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ENT1, a ribavirin transporter, plays a pivotal role in antiviral efficacy of ribavirin in a hepatitis C virus replication cell system.

ABSTRACT: We previously showed that equilibrative nucleoside transporter 1 (ENT1) is a primary ribavirin transporter in human hepatocytes. However, because the role of this transporter in the antiviral mechanism of the drug remains unclear, the present study aimed to elucidate the role of ENT1 in ribavirin antiviral action. OR6 cells, a hepatitis C virus (HCV) replication system, were used to evaluate both ribavirin uptake and efficacy. The ribavirin transporter in OR6 cells was identified by mRNA expression analyses and transport assays. Nitrobenzylmercaptopurine riboside (NBMPR) and micro-RNA targeted to ENT1 mRNA (miR-ENT1) were used to reduce the ribavirin uptake level in OR6 cells. Our results showed that ribavirin antiviral activity was associated with its accumulation in OR6 cells, which was also closely associated with the uptake of the drug. It was found that the primary ribavirin transporter in OR6 cells was ENT1 and that inhibition of ENT1-mediated ribavirin uptake by NBMPR significantly attenuated the antiviral activity of the drug as well as its accumulation in OR6 cells. The results also showed that even a small reduction in the ENT1-mediated ribavirin uptake, achieved in this case using miR-ENT1, caused a significant decrease in its antiviral activity, thus indicating that the ENT1-mediated ribavirin uptake level determined its antiviral activity level in OR6 cells. In conclusion, our results show that by facilitating its uptake and accumulation in OR6 cells, ENT1 plays a pivotal role in the antiviral effectiveness of ribavirin and therefore provides an important insight into the efficacy of the drug in anti-HCV therapy.

SUBMITTER: Iikura M

PROVIDER: S-EPMC3294941 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundWhile ribavirin mono-therapy regimens have minimal effect on patients with chronic hepatitis C virus (HCV) infections, they can be efficacious when combined with interferon. Clinical studies show that interferon-free combination therapies containing ribavirin are also efficacious, suggesting that an interferon-free therapy could be adopted in the near future. However, generation of drug resistant mutants and cross resistance to other drugs could impair the efficacy of the treatment. Therefore, understanding the mechanism of HCV resistance to ribavirin and cross resistance to other antiviral drugs could be of major importance.MethodsWe tested the ability of a J6/JFH1 derived HCV ribavirin resistant mutant to grow in tissue cultured Huh7D cells in the presence of the mutagen 5-Fluorouracil and the nucleoside analog 2'-C-Methylcytidine. Virus replication was assessed by detecting HCV antigens by immunofluorescence and by titrating virus present in the supernatants. Recovered viruses were amplified by RT-PCR and sequenced.ResultsThe sensitivity of HCV-RR relative to parental J6/JFH1 to the tested drugs varied. HCV-RR was more resistant than J6/JFH1 to 5-Fluorouracil but was not more resistant than J6/JFH1 to 2'-C-Methylcytidine. Growth of HCV-RR in 5-Fluorouracil allowed the selection of an HCV-RR derived mutant resistant to 5-Fluorouracil (HCV-5FU). HCV-5FU grows to moderate levels in the presence of high concentrations of 5-Fluorouracil and to parental levels in the absence of the drug. Sequence of its genome shows that HCV-5FU accumulated multiple synonymous and non-synonymous mutations.ConclusionsThese results indicate that determinants of resistance to ribavirin could also confer resistance to other anti-HCV drugs, shedding light toward understanding the mechanism of action of ribavirin and highlighting the importance of combination drug selection for HCV treatment. The results also show that it is possible to select a 5-Fluorouracil HCV resistant mutant that replicates to levels similar to parental virus when grown in the absence of 5-Fluorouracil.

Project description:Time series of gene expression arrays before and during treatment of Hepatitis C; days 0, 1, 2, 7, 14 and 28 for 69 participants (IDs 1 through 69 are used). Treating chronic hepatitis C using peginterferon alpha and ribavirin leads to sustained clearance of virus and clinical improvement in approximately 50% of patients. Response rates are lower among patients with genotype 1 than with genotypes 2 and 3 and among African American ( AA) compared to Caucasian (CA) patients. Using DNA microarrays, gene expression was assessed in a group of 33 African American and 36 Caucasian American patients with chronic hepatitis C, genotype 1 during the first 28 days of treatment. Results were examined with respect to treatment responses and to race. Patients showed a response to treatment at the gene expression level in RNA isolated from PBMC irrespective of degree of decrease in hepatitis C virus (HCV) RNA levels. However, gene expression responses were relatively blunted in patients with poor viral response (< 1.5 log10 IU/ml decrease at 28 days) compared to those in patients with a marked (> 3.5 log10 decrease) or intermediate (1.5 to 3.5 log10 decrease) response. The number of genes that were up- or down-regulated by peginterferon and ribavirin treatment was fewer in patients with a poor response compared to those with an intermediate or marked viral response. However AA patients had a stronger interferon response than CA patients in general. The induced levels of known ISGs such as OAS, MX1, IRF-7 and the toll like receptor TLR-7 was lower in poor response patients than in marked or intermediate response patients. Thus, the relative lack of viral response to interferon therapy of hepatitis C is associated with blunted interferon cell signaling. No specific regulatory gene could be identified as responsible for this global blunting or racial differences. Experiment Overall Design: Eligible patients were naÃ¯ve to interferon and ribavirin treatment, had detectable HCV RNA in serum, and nearly all had a liver biopsy performed within the previous 18 months showing chronic hepatitis. Only patients who were born in the United States and designated themselves as âAfrican American/Blackâ or âCaucasian/Whiteâ were eligible. The clinical protocol called for participants to be treated for up to 48 weeks with peginterferon alpha-2a (Pegasysâ¢, Roche Pharmaceuticals Nutley, NJ) in a dose of 180 Âµg weekly by self-administered subcutaneous injection and ribavirin (Copegusâ¢, Roche) orally in a dose of 1000 or 1200 mg daily based on body weight of less than 75 kg or equal to or greater than 75 kg. Peripheral blood mononuclear cells (PBMC) were collected from patients before treatment (day 0) and on days 1 (after the initial supervised injection of peginterferon), 2, 7, 14 and 28. From the Virahep-C cohort, 72 patients who did not have dose reductions of either peginterferon or ribavirin in the first 28 days of treatment were selected such that 12 patients were included in each race (CA, AA) by virological response category. The three categories of response were: marked, defined as a decrease in HCV RNA levels of more than 3.5 log10 IU/ml or to undetectable on day 28; intermediate, defined as a decrease of 1.4 to 3.5 log10 IU/ml on day 28; and poor, defined as less than 1.4 log10 IU/ml decline on day 28 relative to baseline. These definitions were made a priori in an attempt to analyze the biological basis for virological responses. Of these, adequate RNA was not obtained from 3 patients to provide gene expression information. Of these 72, 69 were analyzed.

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ENT1, a ribavirin transporter, plays a pivotal role in antiviral efficacy of ribavirin in a hepatitis C virus replication cell system.

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