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.
The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is the catalytic subunit of the viral RNA amplification machinery and is an appealing target for the development of new therapeutic agents against HCV infection. Nonnucleoside inhibitors based on a benzimidazole scaffold have been recently reported. Compounds of this class are efficient inhibitors of HCV RNA replication in cell culture, thus providing attractive candidates for further development. Here we report the detailed analysis of the mechanism of action of selected benzimidazole inhibitors. Kinetic data and binding experiments indicated that these compounds act as allosteric inhibitors that block the activity of the polymerase prior to the elongation step. Escape mutations that confer resistance to these compounds map to proline 495, a residue located on the surface of the polymerase thumb domain and away from the active site. Substitution of this residue is sufficient to make the HCV enzyme and replicons resistant to the inhibitors. Interestingly, proline 495 lies in a recently identified noncatalytic GTPbinding site, thus validating it as a potential allosteric site that can be targeted by small-molecule inhibitors of HCV polymerase.Hepatitis C virus (HCV) is the causative agent of the majority of chronic liver disease throughout the world. More than 170 million individuals are estimated to be infected with this virus (27). The size of the HCV epidemic and the limited efficacy of current therapy (based on the use of alpha interferon) have stimulated intense research efforts towards the development of antiviral drugs that are both better tolerated and more effective. The most widely established strategy for developing novel anti-HCV therapeutics aims at the identification of low-molecular-weight inhibitors of essential HCV enzymes.RNA-dependent RNA polymerase (RdRP) activity, carried out by the NS5B protein, is essential for virus replication (13) and has no functional equivalent in uninfected mammalian cells. It is thus likely that specific inhibitors of this enzyme can be found that block HCV replication with negligible associated toxicity. The NS5B RdRP has been expressed in a variety of recombinant forms (2, 4). The production of highly soluble forms of the enzyme (12, 24), devoid of the C-terminal membrane anchoring domain (23), has allowed considerable progress toward the determination of the enzyme's three-dimensional structure and mechanism of action. The crystal structure of NS5B revealed a classical "right hand" shape, showing the characteristic fingers, palm, and thumb subdomains (1,7,14). More recently, the three-dimensional structure of the HCV polymerase was solved in complex with RNA (20) as well as in a complex with nucleoside triphosphates (6). Three distinct nucleotide-binding sites were observed in the catalytic center of HCV RdRP whose geometry was remarkably similar to that observed in the initiation complex of the RNA phage ⌽6 RdRP (8), strengthening the proposal that the two enzymes initiate replication de novo by similar ...
The RNA-dependent RNA polymerase of hepatitis C virus (HCV) is necessary for the replication of viral RNA and thus represents an attractive target for drug development. Several structural classes of nonnucleoside inhibitors (NNIs) of HCV RNA polymerase have been described, including a promising series of benzothiadiazine compounds that efficiently block replication of HCV subgenomic replicons in tissue culture. In this work we report the selection of replicons resistant to inhibition by the benzothiadiazine class of NNIs. Four different single mutations were identified in separate clones, and all four map to the RNA polymerase gene, validating the polymerase as the antiviral target of inhibition. The mutations (M414T, C451R, G558R, and H95R) render the HCV replicons resistant to inhibition by benzothiadiazines, though the mutant replicons remain sensitive to inhibition by other nucleoside and NNIs of the HCV RNA polymerase. Additionally, cross-resistance studies and synergistic inhibition of the enzyme by combinations of a benzimidazole and a benzothiadiazine indicate the existence of nonoverlapping binding sites for these two structural classes of inhibitors.Hepatitis C virus (HCV) chronically infects about 3% of the human population, causing a slowly evolving liver disease that leads to cirrhosis, liver failure, and occasionally hepatocellular carcinoma (39). Given the size of the HCV epidemic and the limited efficacy of the present therapy based on alpha interferon (16), the development of new, safer, and more effective drugs is of paramount importance and is presently an area of intensive research. The strategy most widely applied for developing novel anti-HCV therapeutics aims at identifying small molecule inhibitors of viral enzymes. The nonstructural protein 5B RNA-dependent RNA polymerase (NS5B RdRp) is an important target of drug discovery activities largely because it is essential for viral replication and also due to the clinical successes of inhibitors of other viral polymerases. In addition, the extensive structural and biochemical characterization of this enzyme provides the basis for drug design efforts as well as for elucidating the mechanism of action of inhibitors and for rapidly optimizing their potency.The NS5B protein was initially identified as an RdRp based on the presence of the signature GDD (Gly-Asp-Asp) motif characteristic for this class of enzymes (11). Its function was confirmed when an active form of the full-length protein was purified from baculovirus-infected insect cells (5). Subsequently, attempts to improve solubility, stability, and activity lead to the expression of C-terminal-truncated forms lacking the hydrophobic membrane anchor contained within the last 21 amino acids (1,17,25,33,35). In vitro, the enzyme shows little, if any, specificity for the HCV genome and can catalyze the synthesis of RNA by using a variety of homo-or heteropolymeric RNA templates both with and without a primer. In the absence of primer, NS5B can initiate RNA synthesis either by using the 3Ј-termi...
