Crystal structure of the Middle East respiratory syndrome coronavirus (MERS-CoV) papain-like protease bound to ubiquitin facilitates targeted disruption of deubiquitinating activity to demonstrate its role in innate immune suppression

Abstract

Middle East respiratory syndrome coronavirus (MERS-CoV) is a newly emerging human pathogen that was first isolated in 2012. MERS-CoV replication depends in part on a virus-encoded papain-like protease (PL(pro)) that cleaves the viral replicase polyproteins at three sites releasing non-structural protein 1 (nsp1), nsp2, and nsp3. In addition to this replicative function, MERS-CoV PL(pro) was recently shown to be a deubiquitinating enzyme (DUB) and to possess deISGylating activity, as previously reported for other coronaviral PL(pro) domains, including that of severe acute respiratory syndrome coronavirus. These activities have been suggested to suppress host antiviral responses during infection. To understand the molecular basis for ubiquitin (Ub) recognition and deconjugation by MERS-CoV PL(pro), we determined its crystal structure in complex with Ub. Guided by this structure, mutations were introduced into PL(pro) to specifically disrupt Ub binding without affecting viral polyprotein cleavage, as determined using an in trans nsp3↓4 cleavage assay. Having developed a strategy to selectively disable PL(pro) DUB activity, we were able to specifically examine the effects of this activity on the innate immune response. Whereas the wild-type PL(pro) domain was found to suppress IFN-β promoter activation, PL(pro) variants specifically lacking DUB activity were no longer able to do so. These findings directly implicate the DUB function of PL(pro), and not its proteolytic activity per se, in the inhibition of IFN-β promoter activity. The ability to decouple the DUB activity of PL(pro) from its role in viral polyprotein processing now provides an approach to further dissect the role(s) of PL(pro) as a viral DUB during MERS-CoV infection.

Keywords: Cysteine Protease; Deubiquitylation (Deubiquitination); Innate Immunity; Middle East Respiratory Syndrome Coronavirus; PLpro; Structural Biology; Viral Immunology; X-ray Crystallography.

FIGURE 1.

In vitro cleavage of Lys⁴⁸- and Lys⁶³-linked poly-Ub chains by recombinant MERS-CoV PL^pro. Purified recombinant MERS-CoV PL^pro was incubated with 2.5 μg of Lys⁴⁸-linked (A) or Lys⁶³-linked (B) poly-Ub chains of different length in each reaction for 2 h at 37 °C in a final volume of 10 μl. A range of 2-fold dilutions starting at 2 μm MERS-CoV wild-type PL^pro per reaction was used. Activity of the PL^pro active site mutant (C1592A) was assessed at a concentration of 2 μm. Isopeptidase T (IsoT; 0.5 μg/reaction) served as a positive control (69).

FIGURE 2.

MERS-CoV PL^pro and PL^pro·Ub structures. A, structure of the MERS-CoV PL^pro domain (2.15 Å resolution). The palm, thumb, fingers, and N-terminal ubiquitin-like (Ubl) domains are indicated by colored panels, and arrows indicate the active site and C₄ zinc ribbon motif. The active site residues are depicted as sticks. B, structure of the MERS-CoV PL^pro bound to Ub (2.8 Å resolution). PL^pro is shown in green, and the covalently bound Ub molecule is orange and shown as tubes. Active site residues are shown as sticks with Gly⁷⁵ and the 3CN linker of Ub covalently linked to Cys¹⁵⁹² of PL^pro. C, superposition showing a ∼6.8-Å movement of the zinc ribbon motif between the open (yellow) and closed (green) PL^pro·Ub structures and a previously reported PL^pro structure (gray) (Protein Data Bank entry 4P16 (49)). Our PL^pro structure is not shown because it is highly similar to the open PL^pro·Ub structure. Movement of the zinc ribbon motif was determined by measuring the distance between the zinc atom of the respective structures. Superpositions were performed in Coot (45). Ub was removed from the closed and open PL^pro·Ub structures for clarity. Figures were created using PyMOL (70).

FIGURE 3.

