Mint3 potentiates TLR3/4- and RIG-I–induced IFN-β expression and antiviral immune responses

Abstract

Type I IFNs (IFN-α/β) play crucial roles in the elimination of invading viruses. Multiple immune cells including macrophages recognize viral infection through a variety of pattern recognition receptors, such as Toll-like receptors (TLRs) and retinoic acid-inducible gene-I (RIG-I)-like receptors, and initiate type I IFN secretion and subsequent antiviral immune responses. However, the mechanisms by which host immune cells can produce adequate amounts of type I IFNs and then eliminate viruses effectively remain to be further elucidated. In the present study, we show that munc18-1-interacting protein 3 (Mint3) expression can be markedly induced during viral infection in macrophages. Mint3 enhances TLR3/4-and RIG-I-induced IRF3 activation and IFN-β production by promoting K63-linked polyubiquitination of TNF receptor-associated factor 3 (TRAF3). Consistently, Mint3 deficiency greatly attenuated antiviral immune responses and increased viral replication. Therefore, we have identified Mint3 as a physiological positive regulator of TLR3/4 and RIG-I-induced IFN-β production and have outlined a feedback mechanism for the control of antiviral immune responses.interferon | viral infection | TLR | RIG-I | TRAF3

USP1–UAF1 deubiquitinase complex stabilizes TBK1 and enhances antiviral responses

“…Mouse macrophage cell line RAW264.7, human THP-1, and human embryonic kidney (HEK293T) cells were obtained from American Type Culture Collection. Mouse primary peritoneal macrophages were prepared as described ( Huai et al, 2016 ). The cells were cultured at 37°C under 5% CO 2 in DMEM supplemented with 10% FCS (Invitrogen-Gibco), 100 U/ml penicillin, and 100 µg/ml streptomycin.…”

“…The IRF3 reporter plasmids are composed of two plasmids, p-55UASGLuc and p-EFGAL4/IRF3 as described ( Zhang et al, 2012 ). Expression plasmids for HA-Ub WT, HA-K48 Ub, HA-K63 Ub, RIG-I, MDA5, MAVS, TRIF, TBK1, IKK-ε, TRAF3, RIP1, IRF3, and IRF3 5D were described before ( Zhang et al, 2012 ; Huai et al, 2016 ). Target sequences for transient silencing were 5′-GGUCGAGACUCCAUCAUAA-3′ (siRNA 1); 5′-GGAACAAAGACUCCAUUUA-3′ (siRNA 2) and 5′-GCCCGACCAAGUUAUUAAA-3′ (siRNA 3) for UAF1; 5′-GGCAAGUUAUGAGCUUAUA-3′ (siRNA 1), 5′-CGGCAAGGUUGAAGAACAA-3′ (siRNA 2), and 5′-GGAGAGCUCUGAAAUUUCU-3′ (siRNA 3) for USP1; 5′-CCUAAUGACAGUCUCCAAA-3′ (siRNA 1), 5′-CCCAAGAAGUUCAUCACAA-3′ (siRNA 2), and 5′-GCUUAAGAGGGUUCAGUAA-3′ (siRNA 3) for USP12; 5′-GGGAACACUCACUAACGAA-3′ (siRNA 1), 5′-GCAUUACAUCACCAUCGUA-3′ (siRNA 2), and 5′-GCUCAAGCCAUUGAGGAAU-3′ (siRNA 3) for USP46; “scrambled” control sequences were 5′-UUCUCCGAACGUGUCACGU-3′.…”

“…Protein concentrations in the extracts were measured with a bicinchoninic acid assay (Pierce). For Western blot analysis, immunoprecipitates or whole-cell lysates were loaded and subjected to SDS-PAGE, transferred onto nitrocellulose membranes, and then blotted as described previously ( Huai et al, 2016 ).…”

“…Luciferase activity was measured with the Dual-Luciferase Reporter Assay system according to the manufacturer’s instructions (Promega) as described previously ( Zhang et al, 2012 ; Huai et al, 2016 ). Data were normalized for transfection efficiency by division of firefly luciferase activity with that of renilla luciferase.…”

Viruses Seen by Our Cells: The Role of Viral RNA Sensors

“…In order to control the levels of inflammation induced by the triggering of TLR3, its signaling pathway is regulated by different molecules. Some act as positive regulators such as serine/threonine kinase receptor-associated protein (STRAP) that interacts with TBK1 and IRF3 [ 80 ], munc18-1-interacting protein 3 (Mint3) that stimulates the K63-linked polyubiquitination of TRAF3 [ 81 ], Src-associated substrate in mitosis of 68 kDa (Sam68) that may balance NF- κ B p65 and c-Rel activation [ 82 ], and finally S100A9 that acts during the early stages of TLR3 activation by easing the maturation of TLR3-containing early endosomes into late endosomes [ 83 ]. Other molecules act as negative regulators, such as Rho proteins that decrease the production of proinflammatory cytokines upon TLR3 triggering [ 84 ], SUMO-specific protease 6 (SENP6) that inhibits the NF- κ B-mediated expression of the proinflammatory genes [ 85 ], and miR-155 that controls TLR3 signaling by repressing molecules such as TAB2, IKK- ε , and RIP [ 86 ].…”