Hepatitis C virus (HCV) nonstructural protein 3
(NS3) has been shown to possess protease and helicase activities
and has also been demonstrated to spontaneously associate
with nonstructural protein NS4A (NS4A) to form a stable
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-NS4A21–32–GSGS-NS33–631)
was constructed in which the NS4A core peptide is fused
to the N-terminus of the NS3 protease domain 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 protein was expressed
to high levels in Escherichia coli, purified to
homogeneity, and examined for NTPase, nucleic acid unwinding,
and proteolytic activities. The single-chain recombinant
NS3-NS4A protein possesses physiological properties equivalent
to those of the NS3/NS4A complex except that this novel
construct is stable, soluble and sixfold to sevenfold more
active in unwinding duplex RNA. Comparison of the helicase
activity of the single-chain 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 protease domain and at least the NS4A core peptide
are required for optimal unwinding activity.