Tetraspanin 6 (TSPAN6) negatively regulates retinoic acid-inducible gene I-like receptor-mediated immune signaling in a ubiquitination-dependent manner

Abstract

The recognition between retinoic acid-inducible gene I-like receptors (RLRs) and viral RNA triggers an intracellular cascade of signaling to induce the expression of type I IFNs. Both positive and negative regulation of the RLR signaling pathway are important for the host antiviral immune response. Here, we demonstrate that the tetraspanin protein TSPAN6 inhibits RLR signaling by affecting the formation of the adaptor MAVS (mitochondrial antiviral signaling)-centered signalosome. We found that overexpression of TSPAN6 impaired RLR-mediated activation of IFN-stimulated response element, NF-κB, and IFN-β promoters, whereas knockdown of TSPAN6 enhanced the RLR-mediated signaling pathway. Interestingly, as the RLR pathway was activated, TSPAN6 underwent Lys-63-linked ubiquitination, which promoted its association with MAVS. The interaction of TSPAN6 and MAVS interfered with the recruitment of RLR downstream molecules TRAF3, MITA, and IRF3 to MAVS. Further study revealed that the first transmembrane domain of TSPAN6 is critical for its ubiquitination and association with MAVS as well as its inhibitory effect on RLR signaling. We concluded that TSPAN6 functions as a negative regulator of the RLR pathway by interacting with MAVS in a ubiquitination-dependent manner.

FIGURE 1.

TSPAN6 inhibits MAVS-mediated activation of IFN-β, ISRE, and NF-κB reporters. 293T cells were transfected with IFN-β-luciferase (A), NF-κB-luciferase (B), and ISRE-luciferase (C) reporters, together with the indicated plasmids, and pCMV-β-gal served as an internal control. The luciferase assays were performed 24 h after transfection. All experiments were repeated three times independently, and graphs show means ± S.D. (n = 3).

FIGURE 2.

TSPAN6 specifically inhibits RLR-mediated activation of the IFN-β promoter at the MAVS level. A, TSPAN6 inhibits influenza A virus genome-triggered IFN-β promoter activation. 293T cells were transfected with the IFN-β-luciferase reporter and pCMV-Myc-TSPAN6, together with RNA extracts from influenza A virus-infected 293T cells (referred to as viral RNA) or RNA extracts from uninfected 293T cells (control). The luciferase assays were performed 24 h after transfection. B, TSPAN6 inhibits SeV-induced IFN-β promoter activation. 293T cells were transfected with IFN-β-luciferase and pCMV-Myc-TSPAN6 for 24 h and then infected with SeV for 8 h. The cells were then lysed for luciferase assay. C–E, TSPAN6 inhibits the RLR pathway at the MAVS level. 293T cells were transfected with the IFN-β-luciferase reporter and the indicated expression plasmids and with pCMV-β-gal as an internal control. The luciferase assays were performed 24 h after transfection. All experiments were repeated twice independently, and graphs show means ± S.D. (n = 3).

FIGURE 3.

Knockdown of TSPAN6 potentiates RLR pathway signaling. A, 293T cells were transfected with control or TSPAN6 (si-TSPAN6) siRNA for 48 h. The cell lysates were harvested and subjected to immunoblotting with the indicated antibodies. Knockdown of TSPAN6 enhanced activation of the IFN-β promoter induced by influenza A virus RNA (B), RIG-I (C), poly(I:C) (D), or SeV (E). 293T cells were transfected with control or TSPAN6 siRNA for 48 h and then transfected with the indicated expression plasmid or RNA for 16 h or infected with SeV for 12 h. The cell lysates were subjected to luciferase assay. For B–E, the experiments were repeated two times independently, and graphs show means ± S.D. (n = 3).

FIGURE 4.

