TANK-Binding Kinase 1 (TBK1) Isoforms Negatively Regulate Type I Interferon Induction by Inhibiting TBK1-IRF3 Interaction and IRF3 Phosphorylation - PubMed (original) (raw)
TANK-Binding Kinase 1 (TBK1) Isoforms Negatively Regulate Type I Interferon Induction by Inhibiting TBK1-IRF3 Interaction and IRF3 Phosphorylation
Yi Wei Hu et al. Front Immunol. 2018.
Abstract
TANK-binding kinase 1 (TBK1) is an important serine/threonine-protein kinase that mediates phosphorylation and nuclear translocation of IRF3, which contributes to induction of type I interferons (IFNs) in the innate antiviral response. In mammals, TBK1 spliced isoform negatively regulates the virus-triggered IFN-β signaling pathway by disrupting the interaction between retinoic acid-inducible gene I (RIG-I) and mitochondria antiviral-signaling protein (MAVS). However, it is still unclear whether alternative splicing patterns and the function of TBK1 isoform(s) exist in teleost fish. In this study, we identify two alternatively spliced isoforms of TBK1 from zebrafish, termed TBK1_tv1 and TBK1_tv2. Both TBK1_tv1 and TBK1_tv2 contain an incomplete STKc_TBK1 domain. Moreover, the UBL_TBK1_like domain is also missing for TBK1_tv2. TBK1_tv1 and TBK1_tv2 are expressed in zebrafish larvae. Overexpression of TBK1_tv1 and TBK1_tv2 inhibits RIG-I-, MAVS-, TBK1-, and IRF3-mediated activation of IFN promoters in response to spring viremia of carp virus infection. Also, TBK1_tv1 and TBK1_tv2 inhibit expression of IFNs and IFN-stimulated genes induced by MAVS and TBK1. Mechanistically, TBK1_tv1 and TBK1_tv2 competitively associate with TBK1 and IRF3 to disrupt the formation of a functional TBK1-IRF3 complex, impeding the phosphorylation of IRF3 mediated by TBK1. Collectively, these results demonstrate that TBK1 spliced isoforms are dominant negative regulators in the RIG-I/MAVS/TBK1/IRF3 antiviral pathway by targeting the functional TBK1-IRF3 complex formation. Identification and functional characterization of piscine TBK1 spliced isoforms may contribute to understanding the role of TBK1 expression in innate antiviral response.
Keywords: IRF3; TANK-binding kinase 1; TANK-binding kinase 1 spliced isoforms; alternative splicing; immune homeostasis; spring viremia of carp virus; type I interferon signaling.
Figures
Figure 1
Identification of two spliced isoforms of zebrafish TANK-binding kinase 1 (TBK1). (A) Genomic and domain organizations of zebrafish TBK1 and its isoforms. Exons are square boxes; introns are straight lines. (B) Multiple alignments of amino acid sequences of zebrafish TBK1 and its isoforms. STKc_TBK1 and UBL_TBK1_like domains were indicated in two-way arrows. The amino acids which were used as immunogen against TBK1 polyclonal antibody are shown in red. (C) Expression of zebrafish TBK1_tv1 and TBK1_tv2 isoforms during development. Total RNA was extracted from development samples including 6, 24, 48, 72, and 120 h postfertilization. Data represent mean ± SEM (n = 3). (D) Expression of zebrafish TBK1 protein during development. Total protein was extracted from development samples including 6, 24, 48, 72, and 120 h postfertilization. Each Western blot is representative of two independent experiments. (E) Protein expression of zebrafish TBK1 and its isoforms in ZF4 cells with/without spring viremia of carp virus (SVCV) infection. ZF4 cells were passaged in six-well plates at 1 × 106 cells per well. Then the cells were infected with SVCV at a multiplicity of infection of 1. The cells were collected at 36 h post infection and used for protein extraction. Cell lysates were immunoblotted using anti-TBK1 antibody (1:5,000, upper panels) and anti-GAPDH antibody (1:2,000, lower panels). Each Western blot is representative of at least two independent experiments.
Figure 1
Identification of two spliced isoforms of zebrafish TANK-binding kinase 1 (TBK1). (A) Genomic and domain organizations of zebrafish TBK1 and its isoforms. Exons are square boxes; introns are straight lines. (B) Multiple alignments of amino acid sequences of zebrafish TBK1 and its isoforms. STKc_TBK1 and UBL_TBK1_like domains were indicated in two-way arrows. The amino acids which were used as immunogen against TBK1 polyclonal antibody are shown in red. (C) Expression of zebrafish TBK1_tv1 and TBK1_tv2 isoforms during development. Total RNA was extracted from development samples including 6, 24, 48, 72, and 120 h postfertilization. Data represent mean ± SEM (n = 3). (D) Expression of zebrafish TBK1 protein during development. Total protein was extracted from development samples including 6, 24, 48, 72, and 120 h postfertilization. Each Western blot is representative of two independent experiments. (E) Protein expression of zebrafish TBK1 and its isoforms in ZF4 cells with/without spring viremia of carp virus (SVCV) infection. ZF4 cells were passaged in six-well plates at 1 × 106 cells per well. Then the cells were infected with SVCV at a multiplicity of infection of 1. The cells were collected at 36 h post infection and used for protein extraction. Cell lysates were immunoblotted using anti-TBK1 antibody (1:5,000, upper panels) and anti-GAPDH antibody (1:2,000, lower panels). Each Western blot is representative of at least two independent experiments.
