Background:  The hepatitis C virus (HCV) NS3/4A serine protease is involved not only in the viral polyprotein processing but also efficiently blocks the RIG-I and TLR3 signaling pathways and contributes to virus persistence by enabling HCV to escape the interferon (IFN) antiviral response. Therefore, the NS3/4A protease has emerged as ideal target toward the control of the disease and the development of new anti-HCV agents. Little is known about both the genetic diversity and the catalytic efficiencies of the different NS3/4A proteases within an individual viral population.

Methods:  In this study, we analyzed, at a high resolution (1%), the HCV NS3/4A protease gene quasispecies from 3 HIV-1 co-infected individuals. We also determined the catalytic efficiency of each variant present in the quasispecies in order to establish the relationships between genotype, phenotype and fitness. Finally, a phylogenetic-fitness landscape map was constructed for each quasispecies.

Results:  A huge range of genetic configurations was found, 50%, 84%, and 91% of different genotypes were identified, respectively, which created a dense net that linked different parts of the viral population. Minority variants having mutations involved in the acquisition of resistance to current NS3/4A protease inhibitors were found. A vast diversity of different catalytic efficiencies was also found. Importantly, 70% of the analyzed enzymes displayed a detectable protease activity. Moreover, 35% of the minority individual variants showed similar or better catalytic efficiency than the master (most abundant) enzyme. Nevertheless, and in contrast to minority variants, when different viral polyprotein cleavage sites were tested, master enzymes always displayed a high catalytic efficiency.

Conclusions:  Common and differential traits were found within the 3 studied quasispecies. In all cases, a huge number of different protein variants with detectable protease activity was observed, including mutants having substitution involved in the acquisition of resistance to current NS3/4A protease inhibitors. Nevertheless, a different genetic diversification and distribution of catalytic efficiencies was detected in the 3 quasispecies, suggesting that different selective forces are acting in different infected individuals. These results demonstrate that the rugged HCV protease quasispecies landscape must be capable to act in front of environmental changes that may threaten its survival.