Zebrafish mylipb is upregulated upon poly I:C treatment and SVCV infection.

(A) Zebrafish mylipb as well as ifn1, but not mylipa mRNA was induced by poly I:C in ZFL cells. ZFL cells were seeded in 60-mm plates overnight and treatment with poly I:C (1 μg/mL) for indicate time. (B) Zebrafish mylipb as well as ifn1, but not mylipa mRNA was induced by SVCV (virus strain OMG067) infection (MOI of 1) in ZFL cells. ZFL cells were seeded in 60-mm plates overnight and infected with SVCV for different time. (C) Zebrafish mylipa but not mylipb mRNA was induced by LXR agonist T0901317 in a dose-dependent manne in ZFL cells. ZFL cells were seeded in 60-mm plates overnight and treatment with T0901317 for 24 h. (D) Zebrafish mylipa but not mylipb mRNA was induced by LXR agonist T0901317 (1 μM) in a indicate time in ZFL cells. ZFL cells were seeded in 60-mm plates overnight and treatment with T0901317 for indicate time. Total RNA was extracted for examining the expression of target genes by quantitative real-time PCR (qRT-PCR). All data are presented as mean values based on three repeated experiments, and error bars indicate the ± SD. *,P < 0.05, **, P<0.01; ***, P < 0.001; ****, P< 0.0001.

Zebrafish mylipb exhibits proviral effects both in vivo and in vitro.

(A) Representative images of mylipb-null zebrafish larvae (mylipb^-/-) (3 dpf) and WT siblings (mylipb^+/+) (3 dpf) infected with or without SVCV for 18 h. Zebrafish larvae (3 dpf) were pooled in a disposable 60-mm cell culture dish filled with 5mL of egg water plus 2 mL SVCV (∼2.5×10⁷TCID₅₀/ml). Dead larvae were identified by lack of movement, absence of blood circulation, and bodily degeneration (indicated by red arrows). (B) mylipb-null zebrafish (mylipb^-/-) (n = 48, 3 dpf) were more resistance to SVCV infection than the WT (mylipb^+/+) (n = 48, 3 dpf) based on the survival ratio. SVCV viruses (∼2.50 × 10⁷ TCID₅₀/mL) were added to mylipb-null and WT larvae, and the numbers of dead larvae were counted every hour from 0 to 24 h. (C) mylipb-null zebrafish adults are more resistance to SVCV infection than WT zebrafish. mylipb-null zebrafish (mylipb^-/-)(3 months postfertilization [3 mpf]) displayed less symptoms of abdominal hemorrhage after SVCV infection than WT zebrafish (mylipb^+/+)(3 mpf). The zebrafish were i.p. injected with either 10 μL cell culture medium or 10 μL SVCV(∼2.50 × 10⁷ TCID₅₀/mL). The red arrows indicate hemorrhage regions. (D) mylipb-null zebrafish (mylipb^-/-) (3 mpf) (n = 10) were more resistance to SVCV infection than the WT (mylipb^+/+) (3 mpf) (n = 10) based on the survival ratio. (E) The virus replication number was lower in mylipb-null zebrafish larvae than in the WT zebrafish after infection with SVCV. mylipb-null larvae (mylipb^-/-) and the WT larvae (mylipb^+/+) are offspring of siblings. 30 larvae (3 dpf) were pooled in a disposable 60-mm cell culture dish filled with 5 mL of egg water and 0.5 mL of SVCV (∼2.5×10⁷TCID₅₀/ml).. After challenge for 24 h, the mRNA expression of N protein, P protein, and G protein of SVCV was detected by qRT-PCR analysis. (F-H) The induction of key antiviral genes, including mxc (F), ifn1 (G), lta (H) upon SVCV infection was higher in mylipb-null larvae (mylipb^-/-) compared with the WT larvae (mylipb^+/+). (I) Overexpression of mylipb reduced cell survival after SVCV infection in EPC cells. EPC cells were transfected with Myc-mylipb or empty vector. At 24 h post-transfection, the cells were infected with SVCV at the dose indicated for 48 h. Then, the cells were fixed with 4% paraformaldehyde and stained with 1% crystal violet. (J) Overexpression of mylipb increased virus titer in SVCV-infected EPC cells. The culture supernatant was collected from EPC cells infected with SVCV (MOI of 1), and the viral titer was measured by plaque assay. The results are representative of three independent experiments. (K) Overexpression of mylipb increased copy number of SVCV-related genes in SVCV-infected EPC cells. EPC cells were transfected with Myc-mylipb or empty vector for 24 h and infected with SVCV (MOI of 1). After 24 h, total RNAs were extracted for examining the mRNA levels of the N, P, and G gene of SVCV by qRT-PCR analysis. Western blotting tests to detect the overexpression of mylipb. All data are presented as mean values based on three repeated experiments, and error bars indicate the ± SD. *,P < 0.05, **, P<0.01; ***, P < 0.001; ****, P< 0.0001.

