Modulation of Hepatitis C Virus NS3 Protease and Helicase Activities through the Interaction with NS4A

Abstract

The hepatitis C virus nonstructural 3 protein (NS3) possesses a serine protease activity in the N-terminal one-third, whereas RNA-stimulated NTPase and helicase activities reside in the C-terminal portion. The serine protease activity is required for proteolytic processing at the NS3-NS4A, NS4A-NS4B, NS4B-NS5A, and NS5A-NS5B polyprotein cleavage sites. NS3 forms a complex with NS4A, a 54-residue polypeptide that was shown to act as an essential cofactor of the NS3 protease. We have expressed in Escherichia coli the NS3-NS4A precursor; cleavage at the junction between NS3 and NS4A occurs during expression in the bacteria cells, resulting in the formation of a soluble noncovalent complex with a sub-nanomolar dissociation constant. We have assessed the minimal ionic strength and detergent and glycerol concentrations required for maximal proteolytic activity and stability of the purified NS3-NS4A complex. Using a peptide substrate derived from the NS5A-NS5B junction, the catalytic efficiency (kcat/Km) of NS3-NS4A-associated protease under optimized conditions was 55 000 s-1 M-1, very similar to that measured with a recombinant complex purified from eukaryotic cells. Dissociation of the NS3-NS4A complex was found to be fully reversible. No helicase activity was exhibited by the purified NS3-NS4A complex, but NS3 was fully active as a helicase upon dissociation of NS4A. On the other hand, both basal and poly(U)-induced NTPase activity and ssRNA binding activity associated with the NS3-NS4A complex were very similar to those exhibited by NS3 alone. Therefore, NS4A appears to uncouple the ATPase/ssRNA binding and RNA unwinding activities associated with NS3.

The Nonstructural Protein 3 Protease/Helicase Requires an Intact Protease Domain to Unwind Duplex RNA Efficiently

“…Although the HCV helicase has been analyzed both as an isolated fragment and as part of the full-length NS3 protein, the few studies that directly compared the HCV helicase with and without its attached protease have not noted clear differences (11,23,24). There are nevertheless several noteworthy differences between studies that utilize full-length NS3 and those that use recombinant HCV helicase lacking the protease domain.…”

“…Most previous studies have not noted differences in helicase (11,23,41), RNA binding (42), or RNA replication assays (24) when NS3 was truncated to delete the protease. In early studies Heilek and Peterson (11), concluded that the protease domain had little or no effect on helicase activity.…”

“…In fact, in their assays, the full-length protein seemed to unwind RNA more slowly. Likewise, Gallinari et al (23) concluded that there were no significant differences between the full-length protein and the isolated helicase domain in either ATPase or RNA unwinding assays. Both full-length NS3 and a truncated helicase fragment have been shown specifically to bind sequences in the HCV genome, and the protease is apparently not required for this interaction (42).…”

Characterization of hepatitis C virus NS3 modifications in the context of replication

“…The RNA-unwinding activity seems to be modulated by other viral proteins, e.g. NS5B (Jennings et al, 2008;Zhang et al, 2005) and NS4A (Gallinari et al, 1999).The helicase domain of NS3 can be methylated posttranslationally at Arg1493 (polyprotein) by protein arginine methyltransferase 1 (PMRT1) (Rho et al, 2001). …”

“…The RNA-unwinding activity seems to be modulated by other viral proteins, e.g. NS5B (Jennings et al, 2008;Zhang et al, 2005) and NS4A (Gallinari et al, 1999).…”