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. 2019 Dec 11;10(12):946.
doi: 10.1038/s41419-019-2178-9.

G3BP1 Inhibits RNA Virus Replication by Positively Regulating RIG-I-mediated Cellular Antiviral Response

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Free PMC article

G3BP1 Inhibits RNA Virus Replication by Positively Regulating RIG-I-mediated Cellular Antiviral Response

Wenping Yang et al. Cell Death Dis. .
Free PMC article

Abstract

Retinoic acid-inducible gene I (RIG-I) is a pattern recognition receptor and is involved in the innate immune response against RNA viruses infection. Here, we demonstrate that the Ras-GTPase-activating protein SH3-domain-binding protein 1 (G3BP1) serves as a positive regulator of the RIG-I-mediated signaling pathway. G3BP1-deficient cells inhibited RNA virus-triggered induction of downstream antiviral genes. Furthermore, we found that G3BP1 inhibited the replication of Sendai virus and vesicular stomatitis virus, indicating a positive regulation of G3BP1 to cellular antiviral responses. Mechanistically, G3BP1 formed a complex with RNF125 and RIG-I, leading to decreased RNF125 via its auto-ubiquitination; thus, promoting expression of RIG-I. Overall, the results suggest a novel mechanism for G3BP1 in the positive regulation of antiviral signaling mediated by RIG-I.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1
Fig. 1. G3BP1 positively regulates SeV-triggered signaling.
ac G3BP1 activates SeV-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 105) were transfected with IFN-β reporter, ISRE, and Nifty (0.1 μg), TK (0.02 μg), and G3BP1 plasmids (0.1 μg) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. df G3BP1 activates poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. HEK293T cells (1 × 105) were transfected with IFN-β reporter, ISRE and Nifty (0.1 μg), TK (0.02 μg) and G3BP1 plasmids (0.1 μg) for 24 h and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. g Effects of G3BP1 overexpression on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. HEK293T cells stably overexpressing G3BP1 were infected or uninfected with SeV for the indicated time and then immunoblotting analysis was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. hj G3BP1 activates the IFN-β promoter, ISRE, and Nifty in a dose-dependent manner. HEK293T cells (1 × 105) were transfected with the IFN-β reporter (0.1 μg) and increasing amounts of G3BP1 plasmid (0, 50, 100, and 200 ng) for 24 h and then uninfected or infected with SeV for 12 h before luciferase assays were performed. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, two-tailed t-test. EV empty vector, Luc luciferase.
Fig. 2
Fig. 2. Effects of G3BP1 knockdown on virus-triggered signaling.
a Effects of G3BP1-RNAi plasmids on exogenous or endogenous G3BP1 expression. bd G3BP1 knockdown inhibits SeV-induced IFN-β promoter, ISRE, and Nifty. Stable G3BP1-RNAi knockdown cells (1 × 105) were transfected with the IFN-β reporter, ISRE and Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then uninfected or infected with SeV for 12 h before luciferase assays. The experiment was repeated in triplicates. eg G3BP1 knockdown inhibits poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. Stable G3BP1-knockdown HEK293T cells (1 × 105) were transfected with the IFN-β reporter, ISRE, Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then transfected with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. The experiment was repeated in triplicates. h Effects of G3BP1 knockdown on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. Stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time before immunoblotting was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. Data are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, two-tailed t-test. Coni control-RNAi, Luc luciferase.
Fig. 3
Fig. 3. G3BP1-knockout suppresses SeV- and poly (I:C)-triggered signaling.
a Deficiency of G3BP1 in the KO clones was confirmed by immunoblotting with anti-G3BP1. The G3BP1-deficient HEK293T clones were generated by the CRISPR-Cas9 method. bg G3BP1 KO inhibits SeV- or poly (I:C)-induced IFN-β promoter, ISRE, and Nifty. G3BP1-deficient HEK293T cells (1 × 105) were transfected with the IFN-β reporter, ISRE, and Nifty (0.1 μg), and TK (0.02 μg) for 24 h, and then stimulated with SeV for 12 h or with poly (I:C) (1 μg/ml) for 18 h before luciferase assays were performed. Meanwhile, the unstimulated cells were used as the controls. The experiment was repeated in triplicates. h Effects of G3BP1 deficiency on SeV-induced phosphorylation of TBK1, IRF3, P65, and Iκbα. G3BP1-deficient HEK293T cells were uninfected or infected with SeV for the indicated time before immunoblotting was performed. For the phosphorylation of TBK1, IRF3, P65, and Iκbα, band intensities were determined by Image J software. i, j G3BP1-deficient HEK293T cells were reconstituted with G3BP1 by retroviral-mediated gene transfer. The experiments were similarly described in b. Data are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, two-tailed t-test. KO knockout, WT wild-type, Luc luciferase.
