Hepatitis C virus NS3 ATPases/helicases from different genotypes exhibit variations in enzymatic properties

Abstract

The NS3 ATPase/helicase was isolated and characterized from three different infectious clones of hepatitis C virus (HCV). One helicase was from a genotype that normally responds to therapy (Hel-2a), and the other two were from more resistant genotypes, 1a (Hel-1a) and 1b (Hel-1b). Although the differences among these helicases are generally minor, all three enzymes have distinct properties. Hel-1a is less selective for nucleoside triphosphates, Hel-1b hydrolyzes nucleoside triphosphates less rapidly, and Hel-2a unwinds DNA more rapidly and binds DNA more tightly than the other two enzymes. Unlike related proteins, different nucleic acid sequences stimulate ATP hydrolysis by HCV helicase at different maximum rates and with different apparent efficiencies. This nucleic acid stimulation profile is conserved among the enzymes, but it does not result entirely from differential DNA-binding affinities. Although the amino acid sequences of the three proteins differ by up to 15%, one variant amino acid that is critical for helicase action was identified. NS3 residue 450 is a threonine in Hel-1a and Hel-1b and is an isoleucine in Hel-2a. A mutant Hel-1a with an isoleucine substituted for threonine 450 unwinds DNA more rapidly and binds DNA more tightly than the parent protein.

FIG. 1.

Steady-state rates of ATP hydrolysis catalyzed by HCV helicases from genotypes 1a, 1b, and 2a. The rate of phosphate release from [γ-³²P]ATP was monitored at different concentrations of ATP in the absence (A) or presence (B) of 2 mg of poly(U) RNA/ml. Rates were obtained by using Hel-1a (squares), Hel-1b (triangles), or Hel-2a (circles). The data were fit to equation 1 by nonlinear regression analysis to yield the constants summarized in Table 2.

FIG. 2.

RNA stimulation of ATP hydrolysis catalyzed by Hel-1a, Hel-1b, or Hel-2a. Steady-state rates of ATP hydrolysis catalyzed by Hel-1a (squares), Hel-1b (triangles), or Hel-2a (circles) were measured in the presence of nine different concentrations of poly(U). The experiments were repeated with three different amounts of enzyme, and average turnover rates (moles of ATP hydrolyzed/moles of enzyme/second) were plotted. The data were fit to equation 2, and the curves were drawn by using the resulting constants (Table 2).

FIG. 3.

Inhibition of ATP hydrolysis catalyzed by Hel-1a, -1b, and -2a. Inhibition of reactions containing 50 μM [γ-³²P]ATP by GTP (triangles), CTP (diamonds), or UTP (circles) is shown. Reactions were catalyzed by Hel-1a (A), Hel-1b (B), or Hel-2a (C). Data were fit to equation 3 to yield the constants summarized in Table 2.

FIG. 4.

Template preferences of the various HCV helicases. In experiments analogous to those shown in Fig. 2, the steady-state rate of hydrolysis of ATP by HCV helicase isolated from three genotypes was monitored in the presence of nine different concentrations of various NA templates. Several rates were obtained at NA concentrations above and below each calculated K_NA. Data were fit to equation 2 to yield the maximum turnover rate k_cat (A) and an apparent affinity, K_NA, of the enzyme for each activator (B). In each panel, the bars represent the values obtained with Hel-1a (white), Hel-1b (gray), or Hel-2a (black). Template sequences are listed in Table 1. Kinetic constants were not determined (n/d) for sequences that did not stimulate ATP hydrolysis.

FIG. 5.

Gel shift analysis of DNA binding by the HCV helicase. (A) In 10-μl reactions, various amounts of Hel-1a were incubated with 200 nM radiolabeled CT9. After 10 min at room temperature, bound (dCT9_B) and free (dCT9_F) DNA were separated by native polyacrylamide gel electrophoresis. The reactions that were analyzed in lanes 1 through 15 in panels A and B contained, respectively, 0, 114, 133, 152, 175, 190, 228, 226, 304, 357, 408, 456, 532, 608, and 760 nM of Hel-1a. (B) The experiment was repeated with Hel-1b and also with Hel-2a with radiolabeled dCT9 and dGA9 to determine the concentration of protein required to shift 50% of the radiolabeled oligonucleotide (EC₅₀).

FIG. 6.