Crystal packing arrangement of the open and closed MERS-CoV PL^pro·Ub structures. The contents of four unit cells are shown, with PL^pro and Ub depicted in gray and orange, respectively. A, the open PL^pro·Ub structure crystallized in space group P6₃, where Ub was found to face the solvent, uninvolved in crystal contacts. B, the closed PL^pro·Ub structure crystallized in space group P6₅22, where Ub no longer faces the solvent, and is involved in crystal contacts. Images were created using PyMOL (70).

FIGURE 4.

Structural comparison of the SARS-CoV PL^pro·Ub and MERS-CoV PL^pro·Ub complexes. A, superposition of the closed MERS-CoV PL^pro·Ub complex (green) and the SARS-CoV PL^pro·Ub complex (purple; Protein Data Bank entry 4M0W) using SSM superpose in Coot (45) (bound Ub molecules were ignored during the superposition). The Ub molecules bound to the MERS-CoV PL^pro domain and SARS-CoV PL^pro domain are depicted as tubes in orange and pale cyan, respectively. The ∼26° shift in the fingers domain between the two respective structures is indicated. B, alternate orientation of the SARS-CoV PL^pro·Ub and MERS-CoV PL^pro·Ub superpositions highlighting the difference in Ub binding. In the MERS-CoV PL^pro·Ub complex, Ub is found shifted toward helix α7 compared with the SARS PL^pro·Ub complex. Helix α7 of MERS-CoV PL^pro is indicated with an arrow. Images were created using PyMOL (70).

FIGURE 5.

Active site of MERS-CoV PL^pro and interactions with the C-terminal RLRGG motif of Ub. Interactions between open PL^pro (green) and the C-terminal RLRGG motif of Ub (orange) are depicted in A and B. A, the main-chain amide of the 3CN linker, which mimics Gly⁷⁶ of Ub, forms a hydrogen bond with the main chain carbonyl of PL^pro residue Gly¹⁷⁵⁸. The main-chain amide of Gly⁷⁵ of Ub forms a hydrogen bond with the carbonyl group of PL^pro Asp¹⁶⁴⁵, and a hydrogen bonding interaction occurs between the main-chain carbonyl of Arg⁷⁴ of Ub and the main-chain amide of Gly¹⁷⁵⁸ of PL^pro. The side-chain η-amino group of Ub residue Arg⁷⁴ is hydrogen-bonded to the main-chain carbonyl group of PL^pro Thr¹⁷⁵⁵. Hydrogen bonds also occur between the side-chain ϵ- and η-amino groups of Ub Arg⁷² and the carboxylate of PL^pro Asp¹⁶⁴⁵ as well as between the main-chain amide of Ub residue Leu⁷³ and side chain PL^pro residue Asp¹⁶⁴⁶. The BL2 loop between strands β15 and β16 is indicated with an arrow. B, alternate orientation of the PL^pro active site showing a hydrogen bonding interaction between the Ub Leu⁷³ main-chain amide group and the side-chain carboxylate of PL^pro residue Asp¹⁶⁴⁶. The side chain of Ub residue Leu⁶³ also undergoes hydrophobic interactions with PL^pro residues Phe¹⁷⁵⁰ and Pro¹⁷³¹. C, the MERS-CoV PL^pro Cys¹⁵⁹²-His¹⁷⁵⁹-Asp¹⁷⁷⁴ catalytic triad residues are shown as well as residues Asn¹⁵⁹⁰ and Asn¹⁵⁹¹, which together with Cys¹⁵⁹² form the oxyanion hole via their backbone amide groups. The covalent 3CN molecule is shown linking the C terminus of Ub to the active site Cys¹⁵⁹² of PL^pro. The active site Leu¹⁵⁸⁷ residue, which is not involved in oxyanion hole formation, is also shown. The electron density is a maximum likelihood weighted 2F_o − F_c map contoured at 1.0σ. Images were created using PyMOL (70).

FIGURE 6.

