Cross-genotypic interaction between hepatitis C virus NS3 protease domains and NS4A cofactors

Background/Aims: Hepatitis C virus (HCV) nonstructural protein 3 (NS3) protease requires NS4A as a cofactor. This cofactor activity has been mapped to the central region of NS4A which interacts with the N-terminus of NS3 protease. To investigate whether this interaction is conserved among different genotypes of HCV, cross-genotypic characterization were performed to delineate the importance of NS4A cofactor function in relation to the molecular evolution of HCV. Methods: Active NS3 protease domains of genotype 1–3 (representing five subtypes: 1a, 1b, 2a, 2b and 3a) were produced and purified from bacterial cells. NS4A cofactor-dependent in vitro trans cleavage assays were established using the in vitro translated recombinant protein substrates. These substrates contained the junction site of NS4A/NS4B, NS4B/NS5A or NS5A/NS5B. Results: Our data revealed that NS3 proteases crossinteracted with NS4A cofactors derived from different genotypes, although the genotype 2 cofactor was less efficient, which could be due to greater genetic variations in this region. Furthermore, the corresponding region in hepatitis G virus (HGV) NS4A was found to provide weak cofactor activity for HCV NS3 protease. Surprisingly, a synthetic substrate peptide from the NS4B/NS5A junction was also found to enhance HCV NS3 protease activity in a dose-dependent manner. Conclusion: Our study suggests that the NS4A cofactor function is well conserved among HCV. It is likely that other HCV-related viruses may have developed similar strategies to regulate their protease activity.