Polyubiquitin conjugation to NEMO by triparite motif protein 23 (TRIM23) is critical in antiviral defense

Abstract

The rapid induction of type I IFN is a central event of the innate defense against viral infections and is tightly regulated by a number of cellular molecules. Viral components induce strong type I IFN responses through the activation of toll-like receptors (TLRs) and intracellular cytoplasmic receptors such as an RNA helicase RIG-I and/or MDA5. According to recent studies, the NF-kappaB essential modulator (NEMO, also called IKKgamma) is crucial for this virus-induced antiviral response. However, the precise roles of signal activation by NEMO adaptor have not been elucidated. Here, we show that virus-induced IRF3 and NF-kappaB activation depends on the K(lys)-27-linked polyubiquitination to NEMO by the novel ubiquitin E3 ligase triparite motif protein 23 (TRIM23). Virus-induced IRF3 and NF-kappaB activation, as well as K27-linked NEMO polyubiquitination, were abrogated in TRIM23 knockdown cells, whereas TRIM23 knockdown had no effect on TNFalpha-mediated NF-kappaB activation. Furthermore, in NEMO-deficient mouse embryo fibroblast cells, IFN-stimulated response element-driven reporter activity was restored by ectopic expression of WT NEMO, as expected, but only partial recovery by NEMO K165/309/325/326/344R multipoints mutant on which TRIM23-mediated ubiquitin conjugation was substantially reduced. Thus, we conclude that TRIM23-mediated ubiquitin conjugation to NEMO is essential for TLR3- and RIG-I/MDA5-mediated antiviral innate and inflammatory responses.

Fig. 1.

Interaction between NEMO and TRIM23. (A and B) Schematic drawings of NEMO, TRIM23, and their derivatives used in this work. CC1 coiled-coil domain 1; CC2, coiled-coil domain 2; LZ, leucine zipper; ZF, zinc finger. RING finger domain, B-box-B-box/CCD (coiled-coil domain), and ARF (ADP ribosylation factor) domain in TRIM23 are shown. X indicates sites of mutagenesis described in the text. (C) 293T cells were cotransfected with plasmids encoding HA-TRIM23 and NEMO-FLAG or its mutants. (D) NEMO CC1 and LZ domains interacted with the TRIM23 ARF region in 293T cells. (E) HeLa cells were transfected with plasmids encoding NEMO-kGC and kGN-Mock, kGN-TRIM23 and Mock-kGC, or NEMO-kGC and kGN-TRIM23. Nuclear localizations were detected by Hoechst 33342. Bifc signal revealed TRIM23-NEMO–specific association. Expression of NEMO and TRIM23 in HeLa cells were also confirmed by immunoblot using anti-kGN and -KGC antibody. (F) Interaction of endogenous NEMO with endogenous TRIM23 in 293T cells 24 h after SV infection. Asterisk denotes nonspecific band.

Fig. 2.

TRIM23 conjugates K27-linked polyubiquitin to NEMO at multiple sites. (A) 293T cells were cotransfected with plasmids encoding NEMO-FLAG, HA-TRIM23, HA-TRIM23C34A (abbreviated as C34A), HA-TRIM23 ΔRING (abbreviated as ΔR), and Myc-Ub as indicated. (B) Ubiquitin conjugation to NEMO is enhanced dose-dependent TRIM23 expression. 293T cells were cotransfected with plasmids encoding NEMO-FLAG, Myc-Ub, or plasmid (0–2 μg) expressing HA-TRIM23. Levels of NEMO and GAPDH mRNA are also shown. (C) 293T cells were cotransfected with plasmids expressing NEMO-FLAG, TRIM23, and HA-Ub (WT, KallR, K6-, 11-, 27-, 29-, 33-, 48-, and 63-only). (D) 293T cells were cotransfected with plasmids encoding NEMO-FLAG, TRIM23, TRIM23C34A, and HA-Ub (WT, K27R, or K63R). (E) The K27-only ubiquitin conjugation enhanced NEMO-TRIM23-mediated NF-κB reporter activity. 293T cells were transfected with plasmids encoding NF-κB-luc, NEMO-FLAG, or HA-TRIM23, together with plasmids expressing HA-K27-only Ub or HA-K27R Ub as indicated. (F) NEMO KR mutants expressing in 293T cells transfected with plasmids encoding TRIM23 and HA-Ub were analyzed for their ubiquitin conjugation.

