845 A Genetic Screen to Monitor the Activity of the Hepatitis C Virus NS3 Serine Protease M. A. Martinez*, B. Clotet Fundacio IrsiCaixa Hosp Univ Germans Trias i Pujol, Barcelona, Spain
Background: Post-translational proteolysis is an essential step in the life cycle of viruses. For that reason, site-specific proteolysis has been an attractive target for antiviral agent development. The emergence of hepatitis C virus (HCV) as a major human pathogen has promoted the study of the virally encoded NS3 serine protease as a potential antiviral target. The development of antivirals for this virus has been hampered by the lack of cell culture or small animal model systems.
Methods: Previously, a genetic screen assay was developed for the characterization of the human immunodeficiency virus type 1 (HIV-1) protease. This genetic screen system is based on the bacteriophage lambda regulatory circuit where the viral repressor cI is specifically cleaved to initiate lysogenic to lytic switch. We have adapted this simple lambda-based genetic assay for the analysis of the activity of HCV NS3 serine protease. An HCV protease-specific target, NS5A-5B, was inserted into the lambda phage cI repressor.
Results: This repressor efficiently repressed the infecting phage. The target specificity of the HCV NS5A-5B repressor was evaluated by co-expression of this repressor with a beta-galactosidase (betagal)-HCV NS3/4 protease construct. The betagal-HCV NS3/4 protease construct contained the NS4 residues 21-34 fused in frame to the amino terminus of the NS3 protease domain (residues 2-181). E. coli cells were then co-transformed with plasmids encoding the cI.HCV5AB-cro an the betagal-HCV NS3/4 protease constructs. Upon infection of this strain, lambda phage replicated up to 8,000-fold more efficiently than in cells that did not express the HCV NS3/4 protease. Moreover, the specificity of this trans-cleavage reaction was further demonstrated by the lack of phage replication in cells expressing mutated forms of the HCV NS3/4 protease that included the substitutions H57A, D81A, or S139A. Likewise, a control mutant cI.HCV5ABmt-cro target site also prevented phage replication.
Conclusions: In summary, we present here a simple and rapid genetic screening system for the characterization of the activity of the HCV NS3 serine protease as well as to search for new protease inhibitors.