Engineering, characterization and phage display of hepatitis C virus NS3 protease and NS4A cofactor peptide as a single-chain protein

The polyprotein encoded by hepatitis C virus (HCV) genomic RNA is processed into functional polypeptides by both host- and virus-encoded proteases, Th e HCV-encoded NS3 protease and its cofactor peptide NS4A form a noncovalent complex, which participates in processing the viral polyprotein. This prot eolytic activity is believed to be essential for virus proliferation and th us the NS3 protease is a prime target for developing anti-HCV pharmacologic al agents, Recent X-ray crystallography structural studies have revealed th e nature of this non-covalent complex between NS3 protease and the 'active' central segment of NS4A, providing the opportunity to design a single-chai n polypeptide. To this end, the DNA sequence encoding for the NS4A peptide (residues 21-34) was genetically fused via a short linker, capable of makin g a beta-turn, to the N-terminus of the NS3 protease domain. This engineere d single-chain NS3-protease (scNS3) is fully active with kinetic parameters virtually identical with those of the NS3/NS4A non-covalent complex, Moreo ver, the scNS3 protease can be displayed on filamentous phage and affinity selected using an immobilized specific inhibitor, The scNS3 expressed as a soluble protein and in a phage-display format facilitates enzyme engineerin g for further structural studies and in vitro selection of potential drug-r esistant mutants. These are important steps towards developing effective an ti-protease compounds.