A novel recombinant single-chain hepatitis C virus NS3-NS4A protein with improved helicase activity

Hepatitis C virus (HCV) nonstructural protein 3 (NS3) has been shown to pos sess protease and helicase activities and has also been demonstrated to spo ntaneously associate with nonstructural protein NS4A (NS4A) to form a stabl e complex. Previous attempts to produce the NS3/NS4A complex in recombinant baculovirus resulted in a protein complex that aggregated and precipitated in the absence of nonionic detergent and high salt. A single-chain form of the NS3/NS4A complex (His-NS4A(21-32)-GSGS-NS3(3-631)) was constructed in which the NS4A con peptide is fused to the N-terminus of the NS3 protease d omain as previously described (Taremi et al., 1998). This protein contains a histidine tagged NS4A peptide (a.a. 21-32) fused to the full-length NS3 ( a.a. 3-631) through a flexible tetra amino acid linker. The recombinant pro tein was expressed to high levels in Escherichia coli, purified to homogene ity, and examined for NTPase, nucleic acid unwinding, and proteolytic activ ities. The single-chain recombinant NS3-NS3A protein possesses physiologica l properties equivalent to those of the NS3/NS4A complex except that this n ovel construct is stable, soluble and sixfold to sevenfold more active in u nwinding duplex RNA. Comparison of the helicase activity of the single-chai n recombinant NS3-NS4A with that of the full-length NS3 (without NS4A) and that of the helicase domain alone suggested that the presence of the protea se domain and at least the NS4A core peptide are required for optimal unwin ding activity.