Figure 2
The intact kinase domain is critical for induction of interferons (IFNs) and phosphorylation of IRF3. (A,B) Effect of zebrafish TANK-binding kinase 1 (TBK1) and its isoforms on the IFNφ1 (DrIFNφ1pro-luc) and IFNφ3 (DrIFNφ3pro-luc) promoters with/without spring viremia of carp virus (SVCV) infection. Epithelioma papulosum cyprini (EPC) cells seeded in 24-well plates were cotransfected with 250 ng various indicated plasmids, together with 25 ng Renilla and 250 ng IFNφ1 or IFNφ3 reporter plasmids. Thirty hours post-transfection, the cells were infected with SVCV at a multiplicity of infection (MOI) of 1 or were left untreated. Another 16 h later, the cells were harvested for the detection of luciferase activity. Data represent mean ± SEM (n = 3) and were tested for statistical significance using a one-way ANOVA followed by a Tukey test. *p < 0.05, **p < 0.01. (C) The role of zebrafish TBK1 and its isoforms in the SVCV infection. EPC cells seeded overnight in 24-well plates at 3 × 105 cells per well were transiently transfected with 500 ng TBK1-FLAG, TBK1_tv1-FLAG, TBK1_tv2-FLAG, or FLAG empty plasmid. Thirty hours post-transfection, the cells were infected with SVCV at an MOI of 5, 0.5, or 0.05, respectively. Another 48 h later, the supernatants were collected for the determination of virus titers by standard plaque assay. The plates fixed in 10% paraformaldehyde for 1 h were stained with 0.5% crystal violet and photographed. Data represent mean ± SEM (n = 3) and were tested for statistical significance using a one-way ANOVA followed by a Tukey test. **p < 0.01. (D) Effect of zebrafish TBK1 and its isoforms on protein expression of IRF3. For Western blotting analysis, EPC cells were plated in six-well plates, incubated overnight, and subsequently transfected with 2 μg various indicated plasmids. After 36 h, the cells were collected and used for protein extraction. Cell lysates were immunoblotted using anti-FLAG antibody (upper panels, 1:5,000), anti-IRF3 antibody (middle panels, 1:5,000), and anti-GAPDH antibody (lower panels, 1:2,000). Each Western blot is representative of at least three independent experiments. (E) Effect of zebrafish TBK1_tv1 and TBK1_tv2 isoforms on expression of antiviral genes. Embryos were microinjected at the one-cell stage with 100 ng of indicated expression construct. At 24 h postfertilization, 15 embryos for each sample were used for RNA extraction. The p3xFLAG-injected group was used for control. Data were expressed as mean ± SEM (n = 3) of three independent experiments. **p < 0.01.
Figure 3
TANK-binding kinase 1 (TBK1) isoforms negatively regulate type I interferon (IFN) signaling mediated by TBK1 (A), retinoic acid-inducible gene I (RIG-I) (B), mitochondria antiviral-signaling protein (MAVS) (C), and IRF3 (D). Epithelioma papulosum cyprini (EPC) cells seeded overnight in 24-well plates at 3 × 105 cells per well were transiently transfected with various indicated plasmids with indicated DNA concentration, together with 25 ng Renilla and 250 ng IFNφ1 or IFNφ3 reporter plasmids. Thirty hours post-transfection, the cells were infected with spring viremia of carp virus (SVCV) at a multiplicity of infection of 1. Another 16 h later, the cells were harvested for the detection of luciferase activity. Data represent mean ± SEM (n = 3) and were tested for statistical significance using a one-way ANOVA followed by a Tukey test. *p < 0.05, **p < 0.01. The asterisk above the error bars indicated statistical significance using the group transfected with empty plasmid with SVCV infection as the control group. The asterisk above the bracket indicated statistical significance between the two groups connected by the bracket.
