Deubiquitinating and interferon antagonism activities of coronavirus papain-like proteases

Abstract

Coronaviruses encode multifunctional proteins that are critical for viral replication and for blocking the innate immune response to viral infection. One such multifunctional domain is the coronavirus papain-like protease (PLP), which processes the viral replicase polyprotein, has deubiquitinating (DUB) activity, and antagonizes the induction of type I interferon (IFN). Here we characterized the DUB and IFN antagonism activities of the PLP domains of human coronavirus NL63 and severe acute respiratory syndrome (SARS) coronavirus to determine if DUB activity mediates interferon antagonism. We found that NL63 PLP2 deconjugated ubiquitin (Ub) and the Ub-line molecule ISG15 from cellular substrates and processed both lysine-48- and lysine-63- linked polyubiquitin chains. This PLP2 DUB activity was dependent on an intact catalytic cysteine residue. We demonstrated that in contrast to PLP2 DUB activity, PLP2-mediated interferon antagonism did not require enzymatic activity. Furthermore, addition of an inhibitor that blocks coronavirus protease/DUB activity did not abrogate interferon antagonism. These results indicated that a component of coronavirus PLP-mediated interferon antagonism was independent of protease and DUB activity. Overall, these results demonstrate the multifunctional nature of the coronavirus PLP domain as a viral protease, DUB, and IFN antagonist and suggest that these independent activities may provide multiple targets for antiviral therapies.

FIG. 1.

HCoV-NL63 PLP2, but not PLP1, has a dose-dependent global deubiquitinating activity in cultured cells. (A) Schematic diagram of the NL63 genomic RNA and the resulting polyprotein 1ab, which contains three viral proteases. PLP1 and PLP2 cleavage sites are indicated, as are the resulting nonstructural proteins. The V5-tagged constructs of PLP2 used in this study are listed, and the catalytic residues numbering from ORF 1a are shown. DNA encoding HCoV-NL63 PLP2 (B), PLP2 C1678A (C), or PLP1 (D) was transfected into HeLa cells along with pcDNA3.1-3×Flag-Ub. Cell lysates were prepared at 24 h posttransfection and analyzed for Flag-Ub-conjugated proteins by Western blotting with an anti-Flag antibody. Mouse anti-V5 was used to confirm the expression of PLP1 and PLP2, and anticalnexin antibody was used to detect calnexin, which serves as a protein loading control. Molecular weight markers shown on the left of each gel are in thousands.

FIG. 2.

Processing of K48- and K63-linked ubiquitin chains by PLP2. NL63 PLP2 was incubated with K48-linked (left) or K63-linked (right) Ub₆ chains for the indicated time points before being analyzed by SDS-PAGE. Uncleaved Ub₆ is run in the first lane of each gel. The expected sizes of the Ub species are indicated to the left of all gels. Molecular weight (MW) markers include 250,000-, 100,000-, 75,000-, 50,000-, 37,000-, 25,000-, 20,000-, 15,000-, and 10,000-molecular-weight bands.

FIG. 3.

NL63 PLP2 has global deISGylating activity in cultured cells. HEK293 cells were transfected with pcDNA3-myc6-mISG15, pcDNA3-Ube1L, and pcDNA3-UbcH8 plus indicated amounts of the PLP2 expression construct (A) or PLP2 expression construct and the corresponding catalytic mutants (B). To ensure that the total amount (2 μg/transfection) of plasmids for transfection was equal under every condition, empty vector pcDNA3.1/V5-HisB (v) was used to standardize the total amount of DNA. After 30 h, cells were harvested, and cell lysates were analyzed for ISG-conjugated proteins by Western blotting with monoclonal anti-myc antibody. Expression levels of V5-tagged PLP2 and mutant enzymes were detected with anti-V5 antibody. Actin was detected with antiactin MAb antibody as a protein loading control.

FIG. 4.

Evaluating interferon-β secretion from human airway epithelial (HAE) cell cultures following HCoV-NL63 infection. Apical washes were collected from HAE cultures at 24, 48, 72, 96, and 120 h postinfection, and secreted IFN-β was measured by ELISA. Supernatant from HAE cells infected with Sendai virus (SeV) for 24 h was used as a positive control.

FIG. 5.

