The papain-like protease of severe acute respiratory syndrome coronavirus has deubiquitinating activity

Abstract

Replication of the genomic RNA of severe acute respiratory syndrome coronavirus (SARS-CoV) is mediated by replicase polyproteins that are processed by two viral proteases, papain-like protease (PLpro) and 3C-like protease (3CLpro). Previously, we showed that SARS-CoV PLpro processes the replicase polyprotein at three conserved cleavage sites. Here, we report the identification and characterization of a 316-amino-acid catalytic core domain of PLpro that can efficiently cleave replicase substrates in trans-cleavage assays and peptide substrates in fluorescent resonance energy transfer-based protease assays. We performed bioinformatics analysis on 16 papain-like protease domains from nine different coronaviruses and identified a putative catalytic triad (Cys1651-His1812-Asp1826) and zinc-binding site. Mutagenesis studies revealed that Asp1826 and the four cysteine residues involved in zinc binding are essential for SARS-CoV PLpro activity. Molecular modeling of SARS-CoV PLpro suggested that this catalytic core may also have deubiquitinating activity. We tested this hypothesis by measuring the deubiquitinating activity of PLpro by two independent assays. SARS CoV-PLpro hydrolyzed both diubiquitin and ubiquitin-7-amino-4-methylcoumarin (AMC) substrates, and hydrolysis of ubiquitin-AMC is approximately 180-fold more efficient than hydrolysis of a peptide substrate that mimics the PLpro replicase recognition sequence. To investigate the critical determinants recognized by PLpro, we performed site-directed mutagenesis on the P6 to P2' residues at each of the three PLpro cleavage sites. We found that PLpro recognizes the consensus cleavage sequence LXGG, which is also the consensus sequence recognized by cellular deubiquitinating enzymes. This similarity in the substrate recognition sites should be considered during the development of SARS-CoV PLpro inhibitors.

FIG. 1.

Defining a core SARS-CoVPLpro domain using a trans-cleavage assay. (A) Schematic diagram of constructs generated to identify an active core domain of SARS-CoV PLpro. Starting with the fragment from residues 1541 to 1924, successive deletions of 20 amino acids were made from the C-terminal and N-terminal ends. The catalytic cysteine (C1651) and histidine (H1812) residues are indicated (17, 54). The constructs are designated by the residues at which they begin and end. (B) Expression of SARS-CoV PLpro deletion constructs. SARS-CoV PLpro deletion constructs were expressed via vaccinia-mediated T7 expression in HeLa cells. The proteins were radiolabeled with Trans-³⁵S, immunoprecipitated with antibody anti-R3, and resolved by SDS-PAGE. (C) trans-cleavage assay to assess PLpro activity. SARS-CoV PLpro deletion mutants were coexpressed with substrate NSP1-3* as shown, via vaccinia-mediated T7 expression in HeLa cells. The proteins were radiolabeled with Trans-³⁵S, immunoprecipitated with antibody anti-R1, which precipitates the uncleaved precursor NSP1-3* and cleavage product nsp1. Products were analyzed by electrophoresis on SDS-10% polyacrylamide gels and visualized by autoradiography. ORF, open reading frame.

FIG.2.

Multiple sequence alignment of coronavirus papain-like protease domains. The papain-like protease domain amino acid sequences (either one domain termed PLpro or two domains termed P1 and P2) of nine different coronaviruses (SARS CoV, MHV-JHM, BCoV, HCoV-OC43, HCoV-229E, HCoV-NL63, TGEV, HCoV-HKU1, and aIBV) were aligned using the ALIGN program (SciEd). The amino acids from 1541 to 1855 of SARS-CoV PLpro, experimentally determined as the core domain, were used as a reference sequence. Amino acid numbers are indicated on the left. The regions of identity are highlighted in yellow. Predicted or experimentally determined catalytic cysteine residues, catalytic histidine residues, and cysteine or histidine residues essential for binding zinc are indicated by black boxes. The predicted catalytic aspartic acid residue and putative oxyanion glutamine residue are boxed in red. Residues proposed to be part of the substrate binding site for deubiquitination are boxed in blue. Accession numbers are as follows: SARS-CoV Urbani strain, AY278741; MHVJ (for MHV-JHM), NC_001846; BCoV, NC_003045; HCoV-OC43, AY585228; HCoV-229E, X69721; HCoV-NL63, NC_ 005831; TGEV,Z34093; aIBV, NC_001451; HCoV-HKU1, NC_006577.

FIG. 3.

Identifying residues essential for proteolytic activity of SARS-CoV PLpro. SARS-CoV PLpro constructs encoding the wild-type sequence or specific alanine substitutions were coexpressed with substrate NSP1-3* as described in the legend of Fig. 1C to identify residues essential for SARS-CoV PLpro activity. The site of the alanine substitution is indicated above each lane. WT, wild type.

FIG. 4.

Purification of SARS-CoV PLpro1541-1855 and in vitro assays for enzymatic activity. (A) Expression and purification of wild-type SARS-CoV PLpro. Samples (20 μg) from each stage of the purification were analyzed on an SDS-10% polyacrylamide gel and stained with Coomassie brilliant blue. Lane 1, soluble, crude cell lysate from E. coli BL21(DE3) cells expressing pET11a-SARS CoV-PLpro; lane 2, resuspended pellet of the 40% ammonium sulfate cut of the lysate; lane 3, pooled peak fractions from the phenyl-Sepharose column; lane 4, pooled peak fractions after elution from the Q Sepharose and Mono Q columns. (B) Kinetic assay of peptide hydrolysis and deubiquitination. The peptide hydrolysis (▪) and deubiquitinating (▴) activity of wild-type SARS-CoV PLpro were tested at different concentrations of substrate in 50 mM HEPES, pH 7.5, at 25°C. Peptide hydrolysis reactions contained 1 μM enzyme, whereas deubiquitination assays contained 60 nM enzyme. PLproD1826A was also assayed for proteolytic activity against the fluorescent peptide substrate (□) under the same conditions listed for the wild-type enzyme. The data were fit to the equation v/[E]_Total = k_app [S] by linear regression and then plotted as log [peptide] (peptide concentration) in order to see the differences in rates with the various substrates. (C) SARS-CoV PLpro cleavage of diubiquitin. Wild-type and alanine substitution mutants of SARS-CoV PLpro were tested for their ability to process diubiquitin in vitro. Purified proteins were incubated at 37°C with diubiquitin in a reaction buffer containing bovine serum albumin (BSA) for the times indicated. Cleavage products were analyzed by electrophoresis on a 10 to 20% gradient acrylamide gel and stained with Coomassie blue. The components in each reaction and the incubation time are indicated above each lane. ub, ubiquitin; di-ub, diubiquitin.

FIG. 5.

SARS-CoV PLpro recognition and processing of LXGG consensus substrate cleavage sites. Alanine (or asparagine, indicated by #) substitutions were made at the P6 to P2′ positions of the three SARS-CoV replicase polyprotein cleavage sites recognized by SARS-CoV PLpro: nsp1/nsp2, nsp2/nsp3 (in pNSP1-3* substrate) and nsp3/nsp4 (in pNSP*3-4 substrate [17]). The wild-type amino acid at each position is indicated above the gel. The substrate with the alanine substitution at each position was coexpressed with SARS-CoV PLpro1541-1855 in the trans-cleavage assay, and cleavage products were detected by immunoprecipitation. Products were analyzed by SDS-PAGE and autoradiography.