Fig 2: CVB3-encoded 3C lowers UBE4A levels to rescue Viperin protein expression.a Immunoprecipitation analysis of ubiquitination of Myc-Viperin in HEK293T cells transfected with Myc-Viperin and HA-Ub, followed by CVB3 infection (MOI = 10) for 12 h. b Western blot analysis of HAT1 and UBE4A levels in HL-1 cells infected with CVB3 (MOI = 10) as indicated. c Western blot analysis of UBE4A levels in mouse primary cardiomyocytes infected with CVB3 (MOI = 10) for 12 and 24 h. d Western blot analysis of Flag-Viperin levels in stable Flag-Viperin-expressing HL-1 cells transfected with shCtrl or shUBE4A and then infected with CVB3 (MOI = 10) for 12 h. e RT-qPCR analysis of Ube4a mRNA levels in HL-1 cells infected with CVB3 (MOI = 10) as indicated. f Western blot analysis of Flag-UBE4A and its cleaved fragment by Flag antibodies in HL-1 cells transfected with Flag-UBE4A, and then infected with CVB3 (MOI = 10) as indicated. g Western blot analysis of Flag-UBE4A levels in HL-1 cells transfected with Flag-UBE4A, together with Flag-tagged CVB3-encoded 2A (left) or 3C (right). h Western blot analysis of Flag-Viperin levels in HL-1 cells co-transfected with Flag-Viperin and Flag-3C, together with shCtrl or shUBE4A. i Multiple alignment of a putative motif targeted by CVB3 3C in different species of UBE4A. j, k Western blot analysis of Flag-UBE4A levels in HL-1 cells transfected with Flag-UBE4A (WT or Q328A), together with Flag-3C transfection (j) or CVB3 infection (MOI = 10, 12 h) (k). l Western blot analysis of STAT1, SGK1 and cytomembrane KCNQ1 levels in Ube4a−/− cells transfected with Flag-UBE4A WT or Q328A, and then infected with or without CVB3 (MOI = 10) for 12 h. m Western blot analysis of UBE4A, Viperin and STAT1 levels in mice without CVB3 infection (Normal, n = 3), or in Ctrl mice (n = 3), or in AHF mice (n = 3). NS, not significant (P > 0.05), two-way analysis of variance (ANOVA). Data are shown as means ± SD of four biological replicates (e), or are representative of three independent experiments (a‒d, f‒h, j‒m).

Fig 3: A proposed model.CVB3-encoded 3C protease cleaves UBE4A at Q328 residue to rescue Viperin, which in turn interacts with STAT1 and promotes STAT1 degradation by UBR5. STAT1 reduction in cardiomyocytes enhances SGK1 signaling and eventually promotes AHF. The IP VS-IP1 prevents CVB3-induced AHF by inhibiting Viperin-STAT1 interaction in vivo.

Fig 4: The IP VS-IP1 restricts CVB3-induced AHF in vivo.a Western blot analysis of Myc-STAT1 levels in HEK293T cells co-transfected with Viperin and Myc-STAT1 (WT or deletion mutants). b Western blot analysis of Myc-STAT1-ΔC1 levels in HL-1 cells co-transfected with Myc-STAT1-ΔC1 and increasing amounts of Myc-Viperin. c Design of the IPs to block Viperin‒STAT1 interaction. d Western blot analysis of STAT1, Viperin and UBE4A levels in HL-1 cells treated with IPs (200 μM) and CVB3 (MOI = 10) for 12 h. e Immunoprecipitation analysis of the interaction between Myc-Viperin and Flag-STAT1 in HL-1 cells transfected with Flag-STAT1 and Myc-Viperin and then treated with VS-IP1 (200 μM) for 12 h. f Immunoprecipitation analysis of STAT1 ubiquitination in HL-1 cells transfected with Flag-STAT1, Myc-Viperin and HA-Ub, and then treated with VS-IP1 (200 μM) for 12 h. g Western blot analysis of STAT1 levels in HL-1 cells transfected with Myc-Viperin and then treated with VS-IP1 (200 μM) for 12 h. h Western blot analysis of STAT1, Viperin and UBE4A in Viperin+/+ or Viperin−/− HL-1 cells treated with VS-IP1 (200 μM) and CVB3 (MOI = 10) for 12 h. i Western blot analysis of SGK1 levels in HL-1 cells transfected with Myc-Viperin and then treated with VS-IP1 (200 μM) for 12 h. j‒l C57BL/6 mice were injected with PBS (n = 6, i.p.) or VS-IP1 (n = 6, i.p., 5 mg/kg) for 12 h and then infected with CVB3 (2 × 105 PFU per gram body, i.p.) for 7 days. The LVEF, LVFS, and E/A ratio in mice were analyzed by echocardiography (j). Serum BNP concentrations were analyzed by ELISA (k). The representative images of the echocardiography were shown (l). ***P < 0.001, two-tailed unpaired Student’s t-test. Data are representative of three independent experiments (a, b, d‒i), or are shown as the means ± SEM for six individual mice (j, k).

Fig 5: Viperin promotes STAT1 ubiquitination and degradation dependently on UBR5.a Western blot analysis of STAT1 levels in HEK293T cells transfected with Flag-Viperin and treated with MG132 (10 μM) as indicated. b Immunoprecipitation analysis of STAT1 ubiquitination in HL-1 cells transfected with or without Myc-Viperin and then treated with MG132 (10 μM, 4 h). c cycloheximide (CHX) pulse chase assay was used to analyze Flag-STAT1 protein stability in HEK293T cells co-transfected with Flag-STAT1 and HA-Viperin, followed by CHX treatment (50 μM) for 12 h and 36 h. d Mass spectrometry analysis of Viperin-binding proteins in HEK293T cells transfected with Flag-Viperin. The putative ubiquitin E3 ligases interacting with Viperin are listed. Western blot was used to analyze Myc-STAT1 levels in cells with knockout of these ubiquitin E3 ligases that were transfected with or without HA-Viperin. e Immunoprecipitation analysis of the interaction between UBR5 and STAT1 in HL-1 cells transfected with or without HA-Viperin. f, g Ubr5+/+ or Ubr5−/− cells were made by CRISPR-Cas9 in both stable Viperin-expressing HEK293T cells (f) and intact HEK293T cells (Viperin protein-deficient) (g). Immunoprecipitation was used to analyze STAT1 ubiquitination. h In vitro ubiquitination assay for analysis of Flag-STAT1 ubiquitination by the recombinant UBR5 proteins. Flag-STAT1 was immunoprecipitated by Flag beads. Flag-STAT1 ubiquitination was determined using an anti-Flag antibody using western blot analysis. i Immunoprecipitation analysis of STAT1 ubiquitination types in stable Viperin-expressing HEK293T cells cotransfected with Myc-STAT1 and different types of HA-Ub. j Western blot analysis of STAT1 levels in Ubr5+/+ or Ubr5−/− cells transfected with HA-Viperin. k Western blot analysis of STAT1, SGK1 and cytomembrane KCNQ1 levels in Ubr5+/+ or Ubr5−/− HL-1 cells infected with or without CVB3 (MOI = 10) for 12 h. l RT-qPCR analysis of Sgk1 mRNA in Ubr5+/+ or Ubr5−/− HL-1 cells infected with or without CVB3 (MOI = 10) for 12 h. NS, not significant (P > 0.05), ***P < 0.001, two-tailed unpaired Student’s t-test. Data are shown as means ± SD of four biological replicates (l) or are representative of three independent experiments (a, b, d‒k).