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 PBS (5 μL/individual), SVCV (5 × 108 TCID50/mL, 5 μL/individual) for 48 h. 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 EPC cells infected with VSV, CyHV-2, or SVCV for the indicated times. (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 h. (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 h. (F) IB of proteins in ZF4 and EPC cells infected with SVCV for the indicated times.

CDK2 deficiency protects fish from 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 H&E-stained liver, spleen, and kidney sections from cdk2+/+ and cdk2-/- zebrafish treated with SVCV for 48 h. (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 h. (D) IB of proteins in the liver, spleen, and kidney sections from cdk2+/+ and cdk2-/- zebrafish (n = 3 per group) treated with SVCV for 48 h. (E) CDK2 regulates antiviral response-relevant target genes, presented as a volcano plot of genes with differential expression after SVCV infection in the liver of cdk2+/+ and cdk2-/- zebrafish. (F) GSEA of differentially expressed genes in the liver of cdk2+/+ and cdk2-/- zebrafish with SVCV infection and enrichment of IFN. FDR (q-value) was shown. (G) Heatmap view of mRNA variations of IFN-mediated 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 h.

CDK2 negatively regulates IFN production and promotes viral replication.

(A and C) Luciferase activity of IFNφ1pro and ISRE in EPC cells transfected with indicated plasmids for 24 h, and then untreated or infected with SVCV (MOI = 1) or transfected with poly I:C (0.5 μg) for 24 h before luciferase assays. (B) IB of proteins in EPC cells transfected with indicated plasmids for 24 h. (D) 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) or transfected with poly I:C (2 μg) for 24 h. (E and F) Plaque assay of virus titers in EPC, CIK, and GiCB cells transfected with indicated plasmids for 24 h, followed by SVCV, GCRV, and CyHV-2 challenge for 24-72 h. (G and H) qPCR and IB analysis of SVCV genes in EPC cells transfected with indicated plasmids for 24 h, followed by SVCV challenge for 24 h. (I) IF analysis of N 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.

CDK2 associates with TBK1 and mediates its degradation.

(A and B) Luciferase activity of IFNφ1pro and ISRE in EPC cells transfected with indicated plasmids for 24 h. (C and D) qPCR analysis of ifn and vig1 in EPC cells transfected with indicated plasmids for 24 h. (E) Confocal microscopy of CDK2 and TBK1 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. (F) IB of WCLs and proteins immunoprecipitated with anti-Myc antibody-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 h. (G) IB of WCLs and proteins immunoprecipitated with anti-TBK1 or anti-CDK2 antibody from EPC cells infected with SVCV for 24 h. (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 h. (J) IB of proteins in EPC cells transfected with indicated plasmids for 24 h. (K and L) IB of proteins in EPC cells transfected with CDK2-HA or shcdk2#1 for 24 h, followed by untreated or infected with SVCV (MOI = 1) or transfected with poly I:C (2 μg) for 24 h. (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 h, followed by SVCV challenge for 24-48 h. (O and Q) IB and qPCR analysis of SVCV genes in EPC cells transfected with indicated plasmids for 24 h, followed by SVCV challenge for 24 h. (P) IF analysis of N 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.

CDK2 increases the K48-linked polyubiquitination of TBK1.

(A) qPCR analysis of epc-tbk1 in EPC cells transfected with indicated plasmids for 24 h, and then untreated or infected with SVCV (MOI = 1) or transfected with poly I:C (2 μg) for 24 h. (B and C) IB 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. (D) IB of proteins in EPC cells transfected with CDK2-HA for 24 h, followed by untreated or infected with SVCV (MOI = 1) or transfected with poly I:C (2 μg) for 24 h. Protein lysates were harvested after MG132 (20 μM) treatments (6 h) for IB analysis. (E) TBK1 ubiquitination assays in EPC cells transfected with indicated plasmids for 18 h, followed by DMSO or MG132 treatments for 6 h. (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 h, followed by MG132 treatments for 6 h.

CDK2 recruits Dtx4 to degrade TBK1.

(A-D) IB of WCLs and proteins immunoprecipitated with anti-Myc or Flag Ab-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 h. (E and J) Luciferase activity of IFNφ1pro in EPC cells transfected with indicated plasmids for 24 h. (F and K) qPCR analysis of ifn and vig1 in EPC cells transfected with indicated plasmids for 24 h. (G and L) IB of proteins in EPC cells transfected with indicated plasmids for 24 h. (H and M) IB of proteins in EPC cells transfected with CDK2-HA and Dtx4-Myc or shdtx4#1 for 24 h, followed by untreated or infected with SVCV (MOI = 1) or transfected with poly I:C (2 μg) for 24 h.

K567 site is critical for the ubiquitination degradation of TBK1.

(A-C) TBK1 ubiquitination assays in EPC cells transfected with indicated plasmids for 18 h, followed by MG132 treatments for 6 h. (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 h, followed by MG132 treatments for 6 h. (I) IB of proteins in EPC cells transfected with indicated plasmids for 24 h. (J) Luciferase activity of IFNφ1pro in EPC cells transfected with indicated plasmids for 24 h. (K) qPCR analysis of ifn in EPC cells transfected with indicated plasmids for 24 h. (L) Plaque assay of virus titers in EPC cells transfected with indicated plasmids for 24 h, followed by SVCV challenge for 24-48 h. (M and N) qPCR and IB analysis of SVCV genes in EPC cells transfected with indicated plasmids for 24 h, followed by SVCV challenge for 24 h. (O) IF analysis of N 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. (P) Schematic representation of full-length Dtx4 and its mutants. (Q) IB of WCLs and proteins immunoprecipitated with anti-Myc or HA Ab-conjugated agarose beads from EPC cells transfected with indicated plasmids for 24 h. (R) TBK1 ubiquitination assays in EPC cells transfected with indicated plasmids for 18 h, followed by MG132 treatments for 6 h.