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. 2012 Sep;86(17):9311-22.
doi: 10.1128/JVI.00722-12. Epub 2012 Jun 20.

Foot-and-mouth disease virus 3C protease cleaves NEMO to impair innate immune signaling

Dang Wang  1 , Liurong Fang, Kui Li, Huijuan Zhong, Jinxiu Fan, Chao Ouyang, Huan Zhang, Erzhen Duan, Rui Luo, Zhongming Zhang, Xiangtao Liu, Huanchun Chen, Shaobo Xiao
Affiliations

Foot-and-mouth disease virus 3C protease cleaves NEMO to impair innate immune signaling

Dang Wang et al. J Virol. 2012 Sep.

Abstract

Foot-and-mouth disease is a highly contagious viral illness of wild and domestic cloven-hoofed animals. The causative agent, foot-and-mouth disease virus (FMDV), replicates rapidly, efficiently disseminating within the infected host and being passed on to susceptible animals via direct contact or the aerosol route. To survive in the host, FMDV has evolved to block the host interferon (IFN) response. Previously, we and others demonstrated that the leader proteinase (L(pro)) of FMDV is an IFN antagonist. Here, we report that another FMDV-encoded proteinase, 3C(pro), also inhibits IFN-α/β response and the expression of IFN-stimulated genes. Acting in a proteasome- and caspase-independent manner, the 3C(pro) of FMDV proteolytically cleaved nuclear transcription factor kappa B (NF-κB) essential modulator (NEMO), a bridging adaptor protein essential for activating both NF-κB and interferon-regulatory factor signaling pathways. 3C(pro) specifically targeted NEMO at the Gln 383 residue, cleaving off the C-terminal zinc finger domain from the protein. This cleavage impaired the ability of NEMO to activate downstream IFN production and to act as a signaling adaptor of the RIG-I/MDA5 pathway. Mutations specifically disrupting the cysteine protease activity of 3C(pro) abrogated NEMO cleavage and the inhibition of IFN induction. Collectively, our data identify NEMO as a substrate for FMDV 3C(pro) and reveal a novel mechanism evolved by a picornavirus to counteract innate immune signaling.

