Papain-Like Protease 2 (PLP2) from Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV):  Expression, Purification, Characterization, and Inhibition

Han, Yu-San; Chang, Gu‐Gang; Juo, Chiun-Gung; Lee, Hong-Jen; Yeh, Shiou–Hwei; Hsu, Ju Wei; Chen, Xin

doi:10.1021/bi0504761

Cited by 149 publications

(144 citation statements)

References 48 publications

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“…Based on the consensus sequence of the three PLpro cleavage sites in the SARS polyprotein and biochemical studies addressing substrate preference, it has been demonstrated that an LXGG motif at the P4-P1 positions of the substrate is essential for recognition and cleavage (17,30). There appear to be no preferences for the PЈ positions or for residues N-terminal to P4.…”

Section: Resultsmentioning

confidence: 99%

Severe acute respiratory syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating enzyme

Ratia

Saikatendu²,

Santarsiero

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

372

526

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Replication of severe acute respiratory syndrome (SARS) coronavirus (SARS-CoV) requires proteolytic processing of the replicase polyprotein by two viral cysteine proteases, a chymotrypsin-like protease (3CLpro) and a papain-like protease (PLpro). These proteases are important targets for development of antiviral drugs that would inhibit viral replication and reduce mortality associated with outbreaks of SARS-CoV. In this work, we describe the 1.85-Å crystal structure of the catalytic core of SARS-CoV PLpro and show that the overall architecture adopts a fold closely resembling that of known deubiquitinating enzymes. Key features, however, distinguish PLpro from characterized deubiquitinating enzymes, including an intact zinc-binding motif, an unobstructed catalytically competent active site, and the presence of an intriguing, ubiquitinlike N-terminal domain. To gain insight into the active-site recognition of the C-terminal tail of ubiquitin and the related LXGG motif, we propose a model of PLpro in complex with ubiquitinaldehyde that reveals well defined sites within the catalytic cleft that help to account for strict substrate-recognition motifs.membrane-associated protease ͉ ubiquitin-like domain

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Section: Resultsmentioning

confidence: 99%

Severe acute respiratory syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating enzyme

Ratia

Saikatendu²,

Santarsiero

et al. 2006

Proc. Natl. Acad. Sci. U.S.A.

372

526

View full text Add to dashboard Cite

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“…Antiviral peptides targeting the HR2 region of the S protein can block the entry of SARS-CoV into target cells [21]. Small molecules targeting SARS-CoV proteases, such as main proteases 3C-like protease (3CLpro) [22], papain-like protease 2 (PLP2) [23] and helicase [24], can inhibit viral replication. In addition to these targets, here we have provided the cleavage of the S protein as another crucial target for the development of vaccines and antiviral agents.…”

Section: Discussionmentioning

confidence: 99%

Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

Kao

Zhou

et al. 2007

Biochemical and Biophysical Research Communications

124

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The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitates fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection. Keywords SARS-CoV; Spike protein; Protease Factor Xa; Protease inhibitor; Cleavage of S proteinSevere acute respiratory syndrome (SARS) is a novel life-threatening infectious disease caused by SARS-coronavirus (SARS-CoV) [1]. Viral spike (S) protein is a multifunctional protein that plays pivotal roles in the biology and pathogenesis of SARS-CoV. The N-terminal region of the S protein has been demonstrated to mediate viral infection, by binding through the receptor-binding domain (RBD) to a cellular receptor angiotensin-converting enzyme 2 (ACE2) [2], and subsequently inducing virus-cell membrane fusion, which involves two domains in the C-terminal region of the S protein named heptad repeat (HR) 1 and 2 [3]. However, other functions of the S protein that are important in viral infection have not yet been defined. * Corresponding author: Fax: +852 2855 1241; E-mail address: bzheng@hkucc.hku.hk. (B.J. Zheng). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. In most other members of the coronavirus family, such as human coronavirus HCoV-OC43 and bovine coronaviruses BCoV, the S protein may be post-translationally cleaved into two fragments, S1 (receptor binding domain) and S2 (membrane fusion domain) [4,5]. Studies in other coronaviruses have further elucidated that conformational changes of the S protein can be induced at 37 °C and pH 8.0, accompanied by the cleavage of S1 and S2, which triggers virus-cell membrane fusion [6,7]. As a new member of the coronavirus family, SARS-CoV shares with other coronaviruses some similarity and common features in the amino acid sequences of the S protein [8]. It...

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“…Recently, Barnard et al 9 have reported the enhanced infectivity of SARS CoV with inosine monophosphate (IMP) dehydrogenase inhibitors, which underscores the need for new antiviral therapies for SARS. Several focused screening efforts have resulted in lead-candidate antiviral drugs against SARS CoV, including protease inhibitors, 10,11 fusion inhibitors, critical to identify new drugs to be prepared for the reemergence of these and other emerging pathogens. Toward this goal, a validated HTS approach can be used to identify lead candidates.…”

mentioning

confidence: 99%

Development and Validation of a High-Throughput Screen for Inhibitors of SARS CoV and Its Application in Screening of a 100,000-Compound Library

et al. 2007

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The authors have developed a high-throughput screen (HTS) that allows for the identification of potential inhibitors of the severe acute respiratory syndrome coronavirus (SARS CoV) from large compound libraries. The luminescent-based assay measures the inhibition of SARS CoV-induced cytopathic effect (CPE) in Vero E6 cells. The assay was validated in 96-well plates in a BSL3 containment facility. The assay is sensitive and robust, with Z values > 0.6, signal to background (S/B) > 16, and signal to noise (S/N) > 3. The assay was further validated with 2 different diversity sets of compounds against the SARS CoV. The "hit" rate for both libraries was approximately 0.01%. The validated HTS assay was then employed to screen a 100,000-compound library against SARS CoV. The hit rate for the library in a single-dose format was determined to be approximately 0.8%. Screening of the 3 libraries resulted in the identification of several novel compounds that effectively inhibited the CPE of SARS CoV in vitro-compounds which will serve as excellent lead candidates for further evaluation. At a 10-µM concentration, 3 compounds with selective indexes (SI 50 ) of > 53 were discovered. (Journal of Biomolecular Screening 2007:33-40)

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Papain-Like Protease 2 (PLP2) from Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV): Expression, Purification, Characterization, and Inhibition

Cited by 149 publications

References 48 publications

Severe acute respiratory syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating enzyme

Severe acute respiratory syndrome coronavirus papain-like protease: Structure of a viral deubiquitinating enzyme

Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

Development and Validation of a High-Throughput Screen for Inhibitors of SARS CoV and Its Application in Screening of a 100,000-Compound Library

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