Zebrafish mylipb negatively regulates IFN-I signaling.

(A-B) Overexpression of mylipb suppressed the activity of ISRE reporter (A), zebrafish IFNφ1 reporter (Dr-IFNφ1-Luc.) (B), induced by poly I:C transfected EPC cells. We transfected EPC cells with the indicated reporters (0.1 μg/well) along with Myc-empty vector or Myc-mylipb vector (0.2 μg/well). After 24 h, we transfected the cells with poly I:C (1 μg/mL) for 24 h and then conducted luciferase reporter activity and western blot assays. (C-D) Overexpression of mylipb suppressed the activity of ISRE reporter (C), zebrafish IFNφ1 reporter (Dr-IFNφ1-Luc.) (D), induced by SVCV infection in EPC cells. We transfected EPC cells with the indicated reporters (0.1 μg/well) along with Myc-empty vector or Myc-mylipb vector (0.2 μg/well). After 24 hours, we transfected the cells with SVCV (MOI of 1) for 24 hours and then conducted luciferase reporter activity and western blot assays. (E) Overexpression of mylipb suppressed expression of ifn, isg15, and viperin induced by poly I:C in EPC cells. EPC cells were transfected with Myc-mylipb or empty vector. After 24 hours, we transfected the cells with poly I:C (1 μg/mL) for 24 hours, total RNAs were extracted for examining the mRNA levels of ifn, isg15, and viperin by qRT-PCR analysis. Western blotting tests to detect the overexpression of mylipb. (F) Overexpression of mylipb suppressed expression of ifn, isg15, and viperin induced by SVCV infection in EPC cells. EPC cells were transfected with Myc-mylipb or empty vector for 24 h and infected with SVCV (MOI of 1). After 24 h, total RNAs were extracted for examining the mRNA levels of ifn, isg15, and viperin by qRT-PCR analysis. Western blotting tests to detect the overexpression of mylipb. All data are presented as mean values based on three repeated experiments, and error bars indicate the ± SD. *,P < 0.05, **, P<0.01; ***, P < 0.001; ****, P< 0.0001.

Mylipb interacts with irf3 and induces autophagic degradation of irf3.

