Characterisation of functional domains in the NS2-3 cysteine protease and the NS3-4A serine protease of hepatitis C virus and their role in the assembly of the viral replicase

The Hepatitis C Virus is one of the best characterized RNA viruses and of high medical relevance. While the serine protease in nonstructural protein 3 (NS3) is well characterized, the NS2 cysteine protease and its activation by NS3 are still ill-defined in part due to the lack of structural data for uncleaved NS2-NS3. Recently we identified a hydrophobic patch on the surface of NS3 which is required for the activation of the NS2 protease: A double mutation of the conserved NS3 amino acids (aa) Y105 and P115 to alanine interferes with cleavage at the NS2/NS3 site but not with NS3/NS4A serine protease activity. In line with these findings, these mutations inhibit an NS2-NS5B replicon but allow the replication of an NS3-NS5B replicon. Accordingly, the surface patch surrounding NS3 aa 105/115 serves a specific and essential function in the activation of the NS2 protease. Furthermore we observed that the proteolytic release of NS2 is pivotal for the hyperphosphorylation of NS5A. This posttranslational modification can be regarded as readout system for the correct assembly of a core replicase consisting of NS3 to NS5A. Interestingly, the identified NS3 surface area is also critical for NS5A hyperphosphorylation. Taken together these findings lead to a model of NS2 release being required for the exposure of the NS3 surface patch which is critical for replicase assembly.Future work shall address the following questions experimentally:(i) Which part of NS2 (in NS2-NS3) does interact with the hydrophobic NS3 surface patch around aa 105/115? A hydrophobic NS2 surface area identified by in silico modeling (Redecke Lab) shall be probed experimentally.(ii) How is activation of the NS2 protease by interaction with NS3 accomplished? Different models based on in silico modeling and mutagenesis shall be challenged.(iii) Is it possible to activate the NS2 protease in the absence of NS3? We observed that a mutation in NS2 leads to activation of the NS2 protease even in the absence of NS3. The goal is to identify the underlying mechanism and additional mutations with analogous phenotypes. NS2 protease mutants displaying high activity in the absence of NS3 will be integrated into the viral genome and tested for effect on viral replication. These experiments shall reveal the importance of the regulation of the NS2 protease for the replication cycle of HCV.(iv) Which individual steps lead to the assembly of the viral replicase? The NS3 protease domain plays an important but so far ill-defined role in NS5A hyperphosphorylation and thus also in replicase assembly. According to our model, NS2 release allows the association of additional parts of NS4A to the NS3 surface resulting in the formation/creation of the platform required for the assembly of the viral replicase. We aim to dissect the individual steps in replicase assembly by site specific mutagenesis and selection of second site revertants.