Inhibition of the Hepatitis C Virus Helicase-associated ATPase Activity by the Combination of ADP, NaF, MgCl2, and Poly(rU)

Abstract

Hepatitis C virus (HCV) helicase has an intrinsic ATPase activity and a nucleic acid (poly(rU))-stimulated ATPase activity. The poly(rU)-stimulated ATPase activity was inhibited by F؊ in a time-dependent manner during ATP hydrolysis. Inhibition was the result of trapping an enzyme-bound ADP-poly(rU) ternary complex generated during the catalytic cycle and was not the result of generating enzyme-free ADP that subsequently inhibited the enzyme. However, catalysis was not required for efficient inhibition by F ؊ . The stimulated and the intrinsic ATPase activities were also inhibited by treatment of the enzyme with F ؊ , ADP, and poly(rU). The inhibited enzyme slowly recovered (t1 ⁄2 ‫؍‬ 23 min) ATPase activity after a 2000-fold dilution into assay buffer. The onset of inhibition by 500 M ADP and 15 mM F ؊ in the absence of nucleic acid was very slow (t1 ⁄2 > 40 min). However, the sequence of addition of poly(rU) to a diluted solution of ADP/NaF-treated enzyme had a profound effect on the extent of inhibition. If the ADP/NaFtreated enzyme was diluted into an assay that lacked poly(rU) and the assay was subsequently initiated with poly(rU), the treated enzyme was not inhibited. Alternatively, if the treated enzyme was diluted into an assay containing poly(rU), the enzyme was inhibited. ATP protected the enzyme from inhibition by ADP/NaF. The stoichiometry between ADP and enzyme monomer in the inhibited enzyme complex was 2, as determined from titration of the ATPase activity ( Hepatitis C virus (HCV)1 genome encodes for an RNA helicase that presumably is essential for viral replication (1). Helicases catalyze the separation of double-stranded nucleic acids into single-stranded nucleic acids with the concomitant hydrolysis of ATP (2). The mechanism of coupling of ATP hydrolysis to double-stranded nucleic acid unwinding is unclear (2, 3). Escherichia coli Rep helicase is the most thoroughly characterized helicase in terms of characterizing the binding of DNA and the associated ATPase activities (3-8). Recently, a crystal structure of the Rep helicase complexed with DNA and ADP has been reported. Helicase motifs Ia, V, and III were implicated in single-stranded DNA binding, motifs I and IV were involved in nucleotide binding, and motifs II and IV possibly functioned in the coupling of nucleotide and single-stranded DNA binding (9). Unfortunately, the protein was crystallized in the absence of a divalent metal cofactor such as Mg 2ϩ , which is required for ATP hydrolysis and nucleic acid unwinding. Crystal structures of DNA helicase from Bacillus stearothermophilus (10) and of RNA helicase from HCV have also been reported. In neither case was the divalent metal cofactor present. Nonetheless, these structures suggested a single nucleotide binding site per monomer.HCV helicase has an intrinsic ATPase activity and an nucleic acid-stimulated ATPase activity. Single-stranded DNA or RNA increased the k cat value of HCV helicase ATPase activity, at most, 50-fold (12). For example, poly(rU) enhanced k cat 30-fold. In contrast,...

ATP Binding Modulates the Nucleic Acid Affinity of Hepatitis C Virus Helicase

“…This is in contrast with a report that proposes that the HCV helicase monomer has two ATP binding sites (10). The stoichiometry of 1:1 in our studies was obtained by direct equilibrium binding measurements, whereas the reported studies used the inhibition of ATPase activity as a function of ADP concentration in the presence of NaF to obtain the 1:2 stoichiometry.…”

“…HCV helicase does not form stable oligomers, and each helicase molecule has one nucleic acid binding site (5)(6)(7). Although sequence and crystallographic data support a single NTP binding site (8,9), it was proposed that the HCV helicase molecule has two ATP binding sites (10). In this paper we explore the ATP binding stoichiometry with direct nucleotide binding assays.…”

Understanding Helicases as a Means of Virus Control

“…The products of ATP hydrolysis, ADP and P i , do not inhibit the HCV NS3 helicase. However, in the presence of NaF and PolyU RNA, ADP inhibits ATP hydrolysis with about two moles of ADP binding per protein monomer [181]. When beryllium fluoride is added to the reaction, ADP inhibits ATP hydrolysis more potently with a K i of about 8.5 µM [136,146].…”

The Hepatitis C Virus NS3 Protein: A Model RNA Helicase and Potential Drug Target

“…Dimerization of NS3 has also been visualized using analytical gel filtration, but only in the presence of an oligonucleotide (Khu et al, 2001). Nucleic acid binding data can sometimes be fit to models that do not take into account subunit interactions (Porter, 1998b;Porter, 1998a;Porter et al, 1998;Levin and Patel, 2002), but under certain conditions, cooperative models fit the data better (Locatelli et al, 2002;Frick et al, 2004b). Taken together, these data suggest that two or more HCV helicase protomers cooperatively assemble onto ssDNA (or RNA) in a controlled manner.…”

“…Porter et al first detected a possible second nucleotide binding site when they studied product inhibition in the presence of NaF. In their studies, about two moles of ADP bound per protein monomer (Porter, 1998a). In contrast, as discussed above, when beryllium fluoride is added to the reaction, only one mole of ADP is bound per protomer (Lam et al, 2003a;Levin et al, 2003).…”