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.

Severe acute respiratory syndrome coronavirus (SARS-CoV

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.The front-line defense of a host cell against virus infection is the innate immune system, which utilizes multiple membrane and cytoplasmic sensors, such as toll-like receptors (TLRs) and RNA helicases, to detect pathogen-associated molecular patterns like viral RNA (3,9,31,47,54). Activation of these sensors by viral RNA intermediates sets off a cascade of signaling events that ultimately turn on transcription factors, such as NF-B, ATF2/c-Jun, IRF-7, and IRF-3. These activated transcription factors translocate to the nucleus and upregulate transcription of interferon (IFN) mRNAs. The translation and subsequent secretion of IFNs activates cells to upregulate interferon-stimulated genes (ISGs) to establish an antiviral state hostile to viral replication. Of importance for this study, many of the signaling events that link the sensors to the transcription factors are mediated by the activities of kinases and ubiquitinating enzymes that modify and activate critical intermediates in the cascade (7,8,20,25). For example, signaling from RIG-I can proceed through MAVS/TRAF3/TANK to TBKI and inducible IB kinase (IKKi), which ultimately phosphorylate IRF-3. Recent studies indicate that both TRAF3 and TANK are modified by lysine-63-linked polyubiquitination and can be inactivated by DUBA, a cellular deubiquitinating (DUB) enzyme (28). Thus, ubiquitinating enzymes and DUBs are critical players in modulating the innate immune response.For positive-strand RNA viruses that replicate in the cytoplasm of the cell using double-stranded RNA intermediates, the cytoplasmic innate i...