Fig. 3.

TRIM23 knockdown impaired both ubiquitin conjugation to NEMO and antiviral responses. (A) Efficiency of TRIM23 knockdown using siRNA and shRNA in 293T cells (Top). Si-TRIM23-1 and sh-TRIM23-1 significantly suppressed levels of TRIM23. Protein and mRNA levels of TRIM23 were also quantified by ImageJ software and real-time RT-PCR, respectively (Top). The mRNA levels of TRIM23 were also visualized by RT-PCR (Top). Under these conditions, NF-κB (Middle) and IFNβ (Bottom) luciferase activities were measured after treatment with PolyI:C (20 μg/mL), TNFα (10 ng/mL), or infected with SV (m.o.i. 10). (B) Control and TRIM23 knockdown 293T cells were infected with or without SV (m.o.i 10) for 16 h. Cell lysates were subject to Western blot with the indicated antibodies. The levels of IFNβ and GAPDH mRNA were also analyzed. (C) 293T cells were treated with si-TRIM23-1 or control si-RNA. At 48 h after siRNA transfection, cells were further transfected with plasmids encoding IFNβ-Luc, RIG-I CARD, or IPS-1. At 72 h after siRNA transfection, luciferase activity was measured. (D) NEMO^−/− MEFs were treated with si-TRIM23-1 or control si-RNA. At 48 h after siRNA transfection, cells were further transfected with plasmids encoding IFNβ-Luc and TBK1. At 72 h after siRNA transfection, luciferase activity was measured. (E) 293T cells treated with sh-TRIM23-1 were transfected with plasmids encoding FLAG-IRF3 or IPS-1-HA, and then cells were mock infected or infected with SV for 16 h as indicated. Dimer formation of IRF3 was analyzed. Phosphorylated IRF3 and the total amount of IRF3 in cell lysates as well as IPS-1-HA are also shown. (F) Suppression of IFNβ production in WT and TRAF6^−/− MEFs treated with si-TRIM23-1. The mRNA levels of murine TRIM23 were visualized by RT-PCR (Upper). IFNβ in culture medium was analyzed by ELISA (Lower). (G) 293T cells were treated with control siRNA, si-TRIM23-1, or si-TRIM23-2. At 48 h after the transfection, cells were mock infected or infected with SV (m.o.i. 10). At 16 h after infection, cell lysates were analyzed for ubiquitin conjugation of NEMO. (H) TRAF6 did not enhance K27-linked ubiquitin conjugation to NEMO, but SV infection did. However, this conjugation was abrogated by TRIM23 knockdown. The sh-control and sh-TRIM23-1–treated cells were transfected with plasmids encoding HA-Ub WT, K27-only, or K27R. Cells were then either transfected with plasmids encoding TRAF6 or infected with SV for 16 h.

Fig. 4.

Ubiquitin conjugation to NEMO by TRIM23 affects host defense. (A) NEMO^−/− MEFs were transfected with plasmids encoding ISRE-Luc, TRIM23, and WT-NEMO or NEMO-5pt. At 8 h after transfection, cells were infected with SV for 16 h. (B) NEMO^−/− MEFs were transfected with plasmids encoding NF-κB–Luc, TRIM23, and WT-NEMO or NEMO-5pt. At 8 h after transfection, cells were treated with SV for 16 h (Left). NEMO^−/− MEFs were transfected with plasmids encoding NF-κB-Luc, TRAF6, and WT-NEMO or NEMO-5pt (Right). Cells were harvested 24 h after transfection, and luciferase activity was measured. The amount of NEMO in cell lysates, as an indicator of transfection efficiency, was measured. (A and B). (C) 293T cells were transfected with plasmids encoding TRIM23, TRAF6, FLAG-NEMO, or NEMO-5pt as indicated. (D) TRIM23 knockdown primary MEF cells allow virus production at a higher level than control cells. The primary MEFs were treated with control si-RNA or si-TRIM23-1 as indicated. At 60 h after transfection, VSV was infected with m.o.i. as indicated. Cells were stained at 24 h after infection (Left). Data are representative of two experiments. At 12 h after infection, virus titer was measured according to TCID₅₀ protocol (Right). Data are mean ± SD (P < 0.001, Student's t test, n = 3).