(A) Overexpression of mylipb suppressed rig-i–, mda5- mavs-, tbk1-, and irf3-induced activation of ISRE luciferase activity in EPC cells. (B) Overexpression of mylipb suppressed rig-i–, mda5- mavs-, tbk1-, and irf3-induced activation of EPC–IFN promoter luciferase reporter (EPC-IFN-luc) activity in EPC cells. (C) Immunofluorescence staining showed the predominant colocalization of mylipb and irf3 protein in the cytoplasm. HEK293T cells were co-transfected with Myc-mylipb and Flag-irf3. After 24 h, the cells were fixed and observed by confocal microscopy. Red signals represent overexpressed irf3, green signals represent overexpressed mylipb. (D) Mylipb associated with irf3. HEK293T cells seeded in 100-mm dishes were transfected with the indicated plasmids (4 μg each). After 24 h, total cell lysates were immunoprecipitated (IP) with anti-Flag antibody conjugated agarose beads. Then, the immunoprecipitates and cell lysates were detected with anti-HA or anti-Flag Ab, respectively. (E) Mylipb induced the degradation of irf3 in a dose-dependent manner. HEK293T cells were transfected with Myc-empty, Myc-mylipb, and Flag-irf3 for 24 h, and then the cells were harvested to perform immunoblotting. (F) The protein level of irf3 and p-irf3 was higher in mylipb-null zebrfish liver compared with that in WT zebrafish. mylipb-null zebrafish (3 mpf) and their WT siblings (3 mpf) were i.p. injected with (+) or without (-) SVCV at 10 μl/individual for 48 h, then their livers were dissected for Western blot analysis using anti-irf3, anti-p-irf3. (G) Immunoblotting of lysates from HEK293T cells transiently transfected with Myc-empty, Myc-mylipb, and HA-irf3 for 24 h and then cultured in the presence of the proteasome inhibitor, MG132 (20 μM), autophagy inhibitor, 3-MA (1 mM),or DMSO for 12 h. (H) Immunoblotting of lysates from HEK293T cells transiently transfected with Myc-empty, Myc-mylipb, and HA-irf3 for 24 h and then cultured in the presence of the autophagy inhibitor, Baf-A1 (100 nM), or DMSO for 12 h. (I) Immunoblotting of lysates from HEK293T cells transiently transfected with Myc-empty, Myc-mylipb, and HA-irf3 for 24 h and then cultured in the presence of the lysosome inhibitor, NH₄Cl (5 mM), or H₂O for 12 h. (J) Immunoblotting of lysates from HEK293T cells transiently transfected with Myc-empty, Myc-mylipb, and HA-irf3 for 24 h. (K)) HEK293T cells were co-transfected with Myc-mylipb or pCMV-Myc plus Flag-irf3 and GFP-LC3. After 24 h, the cells were fixed and observed by confocal microscopy. Red signals represent overexpressed irf3, green signals represent LC3 positive autophagosome accumulation.

Mylipb negatively regulates irf3 by promoting K6-linkd ubquitination of irf3 at lysine 352.

(A-B) The interaction between mylipb and irf3 protein mostly depended on the ZF domain of mylipb protein. HEK293T cells seeded in 100-mm dishes were transfected with the indicated plasmids (4 μg each). After 24 h, the cells were treated with 3-MA for 8 h. Total cell lysates were immunoprecipitated (IP) with anti-HA antibody conjugated agarose beads. Then, the immunoprecipitates and cell lysates were detected with anti-HA or anti-Flag Ab, respectively. (C) Mylipb promoted irf3 ubiquitination depended on ZF domain and ubiquitin ligase activity of mylipb. HEK 293T cells were transfected with Flag-irf3, Myc-mylipb-WT, Myc-mylipb-Δ381–416, Myc-mylipb-C381S, or empty vector, together with His-ubiquitin. At 24 h posttransfection, the cells were treated with 3-MA for 8 h. The cells were lysed using guanidinium chloride, and purifified with Ni²⁺-NTA agarose. (D) Mylipb induced the degradation of irf3 dependent of its ZF domain. HEK293T cells were transfected with Myc-mylipb-WT or its mutants, together with HA-irf3 for 24 h. The lysates were then subjected to IB with the indicated Abs. (E) Mylipb induced the degradation of irf3 dependent of its ubiquitin ligase activity. HEK293T cells were transfected with Myc-mylipb-WT or Myc-mylipb-C381S, together with HA-irf3 for 24 h. The lysates were then subjected to IB with the indicated Abs. (F) Ubiquitination assay of irf3 in mylipb-null (mylipb^−/−) zebrafish liver and their WT (mylipb^+/+) siblings. mylipb-null zebrafish (3 mpf) and their WT siblings (3 mpf) were i.p. injected with (+) or without (-) SVCV(∼2.50 × 10⁷ TCID₅₀/mL) at 10 μl/individual for 48 h, then their livers were dissected, and lysed with lysis buffer (100 μl). The supernatants were denatured at 95°C for 5 min in the presence of 1% SDS. The denatured lysates were diluted with lysis buffer to reduce the concentration of SDS (<0.1%). Denature-IP was conducted using anti-irf3 antibody and then subjected to immunoblotting with anti-Ubiquitin antibody. (G-H) Mylipb promoted K6-linked poly-ubiquitination of irf3. (I) Mylipb promoted K6-linked poly-ubiquitination at lysine 352 of irf3. (G-I) HEK 293T cells were transfected with Flag-irf3 or HA-irf3, Myc-mylipb, or empty vector, together with His-ubiquitin or its mutants. At 24 h posttransfection, the cells were treated with 3-MA for 8 h. The cells were lysed using guanidinium chloride, and purifified with Ni²⁺-NTA agarose. IB, immunoblot; KO, K-only; KR, K is mutated to R. (J) K352R mutation of irf3 abolished the inhibitory effect of mylipb on ISRE reporter activities in EPC cells. EPC cells were transfected with ISRE reporter and HA-irf3 or its mutants together with Myc-mylipb or empty vector for 24 h, and then conducted luciferase reporter activity assays. (K) K352R mutation abolished degradation of irf3 induced by mylipb. HEK293T cells were transfected with Myc-mylipb or empty vector, together with HA-irf3 or HA-irf3-K352R for 24 h. The lysates were then subjected to IB with the indicated Abs.

