Contribution of a mutational bias in hepatitis C virus replication to the genetic barrier in the development of drug resistance

Abstract

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...

Response of Hepatitis C Virus to Long-Term Passage in the Presence of Alpha Interferon: Multiple Mutations and a Common Phenotype

“…1). The "low" lineage was further passaged in the presence of IFN-␣, with the concentration increased by 1 IU/ml after passages 45,55,60,65,70,80,85,90, and 95, reaching a final IFN-␣ concentration of 10 IU/ml (Fig. 1A).…”

A hepatitis C-vírus-bázispolimorfizmus jelentősége a kezelésben

“…A genetikai korlát azt jelenti, hogy hány nukleotid mutációja és ennek következtében hány aminosav cseréje szükséges ahhoz, hogy az adott gyógyszerre rezisztencia alakuljon ki. Alacsony genetikai korlát esetén 1-2 mutáció, illetve 1-2 aminosav cseréje elegendő, míg a magas genetikai korlát esetén legalább 3-4 nukleotid együttes mutációja szük-séges a rezisztencia kialakulásához [10].…”

Baseline dasabuvir resistance in Hepatitis C virus from the genotypes 1, 2 and 3 and modeling of the NS5B-dasabuvir complex by thein silicoapproach

“…The emergence of resistance mutations in NS5B can occur in two ways; by a transition or by a transversion substitution. In general, a transition substitution has a lower energy and evolutionary barrier, and therefore occur more frequently than a transversion substitution [18,19]. The resistance-associated substitutions (RASs) against dasabuvir have been extensively characterized in the Kati et al study [20].…”