TSPAN6 interacts with MAVS. A, 293T cells were transfected with the indicated plasmids for 24 h. The cell lysates were immunoprecipitated (IP) with anti-FLAG antibody, followed by immunoblotting (IB) with anti-Myc antibody. The total cell lysate (TCL) was immunoblotted with anti-FLAG and anti-Myc antibodies. B, TSPAN6 co-localizes with MAVS. 293T cells were transfected with pCMV-Myc-TSPAN6 and pCMV-FLAG-MAVS for 16 h. Immunofluorescence was performed as described under “Experimental Procedures.” C, 293T cells were mock-treated or infected with SeV for 16 h, and the total cell lysate was detected with anti-TSPAN6 or anti-β-actin antibody (upper panel), or the cells were subjected to cell fractionation as indicated, and the mitochondrial fraction was subjected to immunoblotting with anti-MAVS, anti-TSPAN6, or anti-cytochrome c oxidase IV (Cox IV; mitochondrial indicator) antibody (lower panel). D, 293T cells were transfected with pCMV-Myc-TSPAN6 or empty vector for 24 h and then mock-treated or infected with SeV for 8 h. The cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-MAVS antibody. IgGH, IgG heavy chain.

FIGURE 5.

TSPAN6 undergoes Lys-63-linked ubiquitination upon RLR signaling activation. A, 293T cells were transfected with the indicated plasmids and the RNA-containing influenza A virus genome or poly(I:C) for 24 h. The cell lysates were immunoprecipitated (IP) with anti-Myc antibody, followed by immunoblotting (IB) with anti-HA antibody. The total cell lysates (TCL) were immunoblotted with the indicated antibodies. B, 293T cells were transfected with the TSPAN6, HA-tagged Lys-63-linked Ub, or HA-tagged Lys-48-linked Ub expression plasmid for 24 h and then infected with SeV for 16 h. The co-immunoprecipitation assay was performed as described for A.

FIGURE 6.

First transmembrane domain of TSPAN6 is critical for its ubiquitination and inhibitory effect on MAVS-mediated activation of the IFN-β promoter. A, scheme of Ndfip1 and its mutants. aa, amino acids. B, 293T cells were transfected with the indicated plasmids for 24 h. The cell lysates were immunoprecipitated (IP) with anti-Myc antibody, followed by immunoblotting (IB) with anti-Myc or anti-HA antibody. The total cell lysates (TCL) were immunoblotted with the indicated antibodies. IgGH, IgG heavy chain. C, 293T cells were transfected with IFN-β-luciferase, pCMV-Myc-TSPAN6, and its deletion mutants for 24 h and then infected with SeV for 8 h. The cell lysates were harvested for luciferase assay. D, 293T cells were transfected with wild-type TSPAN6 and its mutants for 24 h and then infected with SeV for 8 h. The cell lysates were immunoprecipitated with anti-Myc antibody, followed by immunoblotting with anti-Myc antibody. E, 293T cells were transfected with the indicated plasmids for 24 h, and the cell lysate were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-Myc antibody. The total cell lysates were immunoblotted with the indicated antibodies. F, 293T cells were transfected with IFN-β-luciferase, MAVS, and TSPAN6 or its mutants. Luciferase assays were performed 24 h after transfection. For C and F, the experiments were repeated twice independently, and graphs show means ± S.D. (n = 3).

FIGURE 7.

TSPAN6 inhibits formation of the MAVS-centered signalosome. A and B, 293T cells were transfected with the indicated plasmid for 24 h. The cell lysates were immunoprecipitated (IP) with anti-FLAG antibody, followed by immunoblotting (IB) with anti-HA antibody. The total cell lysates (TCL) were immunoblotted with the indicated antibodies. C, 293T cells were transfected with the indicated plasmid for 24 h. The cell lysates were immunoprecipitated with anti-FLAG antibody, followed by immunoblotting with anti-TBK1 antibody. The total cell lysates were immunoblotted with the indicated antibodies. D, working model of the negative regulation of the RLR pathway by TSPAN6. Details are as described under “Results.”