Fig. 4
Fig. 4. G3BP1 positively regulates the cellular antiviral response.
a, b G3BP1-overexpressed HEK293T cell lines a or G3BP1-deficient HEK293T cells b were infected with SeV or VSV-GFP (MOI = 0.1) for the indicated time, and then the cell lysates were analyzed by immunoblotting with the antibodies against SeV, GFP, or β-actin. c Effects of G3BP1 on SeV and VSV infection. G3BP1-overexpressed or G3BP1-deficient and control HEK293T cells were infected with SeV for 12 h or with VSV-GFP (MOI = 0.1) for 4 h. The mRNA level of the SeV P and VSV P proteins in cells was determined by qRT-PCR. The experiment was repeated in triplicates. d Effects of G3BP1-overexpressed on VSV titer. G3BP1-overexpressed HEK293T cells were transfected with 1 μg/ml poly (I:C) for 16 h and infected with VSV-GFP (MOI = 0.1) for 18 h. Supernatants were then analyzed for VSV production by standard plaque assays. The experiment was repeated in triplicates. e G3BP1-overexpressed HEK293T cells were infected with VSV-GFP (MOI = 0.1) for 4 h. Images were captured by fluorescence microscopy. In addition, the GFP fluorescence levels in VSV-GFP-infected cells were analyzed by flow cytometry. The experiment was repeated in triplicates. f Effects of G3BP1-deficient on VSV titer. The experiments were similarly to those described in c. The experiment was repeated in triplicates. g G3BP1-deficient HEK293T cells were infected with VSV-GFP (MOI = 0.1) for 2 h. Images were then captured by fluorescence microscopy. In addition, the GFP fluorescence levels in VSV-GFP-infected cells were analyzed by flow cytometry. The experiment was repeated in triplicates. qRT-PCR, quantitative real-time polymerase chain reaction. The experiments were similarly described in b. Data are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, two-tailed t-test.
Fig. 5
Fig. 5. G3BP1 targets at upstream of MAVS.
a, b Effects of G3BP1 knockdown on the IFN-β promoter and ISRE activation. The control or stable G3BP1-knockdown HEK293T cells (1 × 105) were transfected with the IFN-β promoter a or ISRE reporter b (0.1 μg), TK (0.02 μg), and the indicated plasmids (0.1 μg each) for 24 h followed by luciferase assays. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, two-tailed t-test. EV empty vector, Luc luciferase, NC control RNAi.
Fig. 6
Fig. 6. G3BP1 interacts with RIG-I.
a G3BP1 interacts with RIG-I but not with MDA5, MAVS, TBK1, or IRF3 in the overexpression system. HEK293T cells (2 × 106) were transfected with the indicated plasmids (5 μg of each) for 24 h. Then Co-IP and immunoblotting analysis were performed with the indicated antibodies. b Endogenous G3BP1 interacted with RIG-I in HEK293T cells. HEK293T cells (5 × 107) were uninfected or infected with SeV for the indicated time. Co-IP and immunoblotting experiments were performed with the indicated antibodies. c, d Domain identification of G3BP1 and RIG-I interaction. HEK293T cells were transfected with the expression plasmids encoding RIG-I and G3BP1 or the corresponding mutants (5 μg each) for 24 h. Co-IP and immunoblotting were performed with the indicated antibodies. e, f Effects of G3BP1 overexpression and its mutants on SeV-triggered IFN-β promoter and ISRE activation. HEK293T cells (1 × 105) were transfected with the IFN-β promoter or ISRE reporter (0.1 μg) and the indicated expression plasmids (0.1 μg) for 24 h. Then cells were uninfected or infected with SeV for 12 h before luciferase assays. The experiment was repeated in triplicates. Data are mean ± SD of three independent experiments. Co-IP Co-immunoprecipitation, EV empty vector, Luc luciferase, αF anti-Flag, αH anti-HA, HC heavy chain, WT wild-type.
Fig. 7
Fig. 7. G3BP1 antagonizes RNF125-mediated degradation of RIG-I.
