BAG6 negatively regulates the RLR signaling pathway by targeting VISA/MAVS

Abstract

The virus-induced signaling adaptor protein VISA (also known as MAVS, ISP-1, Cardif) is a critical adaptor protein in the innate immune response to RNA virus infection. Upon viral infection, VISA self-aggregates to form a sizeable prion-like complex and recruits downstream signal components for signal transduction. Here, we discover that BAG6 (BCL2-associated athanogene 6, formerly BAT3 or Scythe) is an essential negative regulator in the RIG-I-like receptor signaling pathway. BAG6 inhibits the aggregation of VISA by promoting the K48-linked ubiquitination and specifically attenuates the recruitment of TRAF2 by VISA to inhibit RLR signaling. The aggregation of VISA and the interaction of VISA and TRAF2 are enhanced in BAG6-deficient cell lines after viral infection, resulting in the enhanced transcription level of downstream antiviral genes. Our research shows that BAG6 is a critical regulating factor in RIG-I/VISA-mediated innate immune response by targeting VISA.

Keywords: BAG6; TRAF2; VISA/MAVS; innate immunity; interferon.

Figure 1

BAG6 interacts with VISA specifically. (A) BAG6 interacts with VISA. HEK 293 cells (2×10⁶) were transfected with indicated plasmids. 12 h after transfection, the cells were infected with SeV (MOI = 1) for 12 h or left untreated before co-immunoprecipitation and Western bolt analysis. (B) Endogenous BAG6 is associated with VISA. HEK 293 cells (2×10⁷) were infected with SeV (MOI = 1) for indicated times or left untreated before endogenous co-immunoprecipitation and Western bolt analysis. (C) A549 or HeLa cells were incubated with anti-VISA (green) and anti-BAG6 (red) antibodies. Mitochondria were stained with Mito-tracker Red CMXRos, and Nuclei were stained with DAPI. Images were acquired with a Leica DMI8 and Leica LAS X software. The size bar represents 50 μm.

Figure 2

Identification of BAG6 as a negative regulator of RIG-I-mediated signaling. (A) BAG6 inhibits the IFN-β promoter, ISRE, and NF-κB in a dose-dependent manner. HEK 293 cells (1×10⁵) were transfected with IFN-β promoter (0.025 μg), ISRE (0.025 μg) and NF-κB (0.05 μg) reporter plasmids and BAG6 plasmids (0, 0.05, 0.1, 0.2, 0.4, 0.8 μg). Twelve hours after transfection, cells were infected with SeV (MOI = 1) for 12 h or untreated before luciferase analysis. (B) Overexpression of BAG6 reduces the transcription of downstream antiviral genes. HEK 293 cells were transfected with control or BAG6 plasmid for 24 h. The cells were infected with SeV (MOI = 1) for 12 h or left untreated, followed by qPCR analysis. (C) Overexpression of BAG6 inhibits RNA virus-induced dimerization of IRF3 and phosphorylation of TBK1, P65, and IRF3. HEK 293 cells (2×10⁵) were transfected with control and BAG6 plasmid for 24 h, and cells were left uninfected or infected with SeV (MOI = 1) for 12 h, followed by immunoblotting analysis. (D) BAG6 inhibits RNA virus-triggered innate immunity by targeting VISA. HEK 293 cells (3×10⁵) were transfected with IFN-β promoter (0.025 μg), ISRE (0.025 μg), BAG6 plasmids (0, 0.1, 0.2, 0.4 μg) and RIG-I-N, VISA, TBK1 or IRF3-5D (0.5 μg) for twenty h before luciferase analysis. (*p < 0.05; **p < 0.01; ***p < 0.001; ns, no significant difference).

Figure 3

BAG6-deficiency significantly increases the transcription and activation of antiviral genes in HeLa and HEK 293 cells. (A) BAG6-deficiency enhances the transcription of downstream antiviral genes in HeLa cells. BAG6-deficient HeLa cells were left uninfected or infected with SeV (MOI = 1) for 8 h before qPCR analysis. (B) BAG6-deficiency enhances the transcription of downstream antiviral genes in HEK 293 cells. BAG6-deficient HEK 293 cells were left uninfected or infected with SeV (MOI = 1) for 8 h before qPCR analysis. (C) BAG6-deficiency inhibits the replication of SeV and VSV in HeLa cells. BAG6-deficiency HeLa cells were infected with SeV or VSV (MOI = 0.1) for twenty hours. The supernatants were collected for plaque assays to determine the viral titer. (D) BAG6-deficiency inhibits the replication of SeV and VSV in HEK 293 cells. BAG6-deficiency HEK 293 cells were infected with SeV or VSV (MOI = 0.1) for twenty hours. The supernatants were collected for plaque assays to determine the viral titer. (E) Effects of BAG6 deficiency on VSV-GFP replication. BAG6-deficient HeLa and control cells were infected with VSV-GFP (MOI = 0.1). The cells were viewed with Fluorescence Microscope after 16 h, and the supernatants were collected to infect Vero cells to determine the VSV-GFP replication. (F, G) BAG6 deficiency enhances RNA virus-induced dimerization of IRF3 and phosphorylation of TBK1, P65, and IRF3. BAG6-deficient HeLa and HEK 293 cells were uninfected or infected with SeV (MOI = 1) for 8 h before immunoblotting. (H) Reconstitution of BAG6 in BAG6-deficient HeLa cells suppresses virus-mediated innate immune responses. HeLa-BAG6-KO cells were transfected with control or BAG6 plasmids for 36 h. Cells then were left uninfected or infected with SeV (MOI = 1) for 8 h before qPCR analysis. (I) Reconstitution of BAG6 in BAG6-deficient HeLa cells enhances the replication of the virus. Similar to (E), HeLa-BAG6-KO cells were transfected with control or BAG6 plasmids for 36 h, and cells then were infected with VSV-GFP (MOI = 0.1). (J) The experimental treatment was the same as (H) before immunoblotting. (**p < 0.01; ***p < 0.001; ns, no significant difference). The size bar represents 500 μm.