Quenching of the intrinsic protein fluorescence of HCV helicase by NAs. Oligonucleotides dCT9 (▪) or dGA9 (•) (0 to 151.6 nM) were added to 12 nM Hel-1a (A), 18 nM Hel-1b (B), or 22 nM Hel-2a (C), and intrinsic protein fluorescence was measured (λ_ex = 280, λ_em = 340). Fractional fluorescence remaining at each NA concentration was determined, and data were fit to equation 5. Resulting changes in intrinsic protein fluorescence and the apparent dissociation constants are summarized in Table 3.

FIG. 7.

Helicase activity of Hel-1a, Hel-1b, and Hel-2a. (A) Helicase action on duplex DNA, RNA, and DNA/RNA heteroduplexes in the presence of an NA trap. One heteroduplex (DNA/3′ RNA) contained a template strand with a single-stranded tail made of RNA, and the other (RNA/3′ DNA) had a DNA template strand and an RNA release stand. Substrate (2 nM) was incubated with 280 nM Hel-1a (white), Hel-1b (gray), or Hel-2a (black) for 30 min and initiated with ATP (5 mM) and trap DNA (3 μM). Reactions were terminated after 10 min at room temperature, and the percentage of DNA unwound was determined by analyzing the products separated on 12% native polyacrylamide gels. (B) Rates of helicase action. DNA/3′ DNA substrate (1 nM) was incubated with 280 nM Hel-1a (▪), Hel-1b (▴), or Hel-2a (•) for 10 min at room temperature before initiating the reaction with ATP (5 mM) and trap DNA (3 μM). Reactions were terminated at various times, and the percentage of DNA unwound was determined. Data were fit to equation 6 to yield the following: for Hel-1a, an A_MAX of 86% and k_obs of 0.099 min⁻¹; for Hel-1b, an A_MAX of 88% and k_obs of 0.073 min⁻¹; and for Hel-2a, an A_MAX of 91% and k_obs of 0.12 min⁻¹.

FIG. 8.

Genetic difference among the helicases from HCV genotypes 1a, 1b, and 2a. (A) Alignment of Hel-1a (NCBI accession no. AAB67036), Hel-1b (NCBI accession no. AAC15722), and Hel-2a (NCBI accession no. AAF01178) (41). Residues in Hel-1b and Hel-2a that were identical to Hel-1a are noted as dots. Conserved motifs (13) are noted within brackets. (B) Variants amino near the NA binding site of the HCV helicase. The side chain hydroxyl of residue T450 was free to rotate to within 2 Å of the phosphate backbone, where it could donate a hydrogen bond. Coordinates are from Protein Data Bank accession code 1A1V.

FIG. 8.

Genetic difference among the helicases from HCV genotypes 1a, 1b, and 2a. (A) Alignment of Hel-1a (NCBI accession no. AAB67036), Hel-1b (NCBI accession no. AAC15722), and Hel-2a (NCBI accession no. AAF01178) (41). Residues in Hel-1b and Hel-2a that were identical to Hel-1a are noted as dots. Conserved motifs (13) are noted within brackets. (B) Variants amino near the NA binding site of the HCV helicase. The side chain hydroxyl of residue T450 was free to rotate to within 2 Å of the phosphate backbone, where it could donate a hydrogen bond. Coordinates are from Protein Data Bank accession code 1A1V.

FIG. 9.

Comparison of Hel-1a and Hel-1a with a T450I substitution. (A) Steady-state rates of ATP hydrolysis catalyzed at different concentrations of poly(U) RNA are given. Data were fit to equation 2 to yield a k_cat of 54 and a K_NA of 0.5 mM. (B) Unwinding of duplex DNA by T450I in a single-turnover assay as described in Fig. 7 are shown. Data were fit to equation 6 to yield an A_max of 86% and k_obs of 0.099 min⁻¹ for Hel-1a and an A_max of 94% and k_obs of 0.15 min⁻¹ for T450I. (C and D) Quenching of intrinsic T450I fluorescence was monitored when the protein was titrated with dCT9 (C) or dGA9 (D). Total protein in the titrations was 12 nM, the data were fit to equation 5, and the resulting K_D and ΔF_MAX values are listed in Table 3. In each panel, data obtained with Hel-1a, the parent of T450I, are shown as a dotted line.