Mylipb negatively regulates cellular antiviral response mainly dependent of its ubiquitin ligase activity.

(A) ISRE reporter activity and EPC-IFN-Luc activity in Myc-empty vector or Myc-mylipb-WT and Myc-mylpib-C381S-transfected EPC cells for 24 h, followed by transfection with or without poly I:C (1μg/mL) for 24 h. Western blotting tests to detect the overexpression of mylipb-WT and mylipb-C381S. (B) ISRE reporter activity and EPC-IFN-Luc activity in Myc-empty vector or Myc-mylipb-WT and Myc-mylipb-C381S-transfected EPC cells with or without SVCV (MOI of 1) infection for 24 h. Western blotting tests to detect the overexpression of mylipb-WT and mylipb-C381S. (C) qRT-PCR of ifn, isg15, and viperin mRNA in EPC cells transfected with Myc-empty or or Myc-mylipb-WT and Myc-mylipb-C381S-transfected EPC cells for 24 h, followed by transfection with or without poly I:C (1μg/mL) for 24 h. Western blotting tests to detect the overexpression of mylipb-WT and mylipb-C381S. (D) qRT-PCR of ifn, isg15, and viperin mRNA in EPC cells transfected with Myc-empty or or Myc-mylipb-WT and Myc-mylipb-C381S-transfected EPC cells for 24 h, followed by infection with or without SVCV (MOI of 1) for 24 h. Western blotting tests to detect the overexpression of mylipb-WT and mylipb-C381S. (E) The plaque assay of EPC cells transfected with Myc-empty or or Myc-mylipb-WT and Myc-mylipb-C381S-transfected EPC cells for 24 h, followed by infection with or without SVCV(MOI:0.1–100) for 48 h. (F) Determination of SVCV titre in culture medium of EPC cells transfected with Myc-empty or or Myc-mylipb-WT and Myc-mylipb-C381S-transfected EPC cells for 24 h, followed by infection with or without SVCV(MOI:1) for 48 h. (G) qRT-PCR of N, P, and G mRNA in EPC cells transfected with Myc-empty or or Myc-mylipb-WT and Myc-mylipb-C381S-transfected EPC cells for 24 h, followed by infection with or without SVCV for 24 h. Western blotting tests to detect the overexpression of mylipb-WT and mylipb-C381S. qRT-PCR data are presented as mean values based on three repeated experiments, and error bars indicate the ± SD. *,P < 0.05, **, P<0.01; ***, P < 0.001; ****, P< 0.0001.

Mylipb decreases tbk1-mediated irf3 phosphorylation and cellular antiviral response.

