Crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus

Stéphane Bressanelli

L Tomei

A Roussel

I Incitti

^{Virologie Moléculaire Structurale, Laboratoire de Génétique des Virus, Centre National de la Recherche Scientifique/Unité Propre de Recherche 9053 1, Avenue de la Terrasse, F-91198 Gif-sur-Yvette Cedex, France; and}^{Department of Biochemistry, Istituto di Ricerche di Biologia Molecolare “Piero Angeletti” Via Pontina Km 30,600 I-00040 Pomezia (Roma), Italy}

^{S.B. and L.T. contributed equally to this work.}

^{To whom reprint requests should be addressed. E-mail:}ti.mbri@ocsecnarfed or rf.fig-srnc.vg@yer.

Communicated by Stephen C. Harrison, Harvard University, Cambridge, MA

Received 1999 Aug 18; Accepted 1999 Sep 15.

Abstract

We report the crystal structure of the RNA-dependent RNA polymerase of hepatitis C virus, a major human pathogen, to 2.8-Å resolution. This enzyme is a key target for developing specific antiviral therapy. The structure of the catalytic domain contains 531 residues folded in the characteristic fingers, palm, and thumb subdomains. The fingers subdomain contains a region, the “fingertips,” that shares the same fold with reverse transcriptases. Superposition to the available structures of the latter shows that residues from the palm and fingertips are structurally equivalent. In addition, it shows that the hepatitis C virus polymerase was crystallized in a closed fingers conformation, similar to HIV-1 reverse transcriptase in ternary complex with DNA and dTTP [Huang H., Chopra, R., Verdine, G. L. & Harrison, S. C. (1998) Science 282, 1669–1675]. This superposition reveals the majority of the amino acid residues of the hepatitis C virus enzyme that are likely to be implicated in binding to the replicating RNA molecule and to the incoming NTP. It also suggests a rearrangement of the thumb domain as well as a possible concerted movement of thumb and fingertips during translocation of the RNA template-primer in successive polymerization rounds.

Abstract

Hepatitis C virus (HCV) is a major human pathogen that has infected an estimated 3% of the population worldwide. The virus is capable of establishing a persistent infection in the majority of cases, leading to chronic hepatitis that often develops into cirrhosis and, in many cases, into hepatocellular carcinoma. There is no vaccine available against HCV, and current therapies are effective only in a minority of cases. There is thus an urgent need to develop HCV-specific antiviral agents to counteract this important public health problem.

HCV is a positive-strand RNA virus in the Flaviviridae family. The viral genome contains a single ORF encoding a polyprotein of ≈3,000 amino acids (1) that is the precursor to all viral proteins. Genome replication proceeds in two steps: synthesis of complementary minus-strand RNA using the genome as template and the subsequent synthesis of genomic RNA using this minus-strand RNA template. The key enzyme involved in both of these steps is a virally encoded RNA-dependent RNA polymerase (RdRp). The HCV NS5B protein, located at the extreme C terminus of the polyprotein, contains motifs shared by all RdRps, such as the GDD motif (2). Indeed, RdRp activity has been demonstrated with recombinant NS5B (3).

The catalytic domain of nucleic acid polymerases is organized around a central cleft in an arrangement that is reminiscent of a right hand, with subdomains that have been termed “palm,” “fingers,” and “thumb,” as originally described in the structure of the Klenow fragment of Escherichia coli DNA polymerase 1 (4). Several three-dimensional structures of polynucleotide polymerases have been determined; they show that the four different classes of polymerases, i.e., DNA-dependent DNA and RNA polymerases and RNA-dependent DNA and RNA polymerases, share the same fold for the palm subdomain, which contains a cluster of catalytically important residues (see ref. 5 for a recent review). An exception is polymerase β, which has a different topology in the palm (6, 7). The similarity of DNA-dependent DNA polymerases of the polymerase 1 family with the other classes of polymerases had actually been suggested earlier by sequence alignments (8). The particular fold adopted by the palm subdomain is shared by many proteins that bind nucleotides and/or nucleic acids (9). It contains two absolutely conserved aspartic acid residues that coordinate two Mg^{ions, which actually carry out the polymerization reaction. Fidelity in replication is achieved by allowing only a nucleotide triphosphate that is correctly base-paired to the matching nucleotide in the template to adopt the right geometry for the esterification reaction to take place (}10).

In the case of RNA-dependent polymerases, the structures of two retroviral polymerases are known: reverse transcriptase (RT) from HIV 1 (for which structures are available in the unliganded form and in complex with several ligands; see ref. 11 for a recent review) and a fragment of RT from murine Moloney leukemia virus (12). The only RdRp for which we have structural data is unliganded poliovirus polymerase (9). This structure shows that the palm is folded as in all of the other classes of polymerases and that the fold of the thumb is different. Large parts of the fingers were disordered in the crystals, so that the complete fold of this subdomain is not known.

The catalytic domain of the HCV RdRp consists of the 531 amino-terminal residues of NS5B. As a key step to developing specific anti-HCV drugs that interfere with viral replication, we have crystallized this catalytic fragment. We describe in the following sections the crystal structure determined by the multiple anomalous diffraction method (13) to a resolution of 2.8 Å.

Wl, wavelength; p, absorption peak; i, inflection point, r, remote Wl; Res, resolution; Nref, number of unique reflections; Red, redundancy.

*Overall value (highest resolution shell).

R_sym = Σ_h,i|I_h,i − 〈I_h〉|/Σ_h,iI_h,i, where I_h,i is the intensity of the ith observation of the reflection of index h, R = Σ|F_o − F_c|/ΣF_o for all reflections, and R_free is R for 6% of the reflections omitted from the refinement (1,985 reflections) (33).

^{Z-score: assessment of the quality of phasing according to the program}solve (15).

Acknowledgments

We thank V. Stojanoff and J. Lescar of the European Synchrotron Radiation Facility for help during data collection, J. Janin for comments on the manuscript, and I. Petitpas for assistance. This work was funded in part through Human Frontier Science Program Grant RG-509 and Association pour la Recherche contre le Cancer Grant 9646 to F.A.R.

Acknowledgments

Abbreviations

HCV	hepatitis C virus
RdRp	RNA-dependent RNA polymerase
RT	reverse transcriptase

Abbreviations

Footnotes

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank, www.rcsb.org (PDB ID code 1csj).

Footnotes

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