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

• Applicant: Professor Dr. Norbert Tautz
• Subject Area: Virology
• Term: since 2015
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 275221303

HCV genome replication and virion morphogenesis take place in dedicated cellular membrane compartments that serve as assembly sites of specialized protein complexes executing these processes. Maturation of these macromolecular protein complexes requires a step-wise succession of protein-protein interactions that must be tightly controlled in a spatio-temporal manner to avoid the formation of non-functional protein complexes. NS3 has a pivotal role in this temporal regulation by providing multipurpose surface areas for the assembly of functionally distinct protein complexes. By interrogating these NS3 surface areas, we show that a set of phylogenetically conserved NS3 residues forms a continuous surface patch across protease and helicase domain that is critical for NS5A hyperphosphorylation, a feature shown to be important for replicase formation to support HCV RNA replication. Furthermore, a helicase residue in close proximity to these residues is pivotal for virion morphogenesis without affecting genome replication. We propose that NS3 provides distinct surface areas to regulate, in a temporal and spatial fashion, the assembly of various, dedicated protein complexes during the different steps of the hepaciviral life cycle that are crucial for replicase assembly and virion formation.In this proposal, the aim is to further characterize the defects caused by the mutations, i.e. which interactions with viral and/or cellular binding partners are abrogated by these mutations. This information will inform us on the composition(s) of dedicated multi-protein complexes required for RNA replication and virion morphogenesis and the temporal and spatial regulation of their formation.To unravel the effect of the individual mutations either interfering with replicase assembly or virion morphogenesis we will follow two strategies (I) identification of second site mutations and (II) identifications of changes in protein complex composition induced by these mutations.

• DFG Programme: Research Grants
• Co-Investigator: Dr. Olaf Isken