Inosine triphosphate pyrophosphatase dephosphorylates ribavirin triphosphate and reduced enzymatic activity potentiates mutagenesis in hepatitis C virus
Journal article, 2018

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.

ITP pyrophosphatase

Mutagenesis

hepatitis C virus

Ribavirin

Mutagenesis

ITPase

ITPA

Inosine triphosphate pyrophosphatase

Author

Kristina Nyström

University of Gothenburg

Paulina Wanrooij

Umeå University

Jesper Waldenström

University of Gothenburg

Ludmila Adamek

University of Gothenburg

Sofia Brunet

University of Gothenburg

Joanna Said

University of Gothenburg

Staffan Nilsson

University of Gