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

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 ϳ8 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 fulllength 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 ϳ10% of NS3 helicase domain and ϳ20% 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 fulllength NS3 is part of an oligomeric species in vitro.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. Helicases are required for virtually all cellular processes involving nucleic acids, including replication, transcription, translation, repair, and recombination (1-4). Several diseases characterized by premature aging and increased incidence of cancer, including Bloom and Werner syndromes, have been linked to mutations in helicase genes (5).The quaternary structure of the active forms of helicases varies considerably, with some requiring dimer or oligomer formation for strand separation activity and others functioning efficiently as monomers. Bacteriophage T4 gp41 helicase, for example, forms a hexameric structure that sequesters singlestranded DNA by encircling it (6). Numerous other helicases function as hexamers (7). 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). Escherichia coli helicase TraI, also called helicase 1, can function as a robust, highly processive helicase as a monomer (10). In order to understand the mechanism by which helicases unwind duplex DNA, the quaternary structure of the active species must be known.More than 170 million people worldwide a...

NS3 Helicase from the Hepatitis C Virus Can Function as a Monomer or Oligomer Depending on Enzyme and Substrate Concentrations

“…The result indicates that, under single turnover conditions, the reduction in observed rate constants was not due solely to competitive interactions for substrate between the wild-type and mutant proteins, indicating that the inactive protein was interfering with an active oligomer in a dominantnegative fashion. This result demonstrates that multiple molecules of NS3 are responsible for product formation under excess enzyme, single turnover conditions, in agreement with previous reports (33,34). Furthermore, this result is similar to investigations of NS3 helicase domain activity (35).…”

“…Work from several laboratories has led to differing proposals regarding the oligomeric state of NS3. These proposals include NS3 functioning as a monomer (29,47), as a dimer (30 -32), and as an oligomer (33,34). The data in this report also appear to provide conflicting results.…”

“…Multiple Turnover ConditionsFormation of NS3 oligomeric structures has been correlated with increasing DNA and RNA unwinding activity by showing that the burst amplitude for product formation increases with increasing enzyme concentration (34). The reported experiments were conducted under single turnover conditions in which the enzyme concentration was similar to or greater than the DNA substrate concentration.…”

“…Results have indicated a dimeric form of the enzyme (30,32), whereas other studies have suggested that larger oligomers can also form (34). As a biophysical method of investigating the interaction of NS3 with itself, a fluorescence anisotropy assay was employed.…”

“…Tackett et al (33) have reported that multiple molecules of NS3 bind to a single DNA duplex and are required for optimal unwinding. Most recently, Sikora et al (34) have shown that NS3 can form oligomeric structures and that the enzyme's optimal DNA unwinding activity correlates with formation of those structures. The question arises as to whether the monomeric form of NS3 also has DNA unwinding activity and, if so, how it relates to the oligomeric forms of the enzyme.…”