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Inosine Triphosphate Pyrophosphatase Dephosphorylates Ribavirin Triphosphate and Reduced Enzymatic Activity Potentiates Mutagenesis in Hepatitis C Virus.

ABSTRACT: A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene (ITPA) that lead to reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been put forward to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction between reduced ITPase activity and ribavirin remain unclear. The in vitro effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with a small interfering RNA (siRNA) directed against ITPA or a negative-control siRNA in the presence or absence of ribavirin in an HCV culture system. Low ribavirin concentrations strikingly depleted intracellular GTP levels in HCV-infected hepatocytes whereas higher ribavirin concentrations induced G-to-A and C-to-U single nucleotide substitutions in the HCV genome, with an ensuing reduction of HCV RNA expression and HCV core antigen production. Ribavirin triphosphate (RTP) was dephosphorylated in vitro by recombinant ITPase to a similar extent as ITP, a naturally occurring substrate of ITPase, and reducing ITPA expression in Huh 7.5 cells by siRNA increased intracellular levels of RTP in addition to increasing HCV mutagenesis and reducing progeny virus production. Our results extend the understanding of the biological impact of reduced ITPase activity, demonstrate that RTP is a substrate of ITPase, and may point to personalized ribavirin dosage according to ITPA genotype in addition to novel antiviral strategies.IMPORTANCE This study highlights the multiple modes of action of ribavirin, including depletion of intracellular GTP and increased hepatitis C virus mutagenesis. In cell culture, reduced ITP pyrophosphatase (ITPase) enzyme activity affected the intracellular concentrations of ribavirin triphosphate (RTP) and augmented the impact of ribavirin on the mutation rate and virus production. Additionally, our results imply that RTP, similar to ITP, a naturally occurring substrate of ITPase, is dephosphorylated in vitro by ITPase.

SUBMITTER: Nystrom K

PROVIDER: S-EPMC6146798 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Inosine Triphosphate Pyrophosphatase Dephosphorylates Ribavirin Triphosphate and Reduced Enzymatic Activity Potentiates Mutagenesis in Hepatitis C Virus.

Nyström Kristina K Wanrooij Paulina H PH Waldenström Jesper J Adamek Ludmila L Brunet Sofia S Said Joanna J Nilsson Staffan S Wind-Rotolo Megan M Hellstrand Kristoffer K Norder Helene H Tang Ka-Wei KW Lagging Martin M

Journal of virology 20180912 19

A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene (<i>ITPA</i>) that lead to reduced enzyme activity. Reduced ITPase activity was earlier reported to protect against ribavirin-induced hemolytic anemia and to diminish relapse following ribavirin and interferon therapy for hepatitis C virus (HCV) genotype 2 or 3 infections. While several hypotheses have been put forward to explain the antiviral actions of ribavirin, details regarding the mechanisms of interaction be ...[more]

PMID: 30045981

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Project description:AimTo investigate and clarify, for the first time, the role of inosine triphosphate pyrophosphatase (ITPA) polymorphism in Egyptian chronic hepatitis C virus (HCV) patients.MethodsThe human genomic DNA of all patients was extracted from peripheral blood cells in order to determine the single nucleotide polymorphism (SNP) of ITPA (rs1127354). SNP genotyping was performed by real time polymerase chain reaction (PCR, ABI TaqMan allelic discrimination kit) for 102 treatment-naive Egyptian patients with chronic HCV. All patients had no evidence of cardiovascular or renal diseases. They received a combination treatment of pegylated interferon α (PEG-IFNα) as a weekly subcutaneous dose plus an oral weight-adjusted dose of ribavirin (RBV). The majority received PEG-IFNα2a (70.6%) while 29.4% received PEG-IFNα2b. The planned duration of treatment was 24-48 wk according to the viral kinetics throughout the course of treatment. Pre-treatment liver biopsy was done for each patient for evaluation of fibrosis stage and liver disease activity. The basal viral load level was detected quantitatively by real time PCR while viral load throughout the treatment course was performed qualitatively by COBAS TaqMan assay.ResultsNinety-three patients (91.2%) had ITPA SNP CC genotype and 9 (8.8%) had non-CC genotype (CA and AA). The percentage of hemoglobin (Hb) decline was higher for CC patients than for non-CC patients, particularly at weeks 4 and 8 (P = 0.047 and 0.034, respectively). During the first 12 wk of treatment, CC patients had significantly more Hb decline > 3 g/dL than non-CC patients: 64.5% vs 22.2% at weeks 8 and 12, respectively, (P = 0.024 and 0.038). Reduction of the amount of the planned RBV dose was significantly higher for CC patients than non-CC patients during the first 12 wk (18% ± 12.1% vs 8.5% ± 10.2%, P = 0.021). The percentage of CC patients with RBV dose reduction was significantly greater than that of non-CC patients (77.4% vs 44.4%, P = 0.044). Multivariate analysis identified only the percentage of RBV dose as a predictor for Hb decline. Platelet decline was significantly higher in non-CC patients than CC patients at weeks 12, 24 and 48 (P = 0.018, 0.009 and 0.026, respectively).ConclusionRs1127354 ITPA polymorphism plays a decisive role in protecting against treatment-induced anemia and the need for RBV dose reduction in Egyptian HCV patients.

Dataset Information

Inosine Triphosphate Pyrophosphatase Dephosphorylates Ribavirin Triphosphate and Reduced Enzymatic Activity Potentiates Mutagenesis in Hepatitis C Virus.

Publications

Inosine Triphosphate Pyrophosphatase Dephosphorylates Ribavirin Triphosphate and Reduced Enzymatic Activity Potentiates Mutagenesis in Hepatitis C Virus.

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