The development of resistance to direct-acting antivirals (DAAs) targeting the hepatitis C virus (HCV) can compromise therapy. However, mechanisms that determine prevalence and frequency of resistance-conferring mutations remain elusive. Here, we studied the fidelity of the HCV RNA-dependent RNA polymerase NS5B in an attempt to link the efficiency of mismatch formation with genotypic changes observed in vivo. Enzyme kinetic measurements revealed unexpectedly high error rates (approximately 10 −3 per site) for G∶U∕U∶G mismatches. The strong preference for G∶U∕U∶G mismatches over all other mistakes correlates with a mutational bias in favor of transitions over transversions. Deep sequencing of HCV RNA samples isolated from 20 treatment-naïve patients revealed an approximately 75-fold difference in frequencies of the two classes of mutations. A stochastic model based on these results suggests that the bias toward transitions can also affect the selection of resistance-conferring mutations. Collectively, the data provide strong evidence to suggest that the nature of the nucleotide change can contribute to the genetic barrier in the development of resistance to DAAs.A pproximately 170 million people worldwide are infected with hepatitis C virus (HCV) (1). Chronically infected individuals are at risk of developing severe liver disease, including cirrhosis and hepatocellular carcinoma (2). Although the infection can be cured with a combination of interferon-α and ribavirin, severe side effects and complicated treatment schedules can compromise therapy (3). Moreover, the response is particularly poor in patients infected with HCV genotype 1, which is the most prevalent genotype in North America and Europe (4).Several direct-acting antivirals (DAAs) targeting important viral and host factors are currently undergoing clinical evaluation. Drugs acting against the viral NS5B RNA-dependent RNA polymerase (RdRp) or NS3 protease are in advanced clinical trials, with the protease inhibitors (PIs) telaprevir and boceprevir recently gaining FDA approval. Combining interferon-α and ribavirin with either PI can produce increases in sustained virological response when compared with the combination of interferon-α and ribavirin without PI (5, 6). However, the emergence of resistance-conferring mutations can lead to treatment failure (7). Like other RNA viruses, HCV shows quasispecies-like characteristics (8). Accordingly, resistant variants are selected from a genetically highly diverse population. Resistance to PIs is rapidly selected in cell-based replicon systems and in vivo (9). Similar observations have been made with the various classes of nonnucleoside analogue inhibitors (NNIs) of NS5B (9). Moreover, mutations that decrease susceptibility to highly potent inhibitors of the RNA binding protein NS5A also emerge rapidly in the replicon system (10). In contrast, the barrier to the development of resistance to nucleoside analogue inhibitors (NIs) appears to be significantly higher. The 2′-C-methylated NIs were shown to select in v...

More than 20 years after the identification of the hepatitis C virus (HCV) as a novel human pathogen, the only approved treatment remains a combination of pegylated interferon-α and ribavirin. This rather non-specific therapy is associated with severe side effects and by far not everyone benefits from treatment. Recently, progress has been made in the development of specifically targeted antiviral therapy for HCV (STAT-C). A major target for such direct acting antivirals (DAAs) is the HCV RNA-dependent RNA polymerase or non-structural protein 5B (NS5B), which is essential for viral replication. This review will examine the current state of development of inhibitors targeting the polymerase and issues such as the emergence of antiviral resistance during treatment, as well as strategies to address this problem.

