Porcine Epidemic Diarrhea Virus 3C-Like Protease Regulates Its Interferon Antagonism by Cleaving NEMO

Abstract

Porcine epidemic diarrhea virus (PEDV) is an enteropathogenic coronavirus causing lethal watery diarrhea in piglets. Since 2010, a PEDV variant has spread rapidly in China, and it emerged in the United States in 2013, posing significant economic and public health concerns. The ability to circumvent the interferon (IFN) antiviral response, as suggested for PEDV, promotes viral survival and regulates pathogenesis of PEDV infections, but the underlying mechanisms remain obscure. Here, we show that PEDV-encoded 3C-like protease, nsp5, is an IFN antagonist that proteolytically cleaves the nuclear transcription factor kappa B (NF-κB) essential modulator (NEMO), an essential adaptor bridging interferon-regulatory factor and NF-κB activation. NEMO is cleaved at glutamine 231 (Q231) by PEDV, and 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 PEDV nsp5 abrogated NEMO cleavage and the inhibition of IFN induction. Structural analysis suggests that several key residues outside the catalytic sites of PEDV nsp5 probably impact NEMO cleavage by modulating potential interactions of nsp5 with their substrates. These data show that PEDV nsp5 disrupts type I IFN signaling by cleaving NEMO. Previously, we and others demonstrated that NEMO is also cleaved by 3C or 3C-like proteinases of picornavirus and artertivirus. Thus, NEMO probably represents a prime target for 3C or 3C-like proteinases of different viruses.

Importance: The continued emergence and reemergence of porcine epidemic diarrhea virus (PEDV) underscore the importance of studying how this virus manipulates the immune responses of its hosts. During coevolution with its hosts, PEDV has acquired mechanisms to subvert host innate immune responses for its survival advantage. At least two proteins encoded by PEDV have been identified as interferon (IFN) antagonists, papain-like protease (PLP) and N protein. Here, we report that the PEDV nsp5 gene, which encodes the 3C-like protease of PEDV, is another IFN antagonist. Mechanistically, the cysteine protease activity of PEDV nsp5 mediates proteolysis of NEMO, the key adaptor for IFN synthesis, and NEMO is cleaved at glutamine 231 (Q231). The new molecular details and determinants impacting NEMO scission by PEDV nsp5 delineated in this study are fundamental to our understanding of critical virus-host interactions that determine PEDV pathogenesis.

FIG 1

PEDV nsp5 inhibits IFN-β promoter activation. (A and B) HEK-293T cells cultured in 48-well plates were transfected with PEDV nsp5 expression plasmid or an empty vector (0.5 μg). Twenty-four hours after the initial transfection, the cells were further infected or mock infected with SEV. The cells and supernatants were collected at 16 h postinfection (hpi) and analyzed for IFN-β levels by real-time RT-PCR (A) and ELISA (B). (C and D) HEK-293T cells or PK-15 cells cultured in 48-well plates were transfected with IFN-β-Luc plasmid (0.1 μg) along with pRL-TK plasmid (0.01 μg, for normalization of transfection efficiency) and increasing quantities of plasmids encoding the indicated PEDV nsp5 (0, 0.05, 0.1, 0.2, or 0.4 μg). Twenty-four hours after the initial transfection, the cells were further infected or mock infected with SEV. Luciferase assays were performed at 16 hpi. (E and F) The experiments were performed similarly to those whose results are shown in panel C, except that the IRF3-Luc (E) or NF-κB-Luc (F) promoter reporter plasmid was used. *, P < 0.05; **, P < 0.01.

FIG 2

The protease activity governs the ability of PEDV nsp5 to suppress virus-induced IFN response. (A) Amino acid alignment of the conserved region surrounding Cys145 and His41 (numbering is based on PEDV nsp5) in CoV nsp5s. Black boxes indicate conserved enzymatic proteolysis residues. The sequences were derived from GenBank entries with the following accession numbers: PEDV, AFQ37597.1; porcine respiratory coronavirus (PRCV), ABG89316.1; feline infectious peritonitis virus (FIPV), AGZ84525.1; human coronaviruses NL63 (HCoV NL63), AFV53147.1; human coronaviruses 229E (HCoV 229E), AGW80947.1; Middle East respiratory syndrome coronavirus (MERS-CoV), AGV08401.1; bat coronavirus HKU4 (BatCoV HKU4), YP_001039952.1; human coronaviruses OC43 (HCoV OC43), AAR01012.1; murine hepatitis virus (MHV), NP_068668.2; SARS-CoV, NP_828850.1. (B) Structural alignment of the conserved residues Cys145 and His41 in CoV nsp5s. Red arrows indicate conserved enzymatic proteolysis residues. The 3D structures were derived from the Protein Data Bank with the following accession numbers: PEDV, 4XFQ; HCoV 229E, 2ZU2; porcine transmissible gastroenteritis virus (TGEV), 2AMP; SARS-CoV, 3IWM; human coronavirus HKU1 (HCoV HKU1), 3D23; BatCoV HKU4, 2YNA. (C) Identification of conserved enzymatic proteolysis residues in PEDV nsp5. (Left) Schematic representation of PEDV nsp5 and its precursors. (Right) HEK-293T cells were transfected with the indicated HA-tagged PEDV nsp5 and Flag-tagged PEDV nsp5 precursors (nsp4-5). Cell lysates were prepared 30 h posttransfection and analyzed by Western blotting. (D) HEK-293T cells were cotransfected with IFN-β-Luc plasmid, pRL-TK plasmid, and PEDV nsp5 expression plasmid (0.4 μg). Twenty-four hours after the initial transfection, the cells were further infected or mock infected with SEV. Luciferase assays were performed 16 h after infection. **, P < 0.01.

