Fig 1.
Identification of PRV UL13 as an inhibitor of cGAS-STING signaling.
(A) Luciferase activities in UL13 PK-15 or control PK-15 cells cotransfected with IFN-β-luc reporter or ISRE-luc reporter. Twenty-four hours after transfection, cells were left untreated or stimulated with B-DNA (2 μg/ml) for 12 h, and cell lysates were analyzed for luciferase activity. (B and C) qPCR analysis of IFNB1 and downstream ISGs (MX1, ISG56) mRNA expression in UL13-PK-15 cells and control cells stimulated with B-DNA (1 μg/ml) (B) or cGAMP (1 μg/ml) (C) for the indicated times. (D and E) Immunoblotting analysis of cGAS, STING, phosphorylated (Ser172)- and total TBK1 and IKKε, phosphorylated (Ser396)- and total IRF3 in whole-cell lysates of UL13-PK-15 cells and control cells stimulated for the various times (above lanes) with B-DNA (1 μg/ml) (D) or cGAMP (1 μg/ml) (E). Data are pooled from three independent experiments (A-C, mean ± SD) or representative of three independent experiments (D, E). *p < 0.05, **p < 0.01 and ***p < 0.001 (Student’s t test).
Fig 2.
Knockdown of UL13 increases PRV-triggered antiviral responses.
(A) qPCR analysis of PRV-induced transcription of Ifnb1 and downstream genes (Mx1, Isg56) in UL13-RNAi stable mouse embryonic fibroblasts (MEFs) and control cells infected with PRV (MOI = 1) infection for the indicated periods. (B) Immunoblotting analysis of cGAS, STING, and phosphorylation of downstream components in UL13-RNAi MEFs and control cells infected with PRV (MOI = 1) for the indicated times. (C and D) qPCR analysis of transcription of Ifnb1 and downstream genes (Mx1, Isg56) (C) or immunoblotting analysis of cGAS, STING, and phosphorylation of downstream components (D) in MEFs infected with wild type PRV (strain Bartha-K61, PRV-WT, MOI = 1) or UL13-deficient PRV (PRV-ΔUL13, MOI = 1) for the indicated times. Cumulative results for densitometric quantitation of STING were normalized relative to the levels of 0 min conditions (D, right). (E) Luciferase activities in UL13-PK-15 cells and control PK-15 cells cotransfected with IFN-β-luc reporter or ISRE-luc reporter and plasmids encoding STING, TBK1, or IRF3-5D or empty vector as indicated. Twenty-four hours after transfection, cell lysates were analyzed for luciferase activity. (F) Immunoblotting analysis of Flag-UL13 and Myc-STING expression in HEK293T cells cotransfected with Myc-STING, Flag-UL13 or UL13 shRNA plasmids as indicated for 24 h. Data are pooled from three independent experiments (A, C, D, right and E, mean ± SD) or representative of three (B, D, left) or two (F) independent experiments. *p < 0.05, **p < 0.01 and ***p < 0.001, ns, not significant (Student’s t test).
Fig 3.
(A-C) Immunoblotting (IB) analysis of indicated proteins in immunoprecipitated (IP) samples and whole-cell lysates of HEK293T cells transfected with Flag-UL13 and HA-STING (A), whole-cell lysates of PK-15 cells transfected with Flag-UL13 (B), or STING-deficient PK-15 cells transfected with Flag-UL13 (C). Anti-HA (A) or anti-STING (B, C) immunoprecipitates were analyzed by immunoblotting with anti-Flag antibodies. Levels of the transfected proteins were analyzed by immunoblotting with anti-HA (A), anti-STING (B, C), or anti-Flag (A-C) antibodies. (D) Colocalization of UL13 and STING. Hela cells were transfected with Flag-UL13 as indicated for 24 h before confocal microscopy. Endogenous STING was labeled by anti-STING antibody. (E) Colocalization of UL13 and endoplasmic reticulum (ER). Hela cells were transfected with Flag-UL13 for 24 h before confocal microscopy. ER was labeled with anti-Calreticulin antibody. (F) Immunoblotting analysis of UL13 and STING in immunoprecipitated samples or whole-cell lysates of HEK293T cells transfected with HA-UL13 and full length or truncated Myc-STING. Anti-Myc immunoprecipitates were analyzed by immunoblotting with an anti-HA or anti-Myc antibody. Levels of the transfected proteins were analyzed by immunoblotting with an anti-HA or anti-Myc antibody. Scale bars, 10 μm. Data are representative of two independent experiments (A-F).