Structure-guided mutagenesis of PL^pro residues involved in Ub recognition. A, surface representation of the closed MERS-CoV PL^pro·Ub complex. PL^pro is shown in green, and Ub is shown in transparent orange. Those residues that were mutated in order to disrupt Ub binding are colored magenta and are indicated with arrows. Colored boxes refer to close-up views of the PL^pro·Ub interactions and are shown to the right. B, hydrophobic interaction is shown between Val¹⁶⁹¹ of PL^pro and Ile⁴⁴ of Ub. C, Thr¹⁶⁵³ of PL^pro is shown hydrogen-bonded to Gln⁴⁹ and Glu⁵¹ of Ub, and Arg¹⁶⁴⁹ of PL^pro is shown interacting with Arg⁷² of Ub. D, hydrogen-bonding interactions are shown between Gln¹⁷⁰⁸ of PL^pro and Gln⁶² of Ub, and a hydrophobic interaction is shown between Val¹⁷⁰⁶ of PL^pro and Phe⁴ of Ub. Asn¹⁶⁷³ and Val¹⁶⁷⁴ of PL^pro, which do not interact with Ub, are also displayed. Images were created using PyMOL (70).

FIGURE 7.

Effect of PL^pro mutations on in trans cleavage of nsp3↓4 and on DUB activity. A, HEK293T cells were co-transfected with plasmids encoding HA-nsp3C-4-V5 (which does not contain PL^pro), PL^pro-V5 (wild type and mutants), and GFP (as a transfection control). As a control, plasmid encoding HA-nsp3-4-V5, which includes the PL^pro domain, was transfected (lanes 1 and 15), and cleavage resulted in the generation of full-length HA-tagged nsp3 and V5-tagged nsp4. Cells were lysed 18 h post-transfection, and expressed proteins were analyzed by Western blotting. Proteolytic cleavage was measured from the generation of N-terminal HA-tagged nsp3C and C-terminal V5-tagged nsp4. B, HEK293T cells were transfected with a combination of plasmids encoding FLAG-Ub, PL^pro-V5 (wild-type and mutants), and GFP (as a transfection control). Cells were lysed 18 h post-transfection, and expressed proteins were analyzed by Western blotting to visualize the deconjugation of FLAG-tagged Ub from a wide range of cellular proteins by MERS-CoV PL^pro wild-type and mutants.

FIGURE 8.

MERS-CoV PL^pro inhibits RIG-I- and MAVS-induced IFN-β promoter activity. HEK293T cells were transfected with a combination of plasmids expressing a firefly luciferase reporter gene under control of the IFN-β promoter, Renilla luciferase; innate immune response inducers RIG-I_(2CARD), MAVS, or IRF3_(5D); and increasing amounts of MERS-CoV PL^pro wild-type, active site mutant C1592A (A, C, and E), or full-length MERS-CoV nsp3 (B, D, and F). Upon induction of the innate immune response with RIG-I_(2CARD) and IRF3_(5D), cells were transfected with the PL^pro (0, 150, 350, or 500 ng) or nsp3 (0, 350, 500, of 1000 ng) constructs. Upon induction with MAVS, cells were transfected with the PL^pro (0, 50, 75, 100 or 150 ng) or nsp3 (0, 150, 350 or 500 ng) constructs. At 16 h post-transfection, cells were lysed, and luciferase activity was measured. All experiments were repeated independently at least four times. Significance was evaluated using an unpaired two-tailed Student's t test; p values of <0.05 were considered significant. Bars, mean; error bars, S.D. Western blotting was used to verify expression of MERS-CoV PL^pro and nsp3.

FIGURE 9.

DUB activity is required for IFN-β promoter antagonism by MERS-CoV PL^pro. HEK293T cells were transfected with plasmids encoding a firefly luciferase reporter gene under control of the IFN-β promoter, Renilla luciferase, innate immune response inducer MAVS (25 ng), and MERS-CoV PL^pro wild type and mutants (75 ng). At 16 h post-transfection, cells were lysed, and luciferase activity was measured. All experiments were repeated independently at least four times. Significance relative to wild type was evaluated using an unpaired two-tailed Student's t test; significant values were indicated as follows: *, p < 0.05; **, p < 0.01. Bars, mean; error bars, S.D. Western blotting was used to verify expression of MERS-CoV PL^pro.