Background:  Hepatitis C virus (HCV) NS3/4A protease is an ideal target for antiviral therapy because its inhibition is expected to block HCV replication both by direct suppression of viral protein production and by restoration of host responsiveness to interferon (IFN). We have developed a bacteriophage lambda-based genetic screen that can be used to characterize site-specific proteases. This genetic screen system is based on the bacteriophage lambda cI-cro regulatory circuit, in which the encoded repressor cI is specifically cleaved to initiate the lysogenic-to-lytic switch. We have adapted this simple, safe and rapid genetic screening system to predict the activities and phenotypes of HCV NS3/4A protease from both different HCV genotypes and cellular or viral substrates (IPS-1, TRIF, NS4B/5A, NS5A/5B).

Methods:  NS3/4A protease coding regions were amplified by polymerase chain reaction (PCR) from 10 individuals co-infected with HIV and HCV and 2 from individuals mono-infected with HCV. NS3/4A proteases from different genotypes (1a, 1b, 3a, 4a, and 4d) were amplified and analyzed. Resistance mutations to HCV protease inhibitors (A156S, A156T, A156V, D168V, D168A) were introduced in the genotype 1b NS3/4A proteases by site-directed mutagenesis and evaluated with NS5A/5B and NS4B/5A cleavage sites. NS4B/5A cleavage site variants were also evaluated with genotype 1b NS3/4A proteases. Finally, NS3/4A proteases from genotypes 1b and 3a were used to screen a protease inhibitor library.

Results:  The genotype 1b NS3/4A proteases displayed the highest catalytic efficiencies. However, within this genotype up to three-fold differences were observed. As expected, different catalytic efficiencies were observed when different mutant NS4B/5A cleavage sites were tested. Resistance mutations A156T and A156V almost abolished catalytic activity of the wild type genotype 1b enzyme in both the NS5A/5B and NS4B/5A cleavage sites. We also identified 4 inhibitors, with IC₅₀ within the µM range.

Conclusions:  Compared with other protease assay methods, this assay has the following advantages:  safety, highly sensitivity, high specificity, easy quantification, and rapid generation of different protease cleavage substrates using molecular cloning and expression. Characterization of the proteolytic activities of individual NS3/4A proteases should provide clues for understanding HCV-host interactions, as well as assisting in the development of new classes of NS3/4A protease inhibitors.