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Journal of Virology, December 2004, p. 14057-14061, Vol. 78, No. 24
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.24.14057-14061.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Genetic Screen for Monitoring Severe Acute Respiratory Syndrome Coronavirus 3C-Like Protease

Mariona Parera, Bonaventura Clotet, and Miguel Angel Martinez*

Fundacio irsiCaixa, Hospital Universitari Germans Trias i Pujol, Badalona, Spain

Received 30 April 2004/ Accepted 3 August 2004

A novel coronavirus (SCoV) is the etiological agent of severe acute respiratory syndrome. Site-specific proteolysis plays a critical role in regulating a number of cellular and viral processes. Since the main protease of SCoV, also termed 3C-like protease, is an attractive target for drug therapy, we have developed a safe, simple, and rapid genetic screen assay to monitor the activity of the SCoV 3C-like protease. This genetic system is based on the bacteriophage lambda regulatory circuit, in which the viral repressor cI is specifically cleaved to initiate the lysogenic-to-lytic switch. A specific target for the SCoV 3C-like protease, P1/P2 (SAVLQ/SGFRK), was inserted into the lambda phage cI repressor. The target specificity of the SCoV P1/P2 repressor was evaluated by coexpression of this repressor with a chemically synthesized SCoV 3C-like protease gene construct. Upon infection of Escherichia coli cells containing the two plasmids encoding the cI. SCoV P1/P2-cro and the ß-galactosidase-SCoV 3C-like protease constructs, lambda phage replicated up to 2,000-fold more efficiently than in cells that did not express the SCoV 3C-like protease. This simple and highly specific assay can be used to monitor the activity of the SCoV 3C-like protease, and it has the potential to be used for screening specific inhibitors.

* Corresponding author. Mailing address: Fundacio irsiCaixa, Hospital Universitari Germans Trias i Pujol, 08916 Badalona, Spain. Phone: 34-934656374. Fax: 34-934653968. E-mail: miguelvilassar{at}yahoo.es.

Journal of Virology, December 2004, p. 14057-14061, Vol. 78, No. 24
0022-538X/04/$08.00+0     DOI: 10.1128/JVI.78.24.14057-14061.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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  • Sabariegos, R., Gimenez-Barcons, M., Tapia, N., Clotet, B., Martinez, M. A. (2006). Sequence Homology Required by Human Immunodeficiency Virus Type 1 To Escape from Short Interfering RNAs. J. Virol. 80: 571-577 [Abstract] [Full Text]  


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