a, b Dose-dependent effects of G3BP1 on the expression of RIG-I or IRF3. HEK293T cells (4 × 105) were transfected with the G3BP1 (0, 1, and 2 μg) and the RIG-I or IRF3 (2 μg) plasmids for 24 h. Then the cell lysates were subjected to immunoblotting with the indicated antibodies. For the HA-RIG-I, HA-G3BP1 and HA-IRF3, band intensities were determined by Image J software. c Effects of inhibitors on G3BP1-mediated stabilization of RIG-I. HEK293T cells (4 × 105) were transfected with the indicated plasmids for 18 h and then cells were treated with the indicated inhibitors for 6 h before immunoblotting analysis. For the Flag-RIG-I, band intensities were determined by Image J software. d, e Effects of G3BP1 on ubiquitination of RIG-I mediated by RNF125 or RNF125 (C72/75 A) mutant. HEK293T cells (2 × 106) were transfected with RIG-I (10 μg), G3BP1 (3 μg), HA-Ub (2 μg), and RNF125 or RNF125 (C72/75 A) (5 μg) for 24 h. Co-IP and immunoblotting analysis were performed with the indicated antibodies. f Effects of G3BP1 knockdown on RNF125-mediated ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were transfected with RIG-I (10 μg), HA-Ub (2 μg), and RNF125 (5 μg) for 24 h. Then Co-IP and immunoblotting were performed with the indicated antibodies. g Effects of G3BP1 on K48-linked ubiquitination of RIG-I mediated by RNF125. The experiments were similarly to those described in d. h Effects of G3BP1 knockdown on K48-linked ubiquitination of RIG-I. The stable G3BP1-knockdown HEK293T cells were infected or uninfected with SeV for the indicated time. Then Co-IP and immunoblotting analysis were performed with the indicated antibodies. i Effects of G3BP1 knockout on K48-linked ubiquitination of RIG-I. The experiments were similarly to those described in h. j G3BP1 binds to 5´ppp-dsRNA and enhances the binding of RIG-I to 5´ppp-dsRNA. HEK293T cells (2 × 106) were transfected with the indicated plasmids (5 μg each). Cell lysates were first incubated with biotinylated-5´ppp-dsRNA and streptavidin-Sepharose. Then conjugated proteins were analyzed by immunoblotting with anti-Flag and anti-HA antibodies. Co-IP Co-immunoprecipitation, αF anti-Flag, Coni control RNAi, KO knockout, WT wild-type.
Fig. 8
Fig. 8. G3BP1 promotes degradation of RNF125 via its auto-ubiquitination.
a Interaction between G3BP1 and RNF125 in the mammalian overexpression system. HEK293T cells were transfected with the indicated plasmids (5 μg of each) for 24 h. Co-IP and immunoblotting were performed with the indicated antibodies. b Endogenous G3BP1 interacted with RNF125 in HEK293T cells. HEK293T cells (5 × 107) were untreated or infected with SeV for the indicated time. Co-IP and immunoblotting experiments were performed with the indicated antibodies. c, d Effects of G3BP1 on the expression of RNF125 or RNF125 (C72/75 A) mutant were evaluated. HEK293T cells were transfected with HA-G3BP1 (0, 0.5, 1.5, and 3 μg) and HA-RNF125 or HA-RNF125 (C72/75 A) plasmids (2 μg) for 24 h. Then the cell lysates were analyzed by immunoblotting with the indicated antibodies. e Effects of SeV infection on the expression of endogenous G3BP1 and RNF125 in HEK293T cells. HEK293T cells were uninfected or infected with SeV for the indicated time. The cell lysates were analyzed by immunoblotting with the indicated antibodies. f Effects of MG132 on G3BP1-mediated destabilization of RNF125. HEK293T cells (4 × 105) were transfected with the indicated plasmids for 18 h and then the cells were treated with DMSO or MG132 for 6 h before immunoblotting analysis. g Interaction between RNF125 and RNF125 in the mammalian overexpression system. HEK293T cells were transfected with the indicated plasmids (5 μg of each). Co-IP and immunoblotting were performed with the indicated antibodies. h Effects of G3BP1 on the interaction between RNF125 and RNF125. The experiments were similarly to those described in g. i Effects of G3BP1 on the ubiquitination of RNF125. HEK293T cells (2 × 106) were transfected with the indicated plasmids for 18 h and then treated with MG132 for 6 h. Co-IP and immunoblotting were performed with the indicated antibodies. j, k Effects of G3BP1 overexpression on RNF125-mediated RIG-I activation were assessed. HEK293T cells (1 × 105) were transfected with the IFN-β reporter, ISRE (0.1 μg), HA-RIG-1 (100 ng), Flag-RNF125 (100 ng) or G3BP1 (0, 100, 200, and 400 ng) expression plasmids for 24 h before luciferase assays were performed. The experiment was repeated in triplicates. l Interaction between G3BP1, RIG-I, and RNF125 in HEK293T cells. HEK293T cells were transfected with the indicated plasmids for 24 h. Co-immunoprecipitation and immunoblotting analysis were performed with the indicated antibodies. m Interaction between G3BP1, G3BP1 mutants, and RNF125. The experiments were similarly to those described in l. Data are mean ± SD of three independent experiments. Co-IP Co-immunoprecipitation, EV, empty vector, Luc luciferase, αH anti-HA tag, HC heavy chain.

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