Abstract
BackgroundGiven the side effects associated with intravenous injections of interferon, an interferon-free regimen for the treatment of HCV infections is highly desirable. Recently published clinical studies show that interferon-free combination therapies containing ribavirin are efficacious, suggesting that an interferon-free therapy could be adopted in the near future. Therefore, understanding HCV resistance to ribavirin could be of major importance. In an approach to understand the effect of ribavirin on HCV replication and HCV resistance, we have selected a ribavirin resistant mutant of HCV in vitro.MethodsWe serially passed the J6/JFH1 strain of HCV in Huh7D cells (a Huh7 cell derivative more permissive to HCV replication) in the presence of different concentrations of ribavirin. Virus replication was assessed by detection of HCV antigens by immunfluorscence of infected cells and titration of recovered virus present in the supernatant. cDNAs from virus RNA grown in 0 or 250 uM concentrations of ribavirin were synthesized by RT-PCR, and sequenced.ResultsA concentration of 125 uM of ribavirin did not have a dramatic effect on HCV replication, while 500 uM of ribavirin lead to viral extinction. Concentrations of 250 uM of ribavirin dramatically reduced virus replication which was sustained over six passages. At passage seven viral resurgence began and over two passages the level of virus reached that of the wild type virus grown without ribavirin. Virus recovered from these cultures were more resistant to 250 uM ribavirin than wild type virus, and showed no difference in replication relative to wild type virus when grown in the absence of ribavirin. The ribavirin resistant virus accumulated multiple synonymous and non-synonymous mutations that are presently being analyzed for their relationship to ribavirin resistance.ConclusionsIt is possible to select a ribavirin resistant mutant of HCV that can replicate to levels similar to wild type virus grown without ribavirin. Analysis of the mutations responsible for the ribavirin resistance may aid in understanding the mechanism of action of ribavirin.
Highlights
Given the side effects associated with intravenous injections of interferon, an interferon-free regimen for the treatment of HCV infections is highly desirable
Understanding how ribavirin suppresses HCV replication and how HCV could escape the effect of ribavirin could be of major importance
Proposed mechanisms of action of ribavirin against HCV include a direct effect against the HCV RNA dependent RNA polymerase (NS5b); induction of misincorporation of nucleotides leading to lethal mutagenesis; depletion of intracellular guanosine triphosphate pools; alteration in the cytokine balance from a Th2 profile to a Th1 profile; and up-regulation of genes involved in interferon
Summary
Given the side effects associated with intravenous injections of interferon, an interferon-free regimen for the treatment of HCV infections is highly desirable. Understanding HCV resistance to ribavirin could be of major importance. In an approach to understand the effect of ribavirin on HCV replication and HCV resistance, we have selected a ribavirin resistant mutant of HCV in vitro. Understanding how ribavirin suppresses HCV replication and how HCV could escape the effect of ribavirin could be of major importance. Clinical studies showed that the efficacy of a interferon-free combination therapy involving a protease and a polymerase inhibitor can be strongly enhanced by adding ribavirin, suggesting a direct antiviral action of ribavirin in vivo [3,4]. We report the selection of a ribavirin resistant HCV mutant that can replicate in vitro to high levels in the presence of high concentrations of ribavirin. The ribavirin resistant virus accumulated multiple mutations distributed throughout the HCV genome that are presently being analyzed
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