The bifunctional NS3 protease-helicase of hepatitis C virus (HCV), together with its cofactor protein NS4A is an important. target for antiviral drugs which can cure HCV infections. HCV strains are divided into six major genotypes based on sequence diversity, and the great majority of it-ports on NS3 have focused exclusively on genotype I proteins Here we report the cloning, expression, and preliminary characterization of NS3-NS4A gene products from HCV genotypes 4, 5, and 6. This work complements our earlier characterization of genotype 2 and 3 proteins [17] We compare NS3-NS4A protease and helicase activities of genotypes 4a, 5a, and 6a to those of common reference strains Con1 (genotype 1b) and JFH1 (genotype 2a). The specific activities of the proteases of the newly isolated proteins were similar to those of the reference proteins Furthermore, the reference inhibitor BILN 2061 had similar activity against all of the proteins except for that of JFH1, which had an apparent K-1 that was 11-fold higher relative to Con 1. RNA and DNA unwinding activities were also similar for genotypes 1, 4, 5. and 6 proteins. but significantly higher for genotype 2JFH1. With the availability of these proteins, inhibitors developed based on their activity against genotype I can be tested against all the either major genotypes, providing a path to improved treatment for all HCV patients. (C) 2009 Elsevier Inc. All rights reserved.