ABSTRACT
Hepatitis C virus (HCV) genotype 3 presents a high level of both baseline and acquired resistance to direct-acting antivirals (DAAs), particularly those targeting the NS5A protein. To understand this resistance we studied a cohort of Brazilian patients treated with the NS5A DAA, daclatasvir and the nucleoside analogue, sofosbuvir. We observed a novel substitution at NS5A amino acid residue 98 [serine to glycine (S98G)] in patients who relapsed post-treatment. The effect of this substitution on both replication fitness and resistance to DAAs was evaluated using two genotype 3 subgenomic replicons. S98G had a modest effect on replication, but in combination with the previously characterized resistance-associated substitution (RAS), Y93H, resulted in a significant increase in daclatasvir resistance. This result suggests that combinations of substitutions may drive a high level of DAA resistance and provide some clues to the mechanism of action of the NS5A-targeting DAAs.
Subject(s)
Antiviral Agents/pharmacology , Carbamates/pharmacology , Drug Resistance, Viral/genetics , Hepacivirus/drug effects , Imidazoles/pharmacology , Pyrrolidines/pharmacology , Valine/analogs & derivatives , Viral Nonstructural Proteins/genetics , Antiviral Agents/therapeutic use , Brazil , Carbamates/therapeutic use , Cell Line, Tumor , Cohort Studies , Drug Resistance, Viral/drug effects , Drug Therapy, Combination , Genotype , Hepacivirus/genetics , Hepacivirus/physiology , Hepatitis C/drug therapy , Hepatitis C/virology , Humans , Imidazoles/therapeutic use , Mutation , Pyrrolidines/therapeutic use , Recurrence , Sofosbuvir/pharmacology , Sofosbuvir/therapeutic use , Valine/pharmacology , Valine/therapeutic use , Viral Nonstructural Proteins/antagonists & inhibitors , Virus Replication/geneticsABSTRACT
Hepatitis C virus (HCV) is an important human pathogen causing 400â¯000 chronic liver disease-related deaths annually. Until recently, the majority of laboratory-based investigations into the biology of HCV have focused on the genotype 2 isolate, JFH-1, involving replicons and infectious cell culture systems. However, genotype 2 is one of eight major genotypes of HCV and there is great sequence variation among these genotypes (>30â% nucleotide divergence). In this regard, genotype 3 is the second most common genotype and accounts for 30â% of global HCV cases. Further, genotype 3 is associated with both high levels of inherent resistance to direct-acting antiviral (DAA) therapy, and a more rapid progression to chronic liver diseases. Neither of these two attributes are fully understood, thus robust genotype 3 culture systems to unravel viral replication are required. Here we describe the generation of robust genotype 3 sub-genomic replicons (SGRs) based on the adapted HCV NS3-NS5B replicase from the DBN3a cell culture infectious clone. Such infectious cell culture-adaptive mutations could potentially promote the development of robust SGRs for other HCV strains and genotypes. The novel genotype 3 SGRs have been used both transiently and to establish stable SGR-harbouring cell lines. We show that these resources can be used to investigate aspects of genotype 3 biology, including NS5A function and DAA resistance. They will be useful tools for these studies, circumventing the need to work under the biosafety level 3 (BSL3) containment required in many countries.