Transmissible Gastroenteritis Virus Papain-Like Protease 1 Antagonizes Production of Interferon- β through Its Deubiquitinase Activity

Abstract

Coronaviruses (CoVs), such as human coronavirus NL63 (HCoV-NL63), severe acute respiratory syndrome CoV (SARS-CoV), murine hepatitis virus (MHV), porcine epidemic diarrhea virus (PEDV), and Middle East Respiratory Syndrome Coronavirus (MERS-CoV), encode papain-like (PL) proteases that inhibit Sendai virus- (SeV-) induced interferon (IFN-β) production. Recently, the crystal structure of transmissible gastroenteritis virus (TGEV) PL1 has been solved, which was similar to that of SARS-CoV PL2^pro, which may antagonize host innate immunity. However, very little is known about whether TGEV PL1 can antagonize host innate immune response. Here, we presented evidence that TGEV PL1 encoded by the replicase gene could suppress the IFN-β expression and inhibit the nuclear translocation of interferon regulatory factor 3 (IRF3). The ability to antagonize IFN-β production was dependent on the intact catalytic activity of PL1. Furthermore, TGEV PL1 exerted deubiquitinase (DUB) activity which strongly inhibited the retinoic acid-induced gene I- (RIG-1-) and stimulator of interferon gene- (STING-) dependent IFN expression. Our data collectively suggest that TGEV PL1 can inhibit the IFN-β expression and interfere with RIG-1- and STING-mediated signaling through a viral DUB activity. Our study has yielded strong evidence for the TGEV PL1 mechanisms that counteract the host innate immunity.

Figure 1

TGEV PL1 inhibits SeV-induced expression of IFN-β-Luc in a dose-dependent manner. (a) HEK-293T cells grown in 24-well plates were transfected with 1 μg plasmid encoding Myc-TGEV PL1 or empty vector and infected with SeV 24 h later (100 hemagglutinating activity units/well). After 10 h infection, cells were lysed, and activities of IFN-β-Luc and pRL-TK reporters were determined according to the manufacturer's protocol. Myc-TGEV PL1 inhibits the activities of IRF3 (b), NF-κB (c), and AP-1 (d). Luciferase activities were assayed as described for (a). Results represent the means and standard deviations of data from three independent experiments. (e) PK-15 cells grown in 12-well plates were transfected with 500 ng plasmid Flag-PL1 and empty vector and infected with SeV 24 h later. After 10 h infection, cell supernatants were collected and analyzed for IFN-β production by ELISA. (f) HEK-293T cells grown in 6-well plates were transfected with 2 μg plasmid encoding Myc-TGEV PL1 or empty vector and infected with SeV 24 h later (100 hemagglutinating activity units/well). After 10 h infection, cells were lysated and detected by Western blot. (g) Immunofluorescence microscopy of HeLa cells expressing Flag-PL1 and IRF3-GFP. Cells were fixed 24 h after transfection and 10 h SeV infection, and Flag-tagged products were visualized using confocal microscopy. Asterisks indicate statistical significance (P < 0.05).

Figure 2

Expression of TGEV PL1 inhibits STING-mediated activation of IFN-β-Luc in a dose-dependent manner. (a) HEK293T cells were cotransfected with certain Flag-PL1, IFN-β-Luc, pRL-TK, and 500 ng Flag-STING or 500 ng empty vector. Asterisks indicate statistical significance (P < 0.05). Proteins were assayed using Western blot with anti-Flag and GAPDH antibodies. (b) HEK293T cells were cotransfected with 500 ng STING and/or 500 ng TGEV PL1, and/or infected with SeV. Cell lysates were separated via SDS-PAGE and subjected to immunoblotting with the relevant antibodies.

Figure 3

Effects of the TGEV PL1 catalytic mutants on expression of the IFN-β-Luc and localization of the protein. (a) HEK293T cells were cotransfected with the 500 ng catalytic mutants C32A, H183A, and D196A, together with reporters of IFN-β-Luc and pRL-TK. Asterisks indicate statistical significance (P < 0.05). (b, c, d, e) HEK293T cells were transfected separately with RIG-1 (500 ng), MAVS (500 ng), STING (500 ng), or TBK-1 (500 ng), together with IFN-β-Luc and pRL-TK. Asterisks indicate statistical significance (P < 0.05). (f) Immunofluorescence microscopy of HeLa cells expressing Myc-TGEV PL1, Flag-STING, and DsRed-Mito. Cells were fixed 24 h after transfection, and Flag-tagged and Myc-tagged products were visualized using confocal microscopy.

Figure 4

TGEV PL1 displays DUB activity that is dependent on its catalytic activity and reduces the ubiquitinated forms of RIG-I and STING. (a) HEK293T cells were transfected with HA-tagged ubiquitin and TGEV PL1 or the catalytic mutants C32A, H183A, and D196A. Proteins were assayed using Western blot with anti-HA and anti-Myc antibodies. ((b) and (c)) HEK293T cells were transfected with Myc-TGEVPL1 together with Flag-RIG-1 (b) and Flag-STING (c) for 24 h, and lysates were subjected to coimmunoprecipitation and Western blot to determine the ubiquitination status of immunoprecipitated proteins. ((d) and (e)) HEK293T cells were transfected with Flag-RIG-1 (d) or Flag-STING (e), together with HA-Ub in the presence or absence of Myc-TGEVPL1. Cells were incubated for 24 h after transfection and then lysates were harvested. Lysates were immunoprecipitated with Flag, HA, and Myc antibodies and the products subjected to immunoblotting with anti-HA to evaluate ubiquitinated proteins.