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig3-data2-v1.zip
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Figure 3—source data 3
Original data for graphs analysis in Figure 3A, C–G and I.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig3-data3-v1.xlsx
Figure 4
CDK2 associates with TBK1 and mediates its degradation.
(A and B) Luciferase activity of IFNφ1pro and ISRE in epithelioma papulosum cyprini (EPC) cells transfected with indicated plasmids for 24 hr. (C and D) qPCR analysis of ifn and vig1 in EPC cells transfected with indicated plasmids for 24 hr. (E) Confocal microscopy of CDK2 and TBK1 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) Immunoblotting (IB) of whole cell lysates (WCLs) and proteins immunoprecipitated with anti-Myc antibody-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 hr. (G) IB of WCLs and proteins immunoprecipitated with anti-TBK1 or anti-CDK2 antibody from EPC cells infected with spring viremia of carp virus (SVCV) for 24 hr. (A–G) Representative experiments are shown (n=3). (H) Schematic representation of full-length TBK1 and its mutants. (I) IB of WCLs and proteins immunoprecipitated with anti-Flag antibody-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 hr. (J) IB of proteins in EPC cells transfected with indicated plasmids for 24 hr. (K and L) IB of proteins in EPC cells transfected with CDK2-HA or shcdk2#1 for 24 hr, followed by untreated or infected with SVCV (MOI = 1) or transfected with poly I:C (2 μg) for 24 hr. (I–L) Representative experiments are shown (n=3). (M) IB of proteins in the liver, spleen, and kidney sections from cdk2+/+ and cdk2-/- zebrafish (n=3 per group). (N) Plaque assay of virus titers in EPC cells transfected with indicated plasmids for 24 hr, followed by SVCV challenge for 24–48 hr. (O and Q) IB and qPCR analysis of SVCV genes in EPC cells transfected with indicated plasmids for 24 hr, followed by SVCV challenge for 24 hr. (P) Interferon (IF) analysis of N 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. (N–P) Representative experiments are shown (n=3).
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Figure 4—source data 1
PDF file containing original western blots for Figure 4F-G, I–M and O indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig4-data1-v1.zip
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Figure 4—source data 2
Original files for western blot analysis displayed in Figure 4F-G, I–M and O.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig4-data2-v1.zip
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Figure 4—source data 3
Original data for graphs analysis in Figure 4A-E, N and P–Q.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig4-data3-v1.xlsx
Figure 5
CDK2 increases the K48-linked polyubiquitination of TBK1.
(A) qPCR analysis of epc-tbk1 in epithelioma papulosum cyprini (EPC) cells transfected with indicated plasmids for 24 hr, and then untreated or infected with spring viremia of carp virus (SVCV) (MOI = 1) or transfected with poly I:C (2 μg) for 24 hr. (B and C) Immunoblotting (IB) 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. (D) IB of proteins in EPC cells transfected with CDK2-HA for 24 hr, followed by untreated or infected with SVCV (MOI = 1) or transfected with poly I:C (2 μg) for 24 hr. Protein lysates were harvested after MG132 (20 μM) treatments (6 hr) for IB analysis. (E) TBK1 ubiquitination assays in EPC cells transfected with indicated plasmids for 18 hr, followed by DMSO or MG132 treatments for 6 hr. (A–E) Representative experiments are shown (n=3). (F) Mass spectrometry analysis of a peptide derived from ubiquitinated TBK1-Myc. (G and H) TBK1 ubiquitination assays in EPC cells transfected with indicated plasmids for 18 hr, followed by MG132 treatments for 6 hr. Representative experiments are shown (n=3).
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Figure 5—source data 1
PDF file containing original western blots for Figure 5B-E and G–H indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig5-data1-v1.zip
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Figure 5—source data 2
Original files for western blot analysis displayed in Figure 5B–E and G–H.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig5-data2-v1.zip
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Figure 5—source data 3
Original data for graphs analysis in Figure 5A.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig5-data3-v1.xlsx
Figure 6
CDK2 recruits Dtx4 to degrade TBK1.