PLP2 inhibits both RIG-I- and TLR3-dependent IFN-β induction. (A) HEK293 cells were transfected with IFN-β-Luc, pRL-TK, and either 300 ng of HCoV-NL63 PLP2 or 300 ng of SARS-CoV PLpro. At 24 h posttransfection, cells were either mock infected or infected with Sendai virus for 16 h. Following infection, cell lysates were prepared and assayed using the Dual-Luciferase reporter assay. (B) HEK293 cells were transfected with IFN-β-Luc, pRL-TK, 200 ng N-RIG, and either 300 ng of HCoV-NL63 PLP2 or 300 ng of SARS-CoV PLpro. At 24 h posttransfection, cell lysates were prepared and assayed using the Dual-Luciferase reporter assay. (C) HEK293-TLR3 cells were transfected with IFN-β-Luc, pRL-TK, and either 300 ng of HCoV-NL63 PLP2 or 300 ng of SARS-CoV PLpro. At 24 h posttransfection, cells were either mock treated or treated with poly(IC) for 6 h. Following poly(IC) treatment, cell lysates were prepared and assayed using the Dual-Luciferase reporter assay. Error bars indicate standard deviations from the means for triplicates.

FIG. 6.

HCoV-NL63 PLP2 and the catalytic mutants inhibit RIG-I-mediated IFN induction in a dose-dependent manner. PLP2 and the C1678A and H1836A catalytic mutants were cotransfected with IFN-β-Luc and pRL-TK reporters into HEK293 cells. A dominant active N-terminal portion of RIG-I was used to stimulate IFN-β induction. At 24 h posttransfection, cell lysates were harvested and assayed for luciferase activity via the Dual-Luciferase reporter assay. Values are expressed as percentages of N-RIG-stimulated luciferase controls set to 100. Error bars indicate standard deviations from the means for triplicates. (B) The cell lysates described above were mixed with 2× sample buffer and subjected to 12.5% SDS-PAGE. Following transfer to nitrocellulose, the membrane was blotted with mouse anti-V5 to detect the proteases and antiactin as a loading control.

FIG. 7.

The transmembrane form of HCoV-NL63 PLP2 and catalytic mutants inhibit RIG-I-mediated IFN-β induction in a dose-dependent manner. The native downstream hydrophobic domain was cloned into the PLP2 plasmid, and the catalytic cysteine or histidine residue was mutated to alanine. (A) HEK293 cells were transfected with the indicated amounts of PLP2-TM, PLP2-TM C1678A, or PLP2-TM H1836A along with the IFN-β-Luc and pRL-TK reporters. N-RIG was used to stimulate IFN-β induction. At 24 h posttransfection, cell lysates were harvested and assayed for luciferase activity via the Dual-Luciferase reporter assay. Values are expressed as percentages of N-RIG-stimulated luciferase controls set to 100. Error bars indicate standard deviations from the means for triplicates. (B) The cell lysates described above were mixed with 2× sample buffer and subjected to 12.5% SDS-PAGE. Following transfer to nitrocellulose, the membrane was blotted with mouse anti-V5 to detect the proteases and antiactin as a loading control.

FIG. 8.

The ubiquitin-like domain of SARS-CoV PLpro is not required for IFN antagonism. (A) Schematic diagram of nsp3 and the various V5-tagged deletion constructs. Numbers above the constructs indicate the amino acid numbers counting from ORF 1a. (B) HEK293 cells were transfected with the indicated amounts PLpro-Sol, ΔUbl-PLpro-Sol, or ΔUbl-PLpro-TM along with the IFN-β-Luc and pRL-TK reporters. A dominant active N-terminal portion of RIG-I was used to stimulate IFN-β induction. At 24 h posttransfection, cell lysates were harvested and assayed for luciferase activity via the Dual-Luciferase reporter assay. Values expressed are relative to results for cells transfected with the reporters alone. Error bars indicate standard deviations from the means for triplicates. (C) The cell lysates described above were mixed with 2× sample buffer and subjected to 12.5% SDS-PAGE. Following transfer to nitrocellulose, the membrane was blotted with mouse anti-V5 to detect the proteases and antiactin as a loading control.

FIG. 9.

SARS-CoV PLpro inhibits IFN-β and ISRE but not NF-κB reporter activity in a dose-dependent manner in the presence or absence of a protease inhibitor. HEK293 cells were transfected with the indicated amounts of PLpro-TM, pRL-TK, nsp2/3-GFP, and either IFN-β-Luc (A), ISRE-Luc (B), or NF-κB-Luc (C). N-RIG was used to stimulate IFN-β and ISRE. TNF-α (10 ng/ml) was used to stimulate the NF-κB-Luc reporter. DMSO vehicle control or 100 μM GRL-0617S was added at the time of transfection. At 24 h posttransfection, cell lysates were harvested and assayed for luciferase activity via the Dual-Luciferase reporter assay. Values are expressed as percentages of N-RIG- or TNF-α-stimulated luciferase controls, set to 100. Error bars indicate standard deviations from the means from triplicates. (D) The cell lysates described above were mixed with 2× sample buffer and subjected to 12.5% SDS-PAGE. Following transfer to nitrocellulose, the membrane was blotted with mouse anti-V5 to detect PLpro-TM and rabbit anti-GFP to detect the nsp2/3-GFP substrate and the nsp3-GFP cleavage product.