The Macroscopic Rate of Nucleic Acid Translocation by Hepatitis C Virus Helicase NS3h Is Dependent on Both Sugar and Base Moieties

Abstract

The NS3 helicase (NS3h) of hepatitis C virus (HCV) is a 3′ to 5′ SF2 RNA and DNA helicase that is essential for the replication of HCV. We have examined the kinetic mechanism of translocation of NS3h along single-stranded nucleic acid with bases rU, dU and dT and have found that the macroscopic rate of translocation is dependent upon both the base and sugar moieties of the nucleic acid, with approximate macroscopic translocation rates of 3 nt/s (oligo-dT), 35 nt/s (oligo-dU), and 42 nt/s (oligo-rU), respectively. We found a strong correlation between the macroscopic translocation rates and the binding affinity of the translocating NS3h protein to the respective substrates such that weaker affinity corresponded to faster translocation. The values of K0.5 for NS3h translocation at a saturating ATP concentration are: (3.3 ± 0.4) μM nucleotide (poly-dT), (27 ± 2) μM nucleotide (poly-dU), and (36 ± 2) μM nucleotide (poly-rU). Furthermore, the results of isothermal titration of NS3h with these oligonucleotides suggest that differences in TΔS° are the principal source of the differences in the affinity of NS3h binding to these substrates. Interestingly, despite the differences in macroscopic translocation rates and binding affinities, the ATP coupling stoichiometry for NS3h translocation was identical for all three substrates, ~0.5 ATP molecules consumed per nucleotide translocated. This similar periodicity of ATP consumption implies a similar mechanism for NS3h translocation along RNA and DNA substrates.

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“…Interestingly, the affinity of NS3h for binding these translocation substrates is inversely proportional to the associated macroscopic rate of translocation (71), similar to what has been observed for the Rep helicase (51). This suggests that the efficiency with which NS3h can couple ATP binding and hydrolysis to nucleic acid translocation provides a constraint that results in a compensatory relationship between the affinity of nucleic acid binding and the macroscopic rate of nucleic acid translocation (71).…”

“…Further confirmation of the correlation between substrate binding affinity and DNA translocation is found in a separate study in which a macroscopic rate of (46 ± 5) nt/s for NS3h translocation along oligo(dT) was determined at higher temperature and higher NaCl concentration (72); an increase in both the temperature and the NaCl concentration would likely decrease the affinity of ssDNA binding by NS3h. This comparison is made slightly problematic, however, by the fact that these two studies used different constructs for NS3h (71,72). Nevertheless, it is worth mentioning that both studies reported similar estimates of the kinetic stepsize of translocation along oligo(dT) [(1.38 ± 0.07) nt (71) vs. (1.7 ± 0.2) nt (72)] and that an additional slow process occurs during the translocation mechanism (71,72).…”

“…Monomers of HCV NS3h have been shown to translocate from 3′ to 5′ along ssDNA and ssRNA with a macroscopic rate that depends upon both the base moiety and sugar moiety of the nucleic acid (71). The macroscopic rate at saturating ATP concentrations is (3.35 ± 0.09) nt/s for oligo(dT) translocation, (35.4 ± 0.6) nt/s for oligo(dU) translocation, and (42.2 ± 1.5) nt/s for oligo(rU) translocation; the corresponding kinetic step-sizes are (1.38 ± 0.07) nucleotides, (1.00 ± 0.07) nucleotides, and (1.05 ± 0.08) nucleotides, respectively.…”

“…This suggests that the efficiency with which NS3h can couple ATP binding and hydrolysis to nucleic acid translocation provides a constraint that results in a compensatory relationship between the affinity of nucleic acid binding and the macroscopic rate of nucleic acid translocation (71). Further confirmation of the correlation between substrate binding affinity and DNA translocation is found in a separate study in which a macroscopic rate of (46 ± 5) nt/s for NS3h translocation along oligo(dT) was determined at higher temperature and higher NaCl concentration (72); an increase in both the temperature and the NaCl concentration would likely decrease the affinity of ssDNA binding by NS3h.…”

“…This comparison is made slightly problematic, however, by the fact that these two studies used different constructs for NS3h (71,72). Nevertheless, it is worth mentioning that both studies reported similar estimates of the kinetic stepsize of translocation along oligo(dT) [(1.38 ± 0.07) nt (71) vs. (1.7 ± 0.2) nt (72)] and that an additional slow process occurs during the translocation mechanism (71,72). Candidates for this slow process are an initiation step proceeding processive translocation or a two-step dissociation from the end of the ssDNA (71,72).…”