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Figures

Fig 1
Fig 1
FMDV 3Cpro independently inhibits type I IFN promoter activation. (A) IBRS-2 cells were cotransfected with IFN-β-Luc and pRL-TK, followed by FMDV infection at 0.1 PFU/cell. As positive controls, 24 h later, the cells were transfected with poly(I·C). Cells were harvested 12 h later and subjected to a dual-luciferase assay. (B) PK-15 cells were transfected with IFN-β-Luc, along with pRL-TK plasmid and the indicated vectors expressing the FMDV proteins (0.8 μg), using Lipofectamine 2000. Twenty-four hours after the initial transfection, the cells were transfected with poly(I·C). Luciferase assays were performed 12 h after the second transfection. (C and D) PK-15 cells were transfected with IFN-β-Luc or IFN-α1-Luc, along with pRL-TK plasmid and increasing quantities (0, 0.125, 0.25, or 0.5 μg) of plasmid encoding 3Cpro, using Lipofectamine 2000. Twenty-four hours after the initial transfection, the cells were transfected with poly(I·C). Luciferase assays and real-time RT-PCR were performed 12 h after the second transfection. The results represent the means and standard deviations of three independent experiments. The firefly luciferase activity was normalized to the Renilla reniformis luciferase, and the untreated empty-vector control value was set to 1. (E and F) RNAs were extracted to measure porcine IFN-β (E) and IFN-α1 (F) mRNA by real-time RT-PCR.
Fig 2
Fig 2
3Cpro significantly reduced the transcription of ISGs and inflammatory cytokines/chemokines. PK-15 cells were transfected with 1 μg of plasmid encoding 3Cpro (HA-3Cpro) or an empty vector (pCMV-HA), and 24 h later, the cells were transfected with 1 μg of poly(I·C). Twelve hours after the second transfection, total RNA was extracted and the expression of 2′,5′-OAS (A), ISG54 (B), IP-10 (C), RANTES (D), TNF-α (E), IL-6 (F), and IL-1β (G) and GAPDH genes was evaluated by quantitative real-time RT-PCR. The results are expressed as increases in mRNA levels relative to those in cells transfected in the absence of poly(I·C) and were normalized by using GAPDH housekeeping gene expression. The results are representative of those from three independent experiments. The error bars indicate standard deviations.
Fig 3
Fig 3
The protease activity of 3Cpro is required to suppress dsRNA-mediated IFN-β induction. PK-15 cells were cotransfected with IFN-β-Luc, along with pRL-TK plasmid, and the designated 3Cpro expression plasmids (1 μg). An empty vector (pCMV-HA) was used as a control. Twenty-four hours after the initial transfection, the cells were transfected with poly(I·C). Luciferase assays were performed 12 h after the second transfection. The error bars indicate standard deviations.
Fig 4
Fig 4
The FMDV 3Cpro protein disrupts RIG-I/MDA5 signaling. (A) PK-15 cells were cotransfected with IFN-β-Luc, pRL-TK plasmid, and 0.5 μg of plasmid encoding 3Cpro, together with the RIG-I-N, MDA5, or IPS-1 expression vector (0.5 μg). Twenty-four hours after the initial transfection, the cells were transfected with 1 μg of poly(I·C). Luciferase assays were performed 12 h after the second transfection. (B to D) The experiments were performed similarly to those in panel A, except that the IFN-α1-Luc (B), 4× PRDIII/I-Luc (C), or 4× PRDII-Luc (D) promoter reporter plasmid was used. **, P < 0.01. The error bars indicate standard deviations.
Fig 5
Fig 5
FMDV 3Cpro cleaves NEMO. (A) PK-15 cells were transfected with Flag-tagged RIG-I, MDA5, IPS-1, or NEMO expression plasmid (2 μg), along with 1 μg of plasmid encoding 3Cpro, using Lipofectamine 2000. Cell lysates were prepared 48 h posttransfection and analyzed by Western blotting. (B) PK-15 cells were transfected with Flag-tagged NEMO expression plasmid (2 μg), along with 1 μg of plasmid encoding 3Cpro. Cell lysates were prepared 48 h posttransfection and analyzed by Western blotting. The lane with protein markers (Bio-Rad; catalog no. 161-0376) includes 250-, 150-, 100-, 75-, 50-, 37-, 25-, and 20-kDa molecular-mass bands. (C) PK-15 cells were transfected with Flag-tagged NEMO expression plasmid (2 μg), along with increasing quantities (0, 0.25, 0.5, 1, and 2 μg) of plasmid encoding 3Cpro. Cell lysates were prepared at 48 h posttransfection and analyzed by Western blotting. (D) IBRS-2 cells cultured in 60-mm dishes were transfected with Flag-tagged NEMO (Flag-NEMO) and infected with FMDV (1 PFU/cell) 24 h posttransfection. The cells were lysed 12 h postinfection and analyzed by Western blotting. Flag-NEMO-conjugated protein was verified with mouse anti-FLAG antibody. Rabbit anti-HA was used to confirm the expression of 3Cpro, and mouse anti-GAPDH antibody was used to detect GAPDH, which served as a protein-loading control.
Fig 6
Fig 6
FMDV 3Cpro disrupts NEMO-mediated type I IFN signaling. (A) PK-15 cells were cotransfected with the indicated promoter reporter plasmid, pRL-TK plasmid, and the wild-type NEMO, NEMO-K277A, or empty expression vector (1 μg). Luciferase assays were performed 36 h after the transfection. (B) PK-15 cells were transfected with Flag-tagged NEMO-K277A expression plasmid (2 μg), along with 1 μg of plasmid encoding 3Cpro. Cell lysates were prepared 48 h posttransfection and analyzed for Flag-NEMO-K277A- and HA-3Cpro-conjugated proteins by Western blotting. Anti-GAPDH antibody was used to detect GAPDH, which served as a protein-loading control. (C) PK-15 cells were transfected with IFN-β-Luc, pRL-TK plasmid, and Flag-tagged NEMO-K277A expression plasmid (0.5 μg), along with increasing quantities (0, 0.02, 0.1, and 0.5 μg) of plasmid encoding 3Cpro. Luciferase assays were performed at 36 h after the transfection. (D) PK-15 cells cultured in 60-mm dishes were transfected with Flag-tagged NEMO expression plasmid (2 μg), along with the indicated 3Cpro expression plasmids (1 μg). Cell lysates were prepared 48 h posttransfection and analyzed for Flag-NEMO- and HA-3Cpro-conjugated proteins by Western blotting. Anti-GAPDH antibody was used to detect beta actin, which served as a protein-loading control. (E) PK-15 cells were cotransfected with Flag-tagged NEMO expression plasmid (2 μg) and 1 μg of plasmids encoding 3Cpro or empty vector. Forty hours after transfection, MG132 or zVAD-FMK was added to a final concentration of 20 nM. Cell lysates were prepared 8 h after treatment and analyzed by Western blotting. (F) PK-15 cells were cotransfected with IFN-β-Luc, pRL-TK plasmid, and Flag-tagged NEMO-K277A expression plasmid (0.5 μg), along with the indicated 3Cpro expression plasmids (0.5 μg). An empty vector (pCMV-HA) was used as a control. Cell extracts were collected 36 h after transfection and analyzed for firefly and Renilla luciferase expression. **, P < 0.01 compared with empty vector plus NEMO-K277A. The error bars indicate standard deviations.
Fig 7
Fig 7
FMDV 3Cpro-mediated NEMO cleavage is involved in the inhibition of type I IFN induction. (A) Schematic representation of wild-type porcine NEMO and its derivatives. The domains of porcine NEMO were annotated analogously to those of human NEMO. (B to D) PK-15 cells were transfected with Flag-tagged wild-type NEMO or NEMO mutants as indicated, along with HA-3Cpro or empty vector. Cell lysates were prepared 48 h posttransfection and analyzed for NEMO by Western blotting with an anti-Flag antibody. Western blots with anti-HA antibody show expression of 3Cpro, and Western blots for GAPDH served as a protein-loading control. (E) Schematic representation of constitutively activated NEMO-K277A and its derivatives. (F) PK-15 cells were cotransfected with IFN-β-Luc, pRL-TK plasmid, and either 0.5 μg of plasmid encoding Flag-fused NEMO-K277A (Full), putative 3Cpro-induced cleavage fragments of NEMO-K277A, or empty vector. Cell extracts were collected at 36 h after transfection and analyzed for firefly and Renilla luciferase expression. *, P < 0.05; **, P < 0.01. The error bars indicate standard deviations.
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