Figure 4
TANK-binding kinase 1 (TBK1) isoforms are negative regulators for mitochondria antiviral-signaling protein (MAVS)-mediated innate immunity. (A) Effects of zebrafish TBK1_tv1 and TBK1_tv2 isoforms on expression of interferons (IFNs) and IFN-stimulated genes induced by MAVS. Embryos were microinjected at the one-cell stage with 100 ng of indicated expression construct. At 48 h postfertilization, 15 embryos for each sample were used for RNA extraction. The p3xFLAG-injected group was used for control. Data were expressed as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01. (B) Effects of zebrafish TBK1 and its isoforms on antiviral activity mediated by MAVS. The upper panels were transfected cells with spring viremia of carp virus (SVCV) infection. The lower panels were transfected cells without infection. (C) Effects of zebrafish TBK1 and its isoforms on SVCV replication mediated by MAVS. Epithelioma papulosum cyprini cells seeded overnight in 24-well plates at 3 × 105 cells per well were transiently transfected with 400 ng various indicated plasmids. Thirty hours post-transfection, the cells were infected with SVCV at a multiplicity of infection of 5. Another 48 h later, the supernatants were collected for the determination of virus titers by standard plaque assay. Plates fixed in 10% paraformaldehyde for 1 h were stained with 0.5% crystal violet and photographed. Data represent mean ± SEM (n = 3) and were tested for statistical significance. *p < 0.05, **p < 0.01.
Figure 5
TANK-binding kinase 1 (TBK1) isoforms are negative regulators for TBK1-mediated innate immunity. (A) Effects of zebrafish TBK1_tv1 and TBK1_tv2 isoforms on the expression of interferons (IFNs) and IFN-stimulated genes induced by TBK1. Embryos were microinjected at the one-cell stage with 100 ng of indicated expression construct. At 24 h postfertilization, 15 embryos for each sample were used for RNA extraction. The p3xFLAG-injected group was used for control. Data were expressed as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01. (B) Effects of zebrafish TBK1_tv1 and TBK1_tv2 isoforms on antiviral activity mediated by TBK1. The upper panels were transfected cells with spring viremia of carp virus (SVCV) infection. The lower panels were transfected cells without infection. (C) Effects of zebrafish TBK1_tv1 and TBK1_tv2 isoforms on SVCV replication mediated by TBK1. Epithelioma papulosum cyprini cells seeded overnight in 24-well plates at 3 × 105 cells per well were transiently transfected with 400 ng various indicated plasmids. Thirty hours post-transfection, the cells were infected with SVCV at a multiplicity of infection of 5. Another 48 h later, the supernatants were collected for the determination of virus titers by standard plaque assay. The plates fixed in 10% paraformaldehyde for 1 h were stained with 0.5% crystal violet and photographed. Data represent mean ± SEM (n = 3) and were tested for statistical significance. *p < 0.05, **p < 0.01.
Figure 6
TANK-binding kinase 1 (TBK1) isoforms inhibit IRF3 phosphorylation mediated by TBK1. (A) Western blot analysis of the effect of TBK1 isoforms on IRF3 phosphorylation mediated by TBK1. (B) Densitometric analysis of the effect of TBK1 isoforms on IRF3 phosphorylation mediated by TBK1. For Western blot analysis, epithelioma papulosum cyprini cells were plated in six-well plates, incubated overnight, and subsequently transfected with 1.5 μg of each of the plasmids. After 36 h, cells were collected and used for protein extraction. Cell lysates were immunoblotted using anti-IRF3 antibody (1:5,000, upper panels), anti-GAPDH antibody (1:2,000, middle panels), and anti-GFP antibody (1:5,000, lower panels). The levels of phosphorylated IRF3 were quantified by measuring the band densitometry using Quantity One software (BioRad), which are normalized to band densitometry of GAPDH. Each Western blot is representative of at least two independent experiments.
Figure 7
TANK-binding kinase 1 (TBK1) isoforms associate with TBK1 and IRF3 to disrupt TBK1-IRF3 interaction in uninfected HEK293T cells. HEK 293T cells seeded in 25 cm2 cell culture flask were transfected with various indicated plasmids. 24 h post transfection, cell lysates were immunoprecipitated with anti-FLAG antibody (covalently conjugated to agarose beads) and immunoblotted with anti-GFP (A) or anti-FLAG (B) antibody. The input proteins were also analyzed by immunoblotting with anti-GFP (C) or anti-FLAG (D) antibody.
Figure 8
TANK-binding kinase 1 (TBK1) isoforms associate with TBK1 and IRF3 to disrupt TBK1-IRF3 interaction in epithelioma papulosum cyprini (EPC) cells infected with spring viremia of carp virus (SVCV). EPC cells seeded in 25 cm2 cell culture flask were transfected with various indicated plasmids. After 24 h post transfection, the cells were infected with SVCV at a multiplicity of infection of 1. Another 24 h later, cell lysates were immunoprecipitated with anti-FLAG antibody (covalently conjugated to agarose beads) and immunoblotted with anti-IRF3 (A) or anti-GFP (B) antibody. The input proteins were also analyzed by immunoblotting with anti-IRF3 (C), anti-FLAG (D), anti-GFP (E), or anti-GAPDH (F) antibody.
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