The RNA-dependent RNA polymerase activity of hepatitis C virus is carried out by the NS5B protein.The full-length protein was previously purified as a non-fusion protein from insect cells infected with a recombinant baculovirus. The characterization is now described of a C-terminal hydrophobic domain deletion mutant of NS5B purified from E. coli. In addition to increased solubility, deletion of this sequence also positively affected the polymerase enzymatic activity. The efficiency of nucleotide polymerization of both the full-length and the C-terminal truncated enzymes were compared on homopolymeric template-primer couples as well as on RNA templates with heteropolymeric sequences. The largest difference in the polymerase activity was observed on the latter. On all the templates, the increased activity could be ascribed, at least in part, to enhanced template turnover of the deletion mutant with respect to the full-length enzyme. The elongation rates of the two enzyme forms were compared under single processive cycle conditions. Under these conditions, both the full-length and the deletion mutant were able to incorporate about 700 nt/min.
In order to find small RNA molecules that are specific and high-affinity ligands of nonstructural 5B (NS5B) polymerase, we screened by SELEX (systematic evolution of ligands by exponential amplification) a structurally constrained RNA library with an NS5B⌬C55 enzyme carrying a C-terminal biotinylation sequence. Among the selected clones, two aptamers appeared to be high-affinity ligands of NS5B, with apparent dissociation constants in the low nanomolar range. They share a sequence that can assume a stem-loop structure. By mutation analysis, this structure has been shown to correspond to the RNA motif responsible for the tight interaction with NS5B. The aptamers appeared to be highly specific for the hepatitis C virus (HCV) polymerase since interaction with the GB virus B (GBV-B) NS5B protein cannot be observed. This is consistent with the observation that the activity of the HCV NS5B polymerase is efficiently inhibited by the selected aptamers, while neither GBV-B nor poliovirus 3D polymerases are affected. The mechanism of inhibition of the NS5B activity turned out to be noncompetitive with respect to template RNA, suggesting that aptamers and template RNA do not bind to the same site. As a matter of fact, mutations introduced in a basic exposed surface of the thumb domain severely impaired both the binding of and activity inhibition by the RNA aptamers.Hepatitis C virus (HCV) is a positive-strand RNA virus of the Flaviviridae family, which affects more than 3% of the world population. About 80% of the infected patients develop liver cirrhosis and, in some cases, hepatocarcinoma (13). Besides interferon-based treatments, effective therapies to counteract this important public health problem are still lacking.The HCV positive-strand RNA viral genome contains a single open reading frame flanked by 5Ј-and 3Ј-untranslated regions. The open reading frame encodes a polyprotein of ca. 3,010 amino acids which is processed into at least 10 mature proteins (C, E1, E2, p7, NS2, NS3, NS4A, NS4B, NS5A, and NS5B) by both host signal peptidases and viral proteases (10,31). In analogy with other positive-strand RNA viruses, HCV replication is supposed to proceed through the synthesis of negative-strand RNA, which is in turn used as a template for the production of genomic RNA molecules. A virally encoded RNA-dependent RNA polymerase (RdRp) is considered one of the key enzymes involved in both steps of HCV replication and is, therefore, a primary target for the development of antiviral drugs. The HCV RdRp activity has been localized in the 66-kDa nonstructural 5B (NS5B) protein (2). In vitro, purified NS5B has been shown to be a processive enzyme (39) capable of transcribing the full-length HCV genome (28) essentially via a snap-back mechanism. Recent studies indicate that NS5B does direct de novo replication, requiring neither an exogenous primer nor a snap-back priming event on a variety of RNA templates (22,30,37,43,44). The lack of specificity toward the HCV RNA genome suggests that NS5B corresponds to the elongation factor...
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