Mutagenesis analysis of the rGTP-specific binding site of hepatitis C virus RNA-dependent RNA polymerase

Abstract

Hepatitis C virus (HCV) nonstructural protein 5B (NS5B) is the virus-encoded RNA-dependent RNA polymerase (RdRp) essential for HCV RNA replication. An earlier crystallographic study identified a rGTP-specific binding site lying at the surface between the thumb domain and the fingertip about 30 A away from the active site of the HCV RdRp (S. Bressanelli, L. Tomei, F. A. Rey, and R. De Francesco, J. Virol 76:3482-3492, 2002). To determine its physiological importance, we performed a systematic mutagenesis analysis of the rGTP-specific binding pocket by amino acid substitutions. Effects of mutations of the rGTP-specific binding site on enzymatic activity were determined by an in vitro RdRp assay, while effects of mutations on HCV RNA replication were examined by cell colony formation, as well as by transient replication of subgenomic HCV RNAs. Results derived from these studies demonstrate that amino acid substitutions of the rGTP-specific binding pocket did not significantly affect the in vitro RdRp activity of purified recombinant NS5B proteins, as measured by their abilities to synthesize RNA on an RNA template containing the 3' untranslated region of HCV negative-strand RNA. However, most mutations of the rGTP-specific binding site either impaired or completely ablated the ability of subgenomic HCV RNAs to induce cell colony formation. Likewise, these mutations caused either reduction in or lethality to transient replication of the human immunodeficiency virus Tat-expressing HCV replicon RNAs in the cell. Collectively, these findings demonstrate that the rGTP-specific binding site of the HCV NS5B is not required for in vitro RdRp activity but is important for HCV RNA replication in vivo.

FIG. 1.

(A) Ribbon structure of the HCV NS5B protein. The catalytic site of the enzyme is highlighted by a nucleotide triphosphate and two metals at the center of the molecule. The amino acid residues (S29, R32, P495, P496, V499, and R503) defining the low-affinity rGTP-specific binding pocket are highlighted by their side chains. (B) Diagram of mutations of the rGTP-specific binding site of NS5B. The name of the mutant RNA is indicated on the left, and the amino acid is indicated by single letters. The amino acid position is shown on the top. Residues involved in the GTP-specific binding are highlighted in boldface type. The underlined residues of the GTP binding site are less conserved among different HCV genotypes.

FIG. 2.

Electrophoretic analysis of purified recombinant NS5B proteins. Wild-type and mutant NS5B proteins with a deletion of the C-terminal 21 amino acids were expressed and purified as described in Materials and Methods. Briefly, 500 ng (each) of purified NS5B proteins was loaded in a 10% SDS-PAGE gel and visualized by Coomassie blue staining. The names of the purified NS5B proteins are indicated on the top, and the sizes of the protein molecule markers are shown on the left.

FIG. 3.

(A) Time course of RNA synthesis by purified HCV NS5B. Wild-type NS5B (100 ng) was incubated with 50 ng of (−)3′UTR RNA template at 30°C for 10, 30, 45, 60, 90, and 120 min, respectively. The radiolabeled RNA products were analyzed in a 6% polyacrylamide-7.7 M urea gel, which was subsequently dried and subjected to autoradiography. (B) Correlation of RNA products with the incubation time of RdRp reaction. The RNA products shown in panel A were quantified by PhosphorImager (Molecular Dynamics). The amounts of RNA products (y axis) are plotted against the incubation time (x axis).

FIG. 4.

Effects of mutations of the low-affinity rGTP-specific binding site on the in vitro RdRp activity. A total of 100 ng of purified NS5B protein was incubated with 50 ng of HCV (−)3′UTR RNA template under the reaction conditions described in Materials and Methods. The RdRp reactions were run for either 120 min (A) or 30 min (B) RNA products were resolved in a 6% polyacrylamide-7.7 M urea gel and visualized by autoradiography. The levels of RNA products were quantified by PhosphorImager analysis (Molecular Dynamics). Purified NS5B proteins are indicated on the top, and the sizes of the RNA markers are indicated on the left. (C) Comparison of relative in vitro RdRp activity between wild-type and mutant NS5B proteins. The amount of RNA synthesized by NS5B was quantified with a PhosphorImager. The RdRp activity of each mutant NS5B relative to wild-type NS5B was calculated as a percentage of wild-type activity, considering wild-type NS5B as 100%. The average percentages of three independent experiments are shown for each mutant NS5B, as indicated at the bottom.

FIG. 5.

Stimulation of RNA synthesis by rGTP. In vitro RdRp experiments were performed as described in Materials and Methods by the addition of increasing concentrations (0, 0.04, 0.2, 1.0, and 5.0 mM) of rGTP. Reaction mixtures were incubated at 30°C for 60 min. (A) Stimulation of wild-type RdRp by rGTP. The concentrations (in millimoles) of rGTP are shown on the top. (B) Effects of mutations of the rGTP binding site on the RdRp stimulation by rGTP. The amounts of RNA synthesized by wild-type and mutant NS5Bs (indicated in the graph) were quantified by PhosphorImager analysis (Molecular Dynamics). The relative increase (n-fold) of the amount of RNA synthesized was calculated using the amount of RNA synthesized by wild-type NS5B at 0.04 mM rGTP, set as 1.

FIG. 6.

Effects of mutations of the rGTP-specific binding site on the efficiency of cell colony formation. Huh7.5 cells were transfected with in vitro T7 transcripts of subgenomic HCV replicons and seeded in 100-mm dishes at the cell density indicated on the top of the panel. Cell colonies were selected by culturing with 500 μg/ml of G418 sulfate for about 3 weeks, stained with a crystal violet solution, and photographed.

FIG. 7.

Effects of mutations of the rGTP-specific binding site on transient HCV RNA replication in the cell. Wild-type and mutant RNAs were transfected into EN5-3 cells. The culture media were sampled at 24-h intervals and assayed for SEAP activity as a measure of the intracellular replicon RNA abundance (see Materials and Methods). The results represent the mean SEAP activities relative to that of the wild-type RNA. Error bars indicate the range of variation in duplicate transfection experiments. Open bars stand for the relative levels of SEAP as determined at 24 h (day 1) posttransfection, and solid bars indicate the relative levels of SEAP at 96 h (day 4) posttransfection. The names of amino acid mutations are indicated at the bottom. (A) Comparison of the relative SEAP activities at day 1 and day 4 between wild-type and mutant HCV RNAs. The percentage of wild-type control was calculated for each mutation considering the wild-type level as 100%. (B) Effects of mutations of the rGTP-specific binding site on transient HCV RNA replication. The levels of SEAP activities at day 1 (A) were used to normalize the input RNA as well as the transfection efficiency. After normalization, the levels of SEAP activities at day 4 are shown as a percentage of the wild-type control (100%).