Figure 4

BAG6-deficiency significantly increases the RLR signaling in primary immune cells. (A) BAG6-deficiency enhances the transcription of downstream antiviral genes in NIH3T3 cells. BAG6-deficient NIH3T3 cells were left uninfected or infected with SeV (MOI = 1) for 8 h before qPCR analysis. (B) BAG6-deficiency enhances the transcription of downstream antiviral genes in mouse BMDMs. Mouse wild-type BMDMs (4×10⁶) were infected with lentivirus containing BAG6-CRISPR CAS9 sgRNA, the supernatants were replaced with fresh medium after 48 h, and the cells were infected with SeV (MOI = 1) for 8 h before qPCR analysis. (C, D) BAG6-deficiency enhances RNA virus-induced phosphorylation of TBK1, P65, and IRF3. BAG6-deficient NIH3T3 and BMDMs were untreated or treated with SeV for 8 h before immunoblotting. (E) Effects of BAG6-deficiency on virus replication. BAG6-deficient and wild-type BMDMs cells were infected with SeV (MOI = 0.1) for twenty hours. The supernatants were collected for plaque assays. (***p < 0.001; ns, no significant difference).

Figure 5

BAG6 regulates the aggregation and recruitment of downstream signaling molecules of VISA. (A) Overexpression of BAG6 inhibits SeV-induced aggregation of VISA. HEK 293 cells (2×10⁶) were transfected with vector or Flag-BAG6 for 20 h, and cells were infected with SeV (MOI = 1) for 8 h before harvest. Then, the lysates were fractionated by SDD-AGE and SDS-PAGE before immunoblotting analysis. (B) BAG6-deficiency increases SeV-induced aggregation of VISA in NIH3T3 cells. Wild type and BAG6-deficient NIH3T3 cells were untreated or treated with SeV (MOI = 1) for 8 h before harvest. Then, the lysates were fractionated by SDD-AGE and SDS-PAGE before immunoblotting analysis. (C) BAG6-deficiency increases SeV-induced aggregation of VISA in BMDMs. Wild-type BMDMs were infected with lentivirus containing BAG6-CRISPR Cas9 sgRNA for 48 h before viral infection. Wild type and BAG6-deficient BMDMs cells were untreated or treated with SeV (MOI = 1) for 8 h before harvest. Then, the lysates were fractionated by SDD-AGE and SDS-PAGE before immunoblotting analysis. (D–G) Overexpression of BAG6 inhibits the interaction of VISA and TRAF2 specifically. HEK 293 cells (2×10⁵) were transfected with the indicated plasmids for 22 h, and cells were untreated or treated with SeV (MOI = 1) for 10 h following co-immunoprecipitation and Western bolting analysis. (H) Overexpression of BAG6 impairs the endogenous interaction of VISA and TRAF2. HEK 293 cells (4×10⁶) were transfected with the indicated plasmids for 22 h, and cells were untreated or treated with SeV (MOI = 1) for 10 h following co-immunoprecipitation and Western bolting analysis. (I) BAG6-deficiency increases the endogenous interaction of VISA and TRAF2. Wild type and BAG6-deficient HeLa cells (1×10⁷) were untreated or treated with SeV (MOI = 1) for 10 h before co-immunoprecipitation and Western bolting analysis.

Figure 6

BAG6 promotes the K48-linked ubiquitination of VISA/TRAF2. (A) Overexpression of BAG6 increases the K48-linked polyubiquitin of VISA. HEK 293 cells (4×10⁶) were transfected with Flag-VISA, HA-ubiquitin (WT, K63R, or K48R), and Myc-BAG6 plasmids for 22 h, and cells were untreated or treated with SeV (MOI = 1) for 10 h before co-immunoprecipitation and Western bolting analysis. (B) BAG6-deficiency inhibits the K48-linked polyubiquitin of VISA. HEK 293 wild type and BAG6-deficient cells (4×10⁶) were transfected with HA-ubiquitin (K63R or K48R) for 22 h, and cells were untreated or treated with SeV (MOI = 1) for 10 h before co-immunoprecipitation and Western bolting analysis. (C) Overexpression of BAG6 increases the K48-linked polyubiquitin and impairs the K63-linked polyubiquitin of TRAF2. HEK 293 cells (4×10⁶) were transfected with Flag-TRAF2, HA-ubiquitin (WT, K63R, or K48R), and Myc-BAG6 plasmids for 22 h. Cells were untreated or treated with SeV (MOI = 1) for 10 h before co-immunoprecipitation and Western bolting analysis. (D) Overexpression of BAG6 increases the K48-linked polyubiquitin and impairs the K63-linked polyubiquitin of endogenous TRAF2. HEK 293 cells (4×10⁶) were transfected with HA-ubiquitin (WT, K63R, or K48R) and Flag-BAG6 plasmids for 22 h, and cells were untreated or treated with SeV (MOI = 1) for 10 h before co-immunoprecipitation and Western bolting analysis.