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Antimicrobial Agents and Chemotherapy, May 2003, p. 1760-1765, Vol. 47, No. 5
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.5.1760-1765.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Genetic Screen for Monitoring Hepatitis C Virus NS3 Serine Protease Activity

Miguel Angel Martinez* and Bonaventura Clotet

Fundacio irsiCaixa, Hospital Universitari Germans Trias i Pujol, Universitat Autònoma de Barcelona, Barcelona, Spain

Received 17 October 2002/ Returned for modification 17 January 2003/ Accepted 11 February 2003

We have developed a genetic system to monitor the activity of the hepatitis C virus (HCV) NS3 serine protease. This genetic system is based on the bacteriophage lambda regulatory circuit where the viral repressor cI is specifically cleaved to initiate the switch from lysogeny to lytic infection. An HCV protease-specific target, NS5A-5B, was inserted into the lambda phage cI repressor. The target specificity of the HCV NS5A-5B repressor was evaluated by coexpression of this repressor with a ß-galactosidase (ßgal)-HCV NS32-181/421-34 protease construct. Upon infection of Escherichia coli cells containing the two plasmids encoding the cI.HCV5AB-cro and the ßgal-HCV NS32-181/421-34 protease constructs, lambda phage replicated up to 8,000-fold more efficiently than in cells that did not express the HCV NS32-181/421-34 protease. This simple, rapid, and highly specific assay can be used to monitor the activity of the HCV NS3 serine 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 Barcelona, Spain. Phone: 34-934656374. Fax: 34-934653968. E-mail: mamartz{at}ns.hugtip.scs.es.

Antimicrobial Agents and Chemotherapy, May 2003, p. 1760-1765, Vol. 47, No. 5
0066-4804/03/$08.00+0     DOI: 10.1128/AAC.47.5.1760-1765.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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