Fig 4.
UL13 targets STING for K27-/K29-linked ubiquitination and degradation.
(A) Immunoblotting analysis of STING protein expression in whole-cell lysates of PK-15 cells transfected with UL13 expression plasmid or empty vector following treatment with cycloheximide CHX (left), MG-132 (middle), or NH4Cl (right) (10 μM) for the indicated times, respectively. Densitometric quantitation of STING was normalized relative to the levels of 0 h conditions (A, lower). (B-D) Immunoprecipitation analysis of HEK293T cells expressing His-Ubiquitin (Ub) and Myc-STING together with or without Flag-UL13 (B), PK-15 cells expressing His-Ub with or without Flag-UL13 (C), or HEK293T cells expressing His-Ub K48R mutant (Lys-to-Arg mutant at position 48 only) and Myc-STING together with or without Flag-UL13 (D). (E) Immunoprecipitation analysis of HEK293T cells expressing Flag-UL13 and Myc-STING together with wild type (WT) His-Ub or His-Ub mutants at K6, K11, K27, K29, K33, K48, and K63 only; or Lys-to-Arg mutants of all Ub lysines, AKR) as indicated. (F) Immunoprecipitation analysis of HEK293T cells expressing Flag-UL13 and Myc-STING together with WT His-Ub, His-Ub at K27 or K29 only or His-K27R, K29R, or K27/29R only mutants as indicated. (G) Immunoprecipitation analysis of HEK293T cells expressing Flag-UL13 and His-Ub together with wild type (WT) Myc-STING or Myc-STING mutants (K20, K137, K150, K224, K236, K289, K338, K347, and K370 only; or Lys-to-Arg mutants of all STING lysines, AKR) as indicated. Anti-Myc (B, D-G) or anti-STING (C) immunoprecipitates were analyzed by immunoblotting with an anti-His, anti-Myc or anti-STING antibody. Levels of the transfected proteins were analyzed by immunoblotting with anti-Flag, anti-His, anti-Myc or anti-STING antibody as indicated. Data are representative of three (A, upper) or two (B-G) independent experiments, or pooled from three independent experiments (A, lower). *p < 0.05, ***p < 0.001 (Student’s t test).
Fig 5.
UL13 recruits E3 ligase RNF5 to induce STING ubiquitination degradation.
(A to C) Immunoprecipitation analysis of HEK293T cells expressing Flag-UL13 together with HA-TRIM21 and HA-RNF5 (A) or HA-RNF5 (B) or Flag-UL13 alone (C). Anti-Flag (A), anti-HA (B), or anti-RNF5 (C) immunoprecipitates were analyzed by immunoblotting with anti-HA (A) and anti-Flag (B, C) antibodies. Levels of the transfected proteins were analyzed by immunoblotting with anti-Flag and anti-HA antibodies (A, B) or anti-RNF5 antibodies. (D) Colocalization of UL13 and RNF5 in Hela cells. Cells were transfected with HA-UL13 for 24 h before confocal microscopy. Endogenous RNF5 was labeled by anti-RNF5 antibody. (E) Immunoprecipitation analysis of HEK293T cells expressing Myc-STING and HA-RNF5 with or without Flag-UL13. Anti-Myc immunoprecipitates were analyzed by immunoblotting with anti-HA and anti-Flag antibodies. The levels of the transfected proteins were analyzed by immunoblotting with anti-Flag, anti-HA, or anti-Myc antibodies. (F, G) Immunoprecipitation analysis of HEK293T cells expressing Flag-UL13, Myc-STING, and His-Ub (WT, K27, or K29 only) with or without HA-RNF5 (F) or HEK293T cells expressing Flag-UL13 and Myc-STING together with His-Ub (WT, K27R, K29R, or K27/29R only mutants) as indicated with or without HA-RNF5 (G). Anti-Myc immunoprecipitates were analyzed by immunoblotting with anti-His, anti-Flag, and anti-HA antibodies (F, G). The levels of the transfected proteins were analyzed by immunoblotting with anti-Flag, anti-HA, or anti-Myc antibodies. Scale bars, 10 μm (D). Data are representative of two independent experiments (A-G).
