Hepatitis C Virus NS3 Helicase Forms Oligomeric Structures That Exhibit Optimal DNA Unwinding Activity in Vitro

Bartek Sikora,Yingfeng Chen,Cheryl F Lichti,Melody K Harrison,Thomas A Jennings,Yong Tang,Alan J Tackett,John B Jordan,Joshua Sakon,Craig E Cameron,Kevin D Raney

doi:10.1074/jbc.m708125200

Abstract

HCV NS3 helicase exhibits activity toward DNA and RNA substrates. The DNA helicase activity of NS3 has been proposed to be optimal when multiple NS3 molecules are bound to the same substrate molecule. NS3 catalyzes little or no measurable DNA unwinding under single cycle conditions in which the concentration of substrate exceeds the concentration of enzyme by 5-fold. However, when NS3 (100 nm) is equimolar with the substrate, a small burst amplitude of approximately 8 nm is observed. The burst amplitude increases as the enzyme concentration increases, consistent with the idea that multiple molecules are needed for optimal unwinding. Protein-protein interactions may facilitate optimal activity, so the oligomeric properties of the enzyme were investigated. Chemical cross-linking indicates that full-length NS3 forms higher order oligomers much more readily than the NS3 helicase domain. Dynamic light scattering indicates that full-length NS3 exists as an oligomer, whereas NS3 helicase domain exists in a monomeric form in solution. Size exclusion chromatography also indicates that full-length NS3 behaves as an oligomer in solution, whereas the NS3 helicase domain behaves as a monomer. When NS3 was passed through a small pore filter capable of removing protein aggregates, greater than 95% of the protein and the DNA unwinding activity was removed from solution. In contrast, only approximately 10% of NS3 helicase domain and approximately 20% of the associated DNA unwinding activity was removed from solution after passage through the small pore filter. The results indicate that the optimally active form of full-length NS3 is part of an oligomeric species in vitro.

Highlights

Helicases are molecular motors that catalyze unwinding of double-stranded DNA or RNA by converting chemical energy from ATP hydrolysis into mechanical energy for nucleic acid strand separation
Bacteriophage T4 Dda helicase is active as a monomer [8] but unwinds DNA substrates more efficiently under conditions that allow binding of multiple helicase molecules per substrate molecule [9]
Current therapy is inadequate in treating all patients afflicted with chronic hepatitis C virus (HCV), and an urgent medical need exists for an effective anti-HCV agent [13]

Summary

EXPERIMENTAL PROCEDURES

Materials—A high pressure liquid chromatography column (Bio-Sil SEC 250-5 column) and gel filtration molecular weight marker standard (molecular weight range 1,350 – 670,000) were purchased from Bio-Rad. Bromphenol blue (0.1%), 0.1% xylene cyanol, and 6% glycerol were added to each, and the double- and single-stranded DNA were resolved via native 20% polyacrylamide gel. Aliquots of the reaction mixture were removed at various time points, quenched by adding 1 M glycine (pH 8.0) and protein sample buffer (62.5 mM Tris-Cl (pH 6.8), 0.45% SDS, 10% glycerol, 5% ␤-mercaptoethanol, and 0.004% bromphenol blue), incubated at 100 °C for 5 min, analyzed by SDS-PAGE (4 –15% gradient gel), and visualized by silver stain-. NS3 or NS3h was incubated with 5 mM ATP in 25 mM MOPS (pH 7.0), 10 mM MgCl2, 10 mM NaCl, 0.1 mg/ml BSA, 4 mM phosphoenolpyruvate, 10 units/ml pyruvate kinase, 15 units/ml lactate dehydrogenase, and 0.9 mM NADH. Hydrolysis rates were calculated using an extinction coefficient of 1,210 MϪ1 cmϪ1 for NADH

RESULTS

Kinetic parameters for DNA unwinding under presteady state conditions

DISCUSSION