(A) Overexpression of mylipb-WT and mylipb-C381S inhibits tbk1-mediated phosphorylation of irf3. HEK293T cells were transfected with Flag-tbk1 and empty vector or Myc-mylipb-WT or mylipb-C381S, together with HA-irf3 for 24 h. At 24 h posttransfection, the cells were treated with 3-MA for 8 h. The lysates were then subjected to IB with the indicated Abs. (B) Mylipb interacts with tbk1. HEK293T cells seeded in 100-mm dishes were transfected with the indicated plasmids (4 μg each). After 24 h, total cell lysates were immunoprecipitated (IP) with anti-Flag antibody conjugated agarose beads. Then, the immunoprecipitates and cell lysates were detected with anti-Myc or anti-Flag Ab, respectively. (C) Overexpression of mylipb does not significantly disrupt the protein level of tbk1 in dose manner. (D-E) Tbk1 phosphorylates mylipb. HEK293T cells co-transfected with HA-tbk1 or Flag-tbk1 and empty vector together with Myc-mylipb for 24 h. The lysates were then subjected to IB with the indicated Abs. (F) Tbk1 phosphorylates mylipb dependent on its phosphokinase activity. HEK293T cells were transfected with Flag-tbk1 or Flag-tbk1-R47H (enzyme inactivated mutation) and empty vector together with Myc-mylipb for 24 h. The lysates were then subjected to IB with the indicated Abs. (G) T318/S319 is the phosphorylation sites of mylipb catalyzed by tbk1. HEK293T cells were transfected with Myc-mylipb mutants together with Flag-tbk1 or empty vector for 24 h. The lysates were then subjected to IB with the indicated Abs. (H) Mylipb-mut4(T318/S319A) has no obvious effect on phosphorylation of irf3. HEK293T cells were transfected with Flag-tbk1 and empty vector or Myc-mylipb-C381S or mylipb-mut4, together with HA-irf3 for 24 h, and then cultured in the presence of 3-MA (1 mM),or DMSO for 12 h. The lysates were then subjected to IB with the indicated Abs. (I-J) Overexpression of mylipb decreases tbk1-mediated decline of viral titer. EPC cells seeded in 12-well plates overnight were transfected with Flag-tbk1 and Myc-mylipb-C381S or empty vector. At 24 h post-transfection, cells were infected with SVCV (MOI = 1) for 48 h. Then, cells were fixed with 4% PFA and stained with 1% crystal violet (I). Culture supernatants from the cells infected with SVCV were collected, and the viral titer was measured (J). (K) Overexpression of mylipb decreases tbk1-mediated decline of copy number of SVCV genes in SVCV-infected EPC cells. EPC cells were transfected with Flag-tbk1 and Myc-mylipb-C381S or empty vector, cells were infected with SVCV (MOI of 1). After 24 h, total RNAs were extracted for examining the mRNA levels of the N, P, and G gene of SVCV by qRT-PCR analysis. Western blotting tests to detect the overexpression of mylipb and tbk1. (L) Overexpression of mylipb blocks the expression of ifn, viperin and isg15 induced by tbk1. EPC cells were transfected with Flag-tbk1 or empty vector together with Myc-mylipb-C381S or empty vector. At 24 h after transfection, total RNAs were extracted for examining the mRNA levels of the ifn, viperin, and isg15 gene of EPC cells by qRT-PCR analysis. Western blotting tests to detect the overexpression of mylipb and tbk1. All data are presented as mean values based on three repeated experiments, and error bars indicate the ± SD. *,P < 0.05, **, P<0.01; ***, P < 0.001; ****, P< 0.0001.

A model on the two synergistic mechanisms of zebrafish mylipb in negative regulation of antiviral innate immunity.

Upon viral infection, zebrafish rig-i/mda5 recognizes the viral RNA and interacts with mavs, leading to the recruitment of tbk1, which phosphorylates irf3 and then induces IFN production. Zebrafish mylipb, an E3 ubiquitin ligase, increases K6-linked polyubiquitination at Lys 352, leading to the induction of autophagic degradation of irf3, while at the same time reducing irf3 phosphorylation by acting as a substrate for tbk1, thereby preventing the expression of IFN.