Identification of NS2 determinants stimulating intrinsic HCV NS2 protease activity

Hepatitis C Virus NS2-NS3 cleavage is mediated by NS2 autoprotease (NS2(pro)) and this cleavage is important for genome replication and virus assembly. Efficient NS2-NS3 cleavage relies on the stimulation of an intrinsic NS2(pro) activity by the NS3 protease domain. NS2(pro) activation depends on conserved hydrophobic NS3 surface residues and yet unknown NS2-NS3 surface interactions. Guided by an in silico NS2-NS3 precursor model, we experimentally identified two NS2 surface residues, F103 and L144, that are important for NS2(pro) activation by NS3. When analyzed in the absence of NS3, a combination of defined amino acid exchanges, namely F103A and L144I, acts together to increase intrinsic NS2(pro) activity. This effect is conserved between different HCV genotypes. For mutation L144I its stimulatory effect on NS2(pro) could be also demonstrated for two other mammalian hepaciviruses, highlighting the functional significance of this finding. We hypothesize that the two exchanges stimulating the intrinsic NS2(pro) activity mimic structural changes occurring during NS3-mediated NS2(pro) activation. Introducing these activating NS2(pro) mutations into a NS2-NS5B replicon reduced NS2-NS3 cleavage and RNA replication, indicating their interference with NS2-NS3 surface interactions pivotal for NS2(pro) activation by NS3. Data from chimeric hepaciviral NS2-NS3 precursor constructs, suggest that NS2 F103 is involved in the reception or transfer of the NS3 stimulus by NS3 P115. Accordingly, fine-tuned NS2-NS3 surface interactions are a salient feature of HCV NS2-NS3 cleavage. Together, these novel insights provide an exciting basis to dissect molecular mechanisms of NS2(pro) activation by NS3. Author summaryThe replication strategy of many viruses involves the translation of a large polyprotein that is processed by viral and/or cellular proteases. This genome organization has many benefits for the virus; it allows the condensation of genetic information, as well as temporal and spatial regulation of protein activity depending on polyprotein cleavage events. Because viral precursor proteins have important functions during all aspects of viral life cycle, including genome replication and virion morphogenesis, a better understanding of polyprotein cleavage regulation and the structure/function of their precursor proteins is necessary. However, many determinants of such regulation are still unknown. Here we define such determinants for NS2(pro) activation by its cofactor NS3, required for efficient NS2-NS3 cleavage of the HCV polyprotein. This protease activation within the HCV polyprotein involves specific NS2:NS3 surface contacts that most likely result in an optimized geometry of the NS2(pro) active site. These results offer novel insights into the modes of HCV polyprotein processing.

Automated summary

The cleavage of Hepatitis C Virus NS2-NS3 is crucial for genome replication and virus assembly, and the activation of NS2(pro) depends on hydrophobic NS3 surface residues and NS2-NS3 surface interactions. In this study, researchers identified two NS2 surface residues that are important for NS2(pro) activation by NS3. These findings provide a basis for understanding the molecular mechanisms of NS2(pro) activation by NS3.