Abstract
Direct-acting antivirals (DAAs) for hepatitis C virus (HCV) have potent anti-HCV effects but may provoke resistance-associated variants (RAVs). In this study, we assessed the characteristics of these RAVs and explored efficacious anti-HCV reagents using recombinant HCV with NS5A from a genotype 1b strain. We replaced the NS5A of JFH1 with that of Con1 (JFH1/5ACon1) and introduced known NS5A inhibitor resistance mutations (L31M, L31V, L31I and Y93H) individually or in combination. Susceptibilities against anti-HCV reagents were also investigated. RAVs with Y93H exhibited high extracellular core antigen levels and infectivity titers. Variants with any single mutation showed mild to moderate resistance against NS5A inhibitors, whereas variants with double mutations at both L31 and Y93 showed severe resistance. The variants with mutations exhibited similar levels of susceptibility to interferon (IFN)-α, IFN-λ1, IFN-λ3 and Ribavirin. Variants with the Y93H mutation were more sensitive to protease inhibitors compared with JFH1/5ACon1. In conclusion, the in vitro analysis indicated that the Y93H mutation enhanced infectious virus production, suggesting advantages in the propagation of RAVs with this mutation. However, these RAVs were susceptible to protease inhibitors. Thus, a therapeutic regimen that includes these reagents is a promising means to eradicate these RAVs.
Highlights
To investigate the effect of resistance-associated NS5A mutations on the virus life cycle, we introduced mutations reported in resistance to NS5A inhibitors (L31M, L31V, L31I and Y93H) into the JFH1 based recombinant virus with the NS5A from Con[1] (JFH1/5ACon1-wt) individually or in combination to generate JFH1/5ACon1-L31M, -L31V, -L31I, -Y93H, -L31M/Y93H, -L31V/Y93H, and -L31I/Y93H, respectively (Fig. 1a)
The hepatitis C virus (HCV) core Ag levels in the culture media of these recombinant virus RNA-transfected cells gradually increased in a time-dependent manner, indicating that these recombinant viruses were capable of replicating in Huh7.5.1 cells (Fig. 1b)
We found higher production of HCV core Ag in the culture medium of cells transfected with the chimeric virus with the Y93H mutation (JFH1/5ACon1-Y93H, -L31M/Y93H, -L31V/Y93H, and -L31I/Y93H) and lower production in the culture medium of cells transfected with chimeric viruses with L31V and L31I (Fig. 1b,c), suggesting that these mutations affected HCV production or propagation
Summary
NS5A inhibitors[13,16]. these polymorphisms have been reported to remain for a long duration (at least 1 year) after the cessation of DCV treatment[9,17,18]. We previously established the cell culture system with JFH1-based recombinant virus by replacement of NS5A with that from genotype 1b strain, Con[1] (JFH1/5ACon1)[21] This HCV cell culture system enabled to evaluate the effects of NS5A of genotype 1b on the HCV life cycle and the susceptibility to the NS5A inhibitor. We used a cell culture system with a JFH1-based recombinant virus generated by the replacement with the NS5A from the genotype 1b strain Con[1] containing resistance-associated NS5A mutations to assess their effects on the HCV life cycle and the susceptibilities of the viruses to various anti-HCV reagents[21]. We found that the Y93H mutation conferred enhanced infectious virus production but was related to the higher susceptibility to protease inhibitors, the susceptibilities to other antiviral reagents (IFN-α, -λ1, -λ3, and RBV) were not changed
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