FIG 3

PEDV nsp5 disrupts RIG-I/MDA5 signaling. HEK-293T cells were cotransfected with IFN-β-Luc plasmid, pRL-TK plasmid, and plasmid encoding PEDV nsp5 (0.2 μg), together with the human RIG-I (A), MDA5 (B), MAVS (C), NEMO-K277A (D), or TBK1 (E) expression vector (0.2 μg). Luciferase assays were performed 36 h after transfection. *, P < 0.05; **, P < 0.01.

FIG 4

PEDV nsp5 cleaves NEMO. (A) HEK-293T cells cultured in 60-mm dishes were transfected with a Flag-tagged human RIG-I, MDA5, MAVS, or NEMO expression plasmid (4 μg) along with an empty vector or plasmid encoding PEDV nsp5 (2 μg). Cell lysates were prepared 30 h posttransfection and analyzed by Western blotting. (B) HEK-293T cells were transfected with a Flag-tagged human NEMO expression plasmid (4 μg), along with increasing quantities of plasmids encoding HA-tagged PEDV nsp5 (0, 0.125, 0.25, 0.5, 1, and 2 μg). Cell lysates were prepared 30 h posttransfection and analyzed by Western blotting. (C) PK-15 cells were transfected with a Flag-tagged human NEMO or porcine NEMO expression plasmid (6 μg) along with an empty vector or plasmid encoding PEDV nsp5 (3 μg). Cell lysates were prepared 30 h posttransfection and analyzed by Western blotting. (D) (Left) HEK-293T cells were infected with PEDV AJ1102 strain (multiplicity of infection [MOI] of 0.2), lysed at different postinfection times, and analyzed by Western blotting. (Right) Vero cells were infected with different doses (MOI of 0.0002, 0.002, 0.02, or 0.2) of PEDV, lysed 24 h postinfection, and analyzed for NEMO protein levels by Western blotting. (E) Vero cells were infected with different doses (MOI of 0.0002, 0.002, 0.02, or 0.2) of PEDV, lysed 24 h postinfection, and analyzed for NEMO mRNA levels by real-time RT-PCR.

FIG 5

Glutamine 231 is the site of PEDV nsp5-mediated cleavage of NEMO. (A) Sequence logos of the polyprotein junctions cleaved by PEDV nsp5. An amino acid sequence logo of the substrate was generated by WebLogo 3 (http://weblogo.threeplusone.com/). (B) Schematic representation of WT NEMO and its derivatives. (C) HEK-293T cells were transfected with Flag-tagged WT NEMO or NEMO mutants as indicated, along with PEDV nsp5. Cell lysates were prepared 30 h posttransfection and analyzed by Western blotting.

FIG 6

PEDV nsp5-mediated NEMO cleavage is involved in the inhibition of type I IFN induction. (A) HEK-293T cells were transfected with a constitutively activated form of NEMO (NEMO-K277A) or its mutants as indicated (4 μg), along with PEDV nsp5 (2 μg). Cell lysates were prepared 30 h posttransfection and analyzed by Western blotting. (B) HEK-293T cells were transfected with the indicated reporter plasmids, pRL-TK plasmid, and Flag-tagged NEMO-K277A expression plasmid (0.2 μg), along with increasing quantities of the plasmid encoding PEDV nsp5 (0.025, 0.05, 0.1, and 0.2 μg). Luciferase assays were performed at 36 h after the transfection. (C) HEK-293T cells were cotransfected with IFN-β-Luc, pRL-TK plasmid, and a plasmid encoding PEDV nsp5 (0.15 μg) together with the empty vector or the NEMO-WT, NEMO-Q231A, NEMO-K277A, or NEMO-Q231A/K277A expression vector (0.25 μg). Twenty-four hours after the initial transfection, the cells were further infected or mock infected with SEV. Luciferase assays were performed at 16 h after infection. (D) HEK-293T cells were transfected with the Flag-tagged NEMO expression plasmid, along with the indicated PEDV nsp5 expression plasmids. Cell lysates were prepared 30 h posttransfection and analyzed by Western blotting. (E) HEK-293T cells were cotransfected with IFN-β-Luc plasmid, pRL-TK plasmid, and Flag-tagged NEMO-K277A expression plasmid along with the designated PEDV nsp5 expression plasmids. Luciferase assays were performed at 36 h after the transfection. (F) HEK-293T cells were cotransfected with the indicated reporter plasmids, pRL-TK plasmid, and either a plasmid encoding Flag-fused NEMO-K277A (Full), a plasmid encoding putative PEDV nsp5-induced cleavage fragments of NEMO-K277A, or an empty vector (0.4 μg). Luciferase assays were performed at 36 h after the transfection. **, P < 0.01.

FIG 7

The modeled structure of PEDV nsp5 in complex with a peptide substrate. (A) Homology model for PEDV and MERS-CoV nsp5 in complex with their peptide substrates based on the structure of SARS-CoV nsp5 (PDB ID 2Q6G). The residues KLAQLQ₂₃₁↓VA (P6 to P2′, numbering based on NEMO) occupy, and thereby define, the subsites S6 to S2′ of PEDV nsp5. (B) The positions of P6 to P2′, S2 to S1′, and the potential residues forming the S2 (blue), S1 (green), and S1′ (red) subsites are labeled. (C) Structure-based sequence alignment of CoV nsp5 from two genera. The α-CoVs were PEDV, PRCV, HCoV 229E, and HCoV NL63; the β-CoVs were MERS-CoV, SARS-CoV, HCoV OC43, and BatCoV HKU4. Secondary structures of PEDV nsp5 are indicated above the sequence. (D) Schematic diagram of the potential van der Waals interactions between P3 residues (NEMO-Q229, NEMO-Q229A, NEMO-Q229K, or NEMO-Q229R) and Glu165 in PEDV nsp5.