MERS-CoV papain-like protease has deISGylating and deubiquitinating activities

Abstract

Coronaviruses encode papain-like proteases (PLpro) that are often multifunctional enzymes with protease activity to process the viral replicase polyprotein and deubiquitinating (DUB)/deISGylating activity, which is hypothesized to modify the innate immune response to infection. Here, we investigate the predicted DUB activity of the PLpro domain of the recently described Middle East Respiratory Syndrome Coronavirus (MERS-CoV). We found that expression of MERS-CoV PLpro reduces the levels of ubiquitinated and ISGylated host cell proteins; consistent with multifunctional PLpro activity. Further, we compared the ability of MERS-CoV PLpro and Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV) PLpro to block innate immune signaling of proinflammatory cytokines. We show that expression of SARS-CoV and MERS-CoV PLpros blocks upregulation of cytokines CCL5, IFN-β and CXCL10 in stimulated cells. Overall these results indicate that the PLpro domains of MERS-CoV and SARS-CoV have the potential to modify the innate immune response to viral infection and contribute to viral pathogenesis.

Keywords: DUB activity; ISG15; MERS-CoV; PLpro; Ubiquitin; deISGylating activity.

© 2013 Published by Elsevier Inc.

Figures

Fig. 1

Modeling MERS-CoV PLpro onto the SARS-CoV PLpro-ubiquitin-aldehyde structure. (A) Schematic diagram of MERS-CoV ORFs and the papain-like protease (PLpro) domain within nonstructural protein 3 (nsp3). Expression plasmid pcDNA-MERS-PLpro (amino acids 1485–1802) and the predicted catalytic cysteine residue 1594 are indicated. (B) Homology model of MERS-CoV PLpro (blue cartoom and gray surface) aligns with the overall structural architecture found in SARS-CoV PLpro-ubiquitin-aldehyde complex PDB:4MM3 (beige cartoon), including the ubiquitn binding domain at the zinc finger and the extended Ub-like (Ubl) domain. Ubiquitin (red) modeled into the zinc finger domain of MERS-CoV PLpro, with its C-terminus reaching the active site. An enlargement of predicted MERS-CoV PLpro active site superimposed onto the SARS-CoV PLpro active site suggests that the MERS-CoV PLpro catalytic triad is composed of C1594–H1761–D1776 and the putative oxyanion hole residue is L1590.

Fig. 2

Enzymatic activites of SARS-CoV PLpro and MERS-CoV PLpro. (A) deISGylating activity of SARS-CoV PLpro and MERS-CoV PLpro, HEK293T cells were transfacted with myc-ISG15, E1, E2, E3 ISGylating machinery plasmids, and wild type (WT) or catalytic mutant (CA) PLpro expression plasmids. At 18 h post-transfection, cells were lysed and analyzed by western blotting. (B) Deubiquitinating activity of SARS-CoV PLpro and MERS-CoV PLpro. HEK293T cells were transfected with Flag-Ub expression plasmid, and wild type (WT) or catalytic mutant (CA) PLpro. Cells were lysed 18 h post-transfection and analyzed by western blotting. Figure shows representative data from at least two independent experiments.

Fig. 3

Interferon antagonism activity of MERS-CoV PLpro. HEK293T cells were transfected with plasmids expressing wild type (WT) or catalytic mutant PLpro (CA), plasmids expressing IFNβ-luc (A, B, and C), or NF-κB-luc (D), Renilla-luc, and the stimulator indicated at the top of the figure. For A–C, at 16 h post-transfection, cells were lysed and luciferase activity was measured. For D, at 10 h post-transfection cells were treated with TFNα for 4 h, lysed and luciferase activity was measured. Experiments were performed in triplicate. Error bars represent standards deviation of the mean.

Fig. 4

Proinflammatory cytokine expression in the presence of SARS-CoV PLpro or MERS-CoV PLpro. HEK293T cells were transfected with plasmids expressing MDA5 and wild type (WT) or catalytic mutants (CA) of MERS-CoV PLpro or SARS-CoV PLpro. At 18 h post-tranfection, cells were lysed and mRNA levels of CCL5, IFNβ and CXCL10 were determined using qRT-PCR. Data represents fold increase of mRNA levels compared to unstimulated cells (2−ΔΔCt). The figure shows the results from representative experiments performed in triplicates and are shown as means, error bars represent SEM. Experiments were performed in duplicate. ⁎p<0.0005, ⁎⁎p<0.001, and ⁎⁎⁎p<0.01.