Characterization of trans- and cis-cleavage activity of the SARS coronavirus 3CLpro protease: basis for the in vitro screening of anti-SARS drugs
| Autor: | Pei Jer Chen, Cheng Wen Lin, Chien-Chen Lai, Chang Hai Tsai, Kuan Hsun Lin, Lei Wan, Hua Hao Chiu, Fuu Jen Tsai |
|---|---|
| Rok vydání: | 2004 |
| Předmět: |
3CLpro
3C-like protease trans- and cis-cleavage medicine.medical_treatment viruses Substrate specificity CoV coronavirus Biophysics Luc luciferase RNA-dependent RNA polymerase Biology medicine.disease_cause Cleavage (embryo) Biochemistry Antiviral Agents Article Microbiology Viral Proteins Structural Biology Chlorocebus aethiops Endopeptidases Genetics medicine Animals SARS severe acute respiratory syndrome skin and connective tissue diseases Molecular Biology Escherichia coli Vero Cells Coronavirus 3C Proteases Coronavirus DNA Primers S-I (II) substrate-I (II) Protease Base Sequence Hydrolysis fungi Cell Biology In vitro body regions Cysteine Endopeptidases Viral replication Severe acute respiratory syndrome-related coronavirus SARS-coronavirus Vero cell 3C-like protease |
| Zdroj: | Febs Letters |
| ISSN: | 0014-5793 |
| Popis: | Severe acute respiratory syndrome (SARS) has been globally reported. A novel coronavirus (CoV), SARS-CoV, was identified as the etiological agent of the disease. SARS-CoV 3C-like protease (3CLpro) mediates the proteolytic processing of replicase polypeptides 1a and 1ab into functional proteins, playing an important role in viral replication. In this study, we demonstrated the expression of the SARS-CoV 3CLpro in Escherichia coli and Vero cells, and then characterized the in vitro trans-cleavage and the cell-based cis-cleavage by the 3CLpro. Mutational analysis of the 3CLpro demonstrated the importance of His41, Cys145, and Glu166 in the substrate-binding subsite S1 for keeping the proteolytic activity. In addition, alanine substitution of the cleavage substrates indicated that Gln-P1 in the substrates mainly determined the cleavage efficiency. Therefore, this study not only established the quantifiable and reliable assay for the in vitro and cell-based measurement of the 3CLpro activity, but also characterized the molecular interaction of the SARS-CoV 3CLpro with the substrates. The results will be useful for the rational development of the anti-SARS drugs. |
| Databáze: | OpenAIRE |
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