Abstract
Treatment for hepatitis C virus (HCV) has improved greatly through the use of direct-acting antivirals (DAAs). However, their effectiveness and potential for drug resistance development in non-genotype 1 variants of HCV remain relatively unexplored, as in vitro assays to assess drug susceptibility are poorly developed and unsuited for a transient-transfection format. In the current study, we have evaluated the effects of dinucleotide frequency changes in the replicon and the use of a SEC14L2-expressing cell line on the replication of HCVs of different genotypes and evaluated the resulting assay formats for measurements of susceptibility to the DAA sofosbuvir. Removal of CpG and UpA dinucleotides from the luciferase gene used in HCV replicons of genotype 1b (Con1) and genotype 2a (JFH-1) achieved between 10- and 100-fold enhancement of replication over that of the wild type posttransfection. Removal of CpG and UpA dinucleotides in the neomycin gene or deletion of the whole gene in replicons of genotype 3a (S52) and genotype 4a (ED43) enhanced replication, but phenotypic effects on altering luciferase gene composition were minimal. A further 10-fold replication enhancement of replicons from all four genotypes was achieved by using a transgenic Huh7.5 cell line expressing SECL14L2, whose expression showed a dose-dependent effect on HCV replication that was reversible by small interfering RNA (siRNA) knockdown of gene expression. By combining these strategies, the 100- to 1,000-fold enhancement of replication allowed the susceptibility of all four genotypes to the RNA polymerase inhibitor sofosbuvir to be robustly determined in a transient-transfection assay format. These methods of replication enhancement provide new tools for monitoring the susceptibility and resistance of a wide range of HCV genotypes to DAAs.
Highlights
Hepatitis C virus (HCV) is a positive-sense RNA virus of the Flaviviridae family first described in 1989(1) and subsequently identified as the principal cause of non-A, non-B hepatitis in blood recipients and haemophiliacs, as well as widely infecting injecting drug users [2,3]
The modified luciferase sequence was mutated to remove all 100 of the CpG dinucleotides present in the native sequence and 64 of the 86 UpA dinucleotides, the maximum possible while retaining identical amino acid coding to the WT sequence (Table 1; Fig. 1A)
Despite only reproducing the intracellular replication steps of the HCV lifecycle, replicons are a valuable tool in HCV research, especially in drug discovery programs and have been instrumental in the discovery of the first direct-acting antivirals (DAAs) [30], including Sofosbuvir [31], Simeprevir
Summary
Hepatitis C virus (HCV) is a positive-sense RNA virus of the Flaviviridae family first described in 1989(1) and subsequently identified as the principal cause of non-A, non-B hepatitis in blood recipients and haemophiliacs, as well as widely infecting injecting drug users [2,3]. The phenotypic diversity of HCV genotypes, as manifested by these major differences in treatment response requires the effectiveness of and resistance development to novel antiviral treatments to be evaluated separately for different HCV genotypes and subtypes Such testing is generally performed using subgenomic replicons in which the structural genes are replaced by a luciferase or other reporter gene to allow replication to be rapidly quantified [11,12]. In the genotype 1b Con 1 replicon, adaptive mutations occur in the NS3, NS4B and NS5A genes [12,14,15] that may enhance HCV protein-protein interactions and viral morphogenesis, the mechanisms remains poorly understood [16] Such mutants, show increases in viral RNA in transient replication assays without antibiotic selection. While transient replication assays are clearly preferable for such testing, many of the currently available replicons do not have the required level of replication needed in this assay format to accurately estimate changes in replication levels in drugs inhibition studies
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