Nonobligate chain terminators, such as 2-C-methylated nucleotides, block RNA synthesis by the RNAdependent RNA polymerase (RdRp) of hepatitis C virus (HCV). Previous studies with related viral polymerases have shown that classical chain terminators lacking the 3-hydroxyl group can be excised in the presence of pyrophosphate (PP i ), which is detrimental to the inhibitory activity of these compounds. Here we demonstrate that the HCV RdRp enzyme is capable of removing both obligate and clinically relevant nonobligate chain terminators. Pyrimidines are more efficiently excised than are purines. The presence of the next complementary templated nucleotide literally blocks the excision of obligate chain terminators through the formation of a dead-end complex (DEC). However, 2-C-methylated CMP is still cleaved efficiently under these conditions. These findings show that a 2-methylated primer terminus impedes nucleotide binding. The S282T mutation, associated with resistance to 2-C-methylated nucleotides, does not affect the excision patterns. Thus, the decreased susceptibility to 2-C-methylated nucleotides appears to be based solely on improved discrimination between the inhibitor and its natural counterpart. In conclusion, our data suggest that the phosphorolytic excision of nonobligate, pyrimidine-based chain terminators can diminish their potency. The templated nucleotide does not appear to provide protection from excision through DEC formation.Hepatitis C virus (HCV) infection is a serious public health concern that affects 170 million people worldwide (33, 42). Among those infected, approximately 20 to 30% develop severe liver disease, such as chronic hepatitis, liver cirrhosis, or hepatocellular carcinoma (2). The combined use of the nucleoside analogue ribavirin and pegylated alpha interferon is the current treatment standard; however, success in treatment depends largely on the viral genotype, and this drug combination has also been associated with severe side effects (30,43). Thus, the development of novel, more potent, specific drugs is urgent.HCV belongs to the Flaviviridae family. The HCV RNA genome consists of approximately 10 kb, encoding a polyprotein which is processed into several smaller polypeptides, including the capsid protein (C), the envelope proteins (E1 and E2), and the nonstructural proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B). Initial cis cleavage through NS2-NS3 releases the NS3 protein, which in turn continues to process the precursor. Promising compounds with the ability to inhibit the viral protease (NS3) and the polymerase (NS5B) have been identified.NS5B is a 65-kDa RNA-dependent RNA polymerase capable of initiating RNA synthesis de novo in the absence of a primer (16,17,25,27,45). Three classes of inhibitors of HCV NS5B have been developed, namely, nucleoside analogue inhibitors, nonnucleoside analogue inhibitors, and pyrophosphate (PP i ) analogues. Nonnucleoside inhibitors and PP i analogues are still under preclinical evaluation, while a 2Ј-modified nucleoside analogue has advanc...

Background:The dynamics associated with RNA binding by the hepatitis C virus (HCV) polymerase remain elusive. Results: Single molecule experiments reveal changing populations of binary RNA-enzyme complexes. Conclusion: Rapid enzyme conformational changes facilitate sliding/wrapping of the polymerase along its RNA substrate. Significance: This study provides novel insight into mechanisms associated with viral replication and its inhibition.

We studied the biochemical mechanisms associated with inhibition and resistance to a 4,5-dihydroxypyrimidine carboxylate that inhibits the hepatitis C virus (HCV) RNA-dependent RNA polymerase NS5B. On the basis of the structure of the pharmacophore, it has been suggested that these compounds may act as pyrophosphate (PP i ) mimics. We monitored nucleotide incorporation events during the elongation phase and showed that the polymerase activity of wild-type NS5B was inhibited by the dihydroxypyrimidine at a 50% inhibitory concentration (IC 50 ) of 0.73 M. Enzymes with the G152E or P156L mutation, either of which confers resistance to this compound, showed four-to fivefold increases in IC 50 s. The inhibitor was competitive with respect to nucleotide incorporation. It was likewise effective at preventing the PP i -mediated excision of an incorporated chain terminator in a competitive fashion. In the absence of the dihydroxypyrimidine, the reaction was not significantly affected by the G152E or P156L mutation. These data suggest that the resistance associated with these two mutations is unlikely due to an altered interaction with the pyrophosphate-mimicking domain of the compound but, rather, is due to altered interactions with its specificity domain at a region distant from the active site. Together, our findings provide strong experimental evidence that supports the notion that the members of this class of compounds can act as PP i mimics that have the potential to mechanistically complement established nucleoside and nonnucleoside analogue inhibitors.Hepatitis C virus (HCV) is a major public health problem, with an estimated 170 million people worldwide being infected with the virus (9). Chronic infection with HCV can lead to the development of severe liver disease, including cirrhosis and hepatocellular carcinoma (HCC) (17). The current standard of care for those who are in need of antiviral therapy consists of a combination of pegylated alpha interferon and the nucleoside analogue ribavirin (21). However, the clinical use of both components is associated with toxic side effects, and by far, not everyone benefits from treatment (4, 11).Nonstructural proteins NS2 through NS5B represent important targets for current drug discovery and development efforts aimed at improving anti-HCV therapy. Various classes of inhibitors of the HCV RNA-dependent RNA polymerase NS5B have been developed. These compounds can be further grouped into nonnucleoside analogue inhibitors (NNIs) and nucleoside analogue inhibitors (NIs). The two classes of inhibitors target different stages of RNA synthesis. The HCV NS5B protein is capable of initiating RNA synthesis de novo, i.e., in the absence of a primer (12,14,23,25,33). At this early stage, productive initiation complexes are fragile and RNA synthesis is distributive. After four to six nucleotide incorporation events, at which point the enzyme switches to the elongation stage, conformational changes render the polymerization process highly processive (18). NNIs were shown to interfere w...