Fig 6.
RNF5-deficiency potentiates PRV-induced antiviral immune responses.
(A) Immunoblotting analysis of indicated proteins in immunoprecipitated samples and whole-cell lysates of HEK293T cells transfected with RNF5 siRNA or control siRNA (50 μM). Twenty-four hours after transfection, cells were further transfected with Flag-UL13, Myc-STING, and His-Ub for another 24 h before analysis. (B) Immunoblotting analysis of protein levels of RNF5, UL13, and STING in whole-cell lysates of HEK293T cells transfected with RNF5 siRNA or control siRNA for 24 h. Cells were then transfected with Flag-UL13 and Myc-STING for the indicated times (above lane). (C) Immunoblotting analysis of RNF5 protein expression in wild type (WT) and RNF5-deficient (RNF5 KO) MEFs. (D) qPCR analysis of transcription of Ifnb1 and downstream genes (Mx1, Isg56) in WT and RNF5 KO MEFs infected with PRV (MOI = 1) for the indicated times. (E) Immunoblotting analysis of cGAS, STING, phosphorylated (Ser172) and total TBK1 and IKKε, phosphorylated (Ser396) and total IRF3, or RNF5 in whole-cell lysates of WT and RNF5 KO MEFs infected with PRV (MOI = 1) for the indicated times. (F) qPCR analysis of mRNA expression of Ifnb1 and downstream genes (Mx1, Isg56) in RNF5 KO MEFs infected with PRV-WT or PRV-ΔUL13 (MOI = 1) for indicated times. Data are representative of two (A, C) or three (B, E) independent experiments, or are pooled from three independent experiments (D, F, mean ± SD). *p < 0.05, **p < 0.01 (Student’s t test).
Fig 7.
UL13 deficiency enhances host defense against PRV infection in vivo.
(A) Survival rate of C57BL/6 mice (n = 7 in each group) infected with a lethal dose (0.5 ×106 PFU, the same below) of PRV-WT or PRV-ΔUL13. (B) ELISA of IFN-β in serum from mice infected with a lethal dose of PRV-WT or PRV-ΔUL13 (n = 5 in each group). Serum was collected 24 h post-infection. (C) qPCR analysis of viral propagation in tissue of mouse brain and lungs at day 6 after infection with a lethal dose of PRV-WT or PRV-ΔUL13 (n = 5 in each group). (D) Hematoxylin and eosin (H&E) staining of sections of brain and lung of mice as in (C); arrows indicate cerebral vascular congestion and hemorrhage in brain; triangles indicate diffuse alveolar hemorrhage or alveolar exudates. Scale bars, 50 μm. Original magnification, ×40. (E) Body weight loss of mice infected with PRV-WT or PRV-ΔUL13 as in A at day 6. (F) A working model of how UL13 regulates the STING-mediated immune response. Data are representative of two independent experiments (A, D) or one independent experiment (B, C, E; mean ± SD). *p < 0.05, **p < 0.01 (Student’s unpaired t test or log-rank test in A).