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.
Highlights
A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene (ITPA) that lead to reduced enzyme activity
We introduced ITPA small interfering RNA (siRNA) in hepatitis C virus (HCV)-infected Huh7.5 cells and achieved ITPA expression levels (20%) comparable to the level of ITPase activity reported in patients carrying genetic variants of ITPA encoding ITPase deficiency, i.e., activity of Ͻ5 to 60% of normal, wild-type ITPase activity
It was observed that the silencing of ITPA (i) promoted ribavirin-induced reduction in HCV RNA and HCV core antigen, (ii) potentiated the mutagenesis noted in the presence of higher concentrations of ribavirin, and (iii) increased the intracellular levels of ribavirin triphosphate (RTP)
Summary
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. Two variants of the ITP pyrophosphatase (ITPase) gene (ITPA), a missense variant in exon 2 (rs1127354, P32T) and a splice-altering single nucleotide polymorphism (SNP) in intron 2 (rs7270101, IVS2), have been demonstrated in a genome-wide association study (GWAS) to protect against ribavirin-induced hemolytic anemia during therapy with peginterferon in combination with ribavirin for hepatitis C virus (HCV) genotype 1 infection [1]. In patients with chronic HCV genotype 2 or 3 infection receiving peginterferon and ribavirin, ITPA variants encoding reduced ITPase activity were found in approximately one-third of patients and were associated with reduced relapse risk following antiviral therapy despite lower plasma ribavirin concentrations [2]. In the setting of decompensated cirrhosis (Child-Pugh B or C), ribavirin likely will remain a component of HCV therapy as protease inhibitors should not be used in these patients [24]
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