This study examines the specificity and mechanism of action of a recently reported hepatitis C virus (HCV) nonstructural protein 3 (NS3) helicase-protease inhibitor (HPI), and the interaction of HPI with the NS3 protease inhibitors telaprevir, boceprevir, danoprevir, and grazoprevir. HPI most effectively reduced cellular levels of subgenomic genotype 4a replicons, followed by genotypes 3a and 1b replicons. HPI had no effect on HCV genotype 2a or dengue virus replicon levels. Resistance evolved more slowly to HPI than telaprevir, and HPI inhibited telaprevir-resistant replicons. Molecular modeling and analysis of the ability of HPI to inhibit peptide hydrolysis catalyzed by a variety of wildtype and mutant NS3 proteins suggested that HPI forms a bridge between the NS3 RNA-binding cleft and an allosteric site previously shown to bind other protease inhibitors. In most combinations, the antiviral effect of HPI was additive with telaprevir and boceprevir, minor synergy was observed with danoprevir, and modest synergy was observed with grazoprevir.