(A–D) Immunoblotting (IB) of whole cell lysates (WCLs) and proteins immunoprecipitated with anti-Myc or Flag Ab-conjugated agarose beads from epithelioma papulosum cyprini (EPC) cells transfected with indicated plasmids for 24 hr. (E and J) Luciferase activity of IFNφ1pro in EPC cells transfected with indicated plasmids for 24 hr. (F and K) qPCR analysis of ifn and vig1 in EPC cells transfected with indicated plasmids for 24 hr. (G and L) IB of proteins in EPC cells transfected with indicated plasmids for 24 hr. (H and M) IB of proteins in EPC cells transfected with CDK2-HA and Dtx4-Myc or shdtx4#1 for 24 hr, followed by untreated or infected with spring viremia of carp virus (SVCV) (MOI = 1) or transfected with poly I:C (2 μg) for 24 hr. All experiments were repeated for at least three times with similar results.
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Figure 6—source data 1
PDF file containing original western blots for Figure 6A-D, G–I and L–M indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig6-data1-v1.zip
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Figure 6—source data 2
Original files for western blot analysis displayed in Figure 6A-D, G–I and L–M.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig6-data2-v1.zip
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Figure 6—source data 3
Original data for graphs analysis in Figure 6E-F and J–K.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig6-data3-v1.xlsx
Figure 7
K567 site is critical for the ubiquitination degradation of TBK1.
(A–C) TBK1 ubiquitination assays in epithelioma papulosum cyprini (EPC) cells transfected with indicated plasmids for 18 hr, followed by MG132 treatments for 6 hr. Representative experiments are shown (n=3). (D) Mass spectrometry analysis to show that K154 and K567 of TBK1 is conjugated with ubiquitin. (E–H) TBK1 ubiquitination assays in EPC cells transfected with indicated plasmids for 18 hr, followed by MG132 treatments for 6 hr. (I) Immunoblotting (IB) of proteins in EPC cells transfected with indicated plasmids for 24 hr. (J) Luciferase activity of IFNφ1pro in EPC cells transfected with indicated plasmids for 24 hr. (K) qPCR analysis of ifn in EPC cells transfected with indicated plasmids for 24 hr. (L) Plaque assay of virus titers in EPC cells transfected with indicated plasmids for 24 hr, followed by spring viremia of carp virus (SVCV) challenge for 24–48 hr. (M and N) qPCR and IB analysis of SVCV genes in EPC cells transfected with indicated plasmids for 24 hr, followed by SVCV challenge for 24 hr. (O) Interferon (IF) analysis of N 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. (E–O) Representative experiments are shown (n=3). (P) Schematic representation of full-length Dtx4 and its mutants. (Q) IB of whole cell lysates (WCLs) and proteins immunoprecipitated with anti-Myc or HA Ab-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 hr. (R) TBK1 ubiquitination assays in EPC cells transfected with indicated plasmids for 18 hr, followed by MG132 treatments for 6 hr. (Q and R) Representative experiments are shown (n=3).
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Figure 7—source data 1
PDF file containing original western blots for Figure 7A-C, E–I, M and Q–R indicating the relevant bands and treatments.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig7-data1-v1.zip
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Figure 7—source data 2
Original files for western blot analysis displayed in Figure 7A-C, E–I, M and Q–R.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig7-data2-v1.zip
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Figure 7—source data 3
Original data for graphs analysis in Figure 7J-L and N-O.
- https://cdn.elifesciences.org/articles/98357/elife-98357-fig7-data3-v1.xlsx
Author response image 1
Additional files
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Supplementary file 1
Primers used in this study.
- https://cdn.elifesciences.org/articles/98357/elife-98357-supp1-v1.docx
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MDAR checklist
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Fish CDK2 recruits Dtx4 to degrade TBK1 through ubiquitination in the antiviral response
eLife 13:RP98357.
https://doi.org/10.7554/eLife.98357.4
