Identification of a C-terminal regulatory motif in hepatitis C virus RNA-dependent RNA polymerase: structural and biochemical analysis

Abstract

The NS5B RNA-dependent RNA polymerase encoded by the hepatitis C virus (HCV) is a key component of the viral replicase. Reported here is the three-dimensional structure of HCV NS5B polymerase, with the highlight on its C-terminal folding, determined by X-ray crystallography at 2.1-A resolution. Structural analysis revealed that a stretch of C-terminal residues of HCV NS5B inserted into the putative RNA binding cleft, where they formed a hydrophobic pocket and interacted with several important structural elements. This region was found to be conserved and unique to the RNA polymerases encoded by HCV and related viruses. Through biochemical analyses, we confirmed that this region interfered with the binding of HCV NS5B to RNA. Deletion of this fragment from HCV NS5B enhanced the RNA synthesis rate up to approximately 50-fold. These results provide not only direct experimental insights into the role of the C-terminal tail of HCV NS5B polymerase but also a working model for the RNA synthesis mechanism employed by HCV and related viruses.

FIG. 1.

Analyses of HCV NS5B proteins. (A) Sequence comparison of the NS5B proteins used in this study. The C-terminal domain deleted from NS5B-540H is highlighted. (B) Analysis of purified HCV NS5B proteins. A 200-ng portion of purified NS5B protein was subject to separation by SDS-PAGE followed by Coomassie blue staining. Lanes: 1, NS5B-570H; 2, NS5B-540H. (C) CD spectrometric analysis of NS5B-570H (solid line) and NS5B-540H (dotted line).

FIG. 2.

Stereo diagram of the 2F_O-F_c electron density map. The map covers residues 559 to 564 from the C-terminal tail that forms a β-sheet with the β-hairpin of residues 450 to 455 and 440 to 445 from the catalytic core. The map was calculated using the native data including reflections from 20 to 2.1 Å at the 1.0 σ contour level.

FIG. 3.

Overall structure of NS5B-570H. (Left) Ribbon representation of the polypeptide chain. The thumb domain is colored in green, the palm domain is in red, the fingers domain is in blue, and the C-terminal tail (residues 531 to 570) is in yellow. The putative RNA-binding groove is also indicated. (Right) Cα trace of NS5B-570H with the same color scheme as in the left panel. The C-terminal tail is highlighted in yellow ribbon. The catalytic-site -GDD- motif is shown as a red cpk ball model. The tip of the C-terminal tail reaches to the -GDD- pocket.

FIG. 4.

Interactions between the C-terminal tail and the catalytic core structure. (Left) Hydrophobic interactions between conserved residues in the C-terminal tail and the thumb domain. Residues Trp550, Phe551, and Leu547 packed with Leu409, Leu459, Leu466, and Ile462 to form a well-defined hydrophobic pocket. Leu545 and Try448 plus Met414 lie in the top and bottom of this pocket. (Right) Hydrogen-bonding network between the β-strand (residues 560 to 565) and β-hairpin (residues 450 to 455). The hydrogen bond pairs are carbonyl oxygen of Ile560 with the backbone amide NH of Cys451, carbonyl oxygen of Cys451 with amide NH of His562, carbonyl oxygen of His562 with amide NH of Ser453, and carbonyl oxygen of Ser453 with amide NH of Leu564. These hydrogen bonds stabilize the β-hairpin structure.

FIG. 5.

RNA-binding efficiency of HCV NS5B proteins. The RNA-binding efficiencies of NS5B-570H and NS5B-540H were compared using the conditions specified in Materials and Methods. Three proteins were evaluated: NS5B-540H (lanes 1 and 4), NS5B-570H (lanes 2 and 5), and bovine serum albumin as control (lanes 3 and 6). The same amounts of ³²P-labeled RNAs (0.4 pmol) and protein (100 ng) were used in each experiment. Experiments were performed using dsRNA (preannealed sym/sub) and ssRNA [³²P-labeled oligo(U₁₂)].

FIG. 6.

RNA elongation catalyzed by HCV NS5B. Reactions were performed using poly(A) as the template and [α-³²P]UTP as the substrate, as described in the text. Shown is the time course for 30 nM NS5B-570H (▪) and NS5B-540H (•).

FIG. 7.

Single-nucleotide incorporation by HCV NS5B. (A) The RNA template, sym/sub, used in this study. (B) Single-nucleotide incorporation catalyzed by purified NS5B proteins. These experiments were performed using [α-³²P]UTP as substrate and cold sym/sub RNA as template, as described in Materials and Methods. Lanes: 1, NS5B-540H; 2, NS5B-570H; 3, ³²P-labeled sym/sub; 4, no-enzyme control. The positions for input sym/sub RNA template and 13-mer product (P) are labeled. (C) Multinucleotide incorporation assays were performed using cold NTP (ATP, GTP, CTP, and UTP) as substrates and ³²P-labeled sym/sub RNA as the template, as described in Materials and Methods. Lanes: 1, NS5B-540H; 2, NS5B-570H; 3, no-enzyme control. The positions for template RNA (sym/sub) and expected 13- to 16-mer products (P) are marked. (D) Time course for single-nucleotide incorporation catalyzed by HCV NS5B. Assays were performed side by side using 0.6 μM NS5B-540H or NS5B-570H as described in Materials and Methods, with cold sym/sub RNA as the template and [α-³²P]UTP as the substrate. (E) Quantitative analysis of UMP incorporation catalyzed by NS5B-540H (•) or NS5B-570H (○) as shown in panel D.

FIG. 8.

RNA initiation catalyzed by HCV NS5B. Primer-independent RNA synthesis reactions were performed using NS5B-540H or NS5B-570H with poly(U) as the template and [α-³²P]ATP as the substrate as described in Materials and Methods. At the time point specified, an aliquot of the reaction mix was taken out and subjected to quantitative filter binding and scintillation counting.

FIG. 9.

Effect of synthetic C-terminal peptides on HCV polymerase activity. Amino acid sequences of the peptides used for this study are shown at the top. Below are shown the results of the primer-dependent RNA synthesis reactions of NS5B-540H that were performed using poly(A) as the template and [α-³²P]UTP as the substrate in the presence of different peptides. Shown is the percent inhibition of NS5B-540H activity at different concentrations of individual peptides: C_545/570 (•), A4C_545/570 (▪), and C_545/559 (▴).

FIG. 10.

Sequence alignment of viral RNA polymerases. Amino acid residues of the listed viral polymerase were aligned to the center of the nucleotide/metal-binding site (solid box). For most polymerases listed, the nucleotide/metal-binding site is referred as the -GDD- motif. The C-terminal regulatory motif found in HCV NS5B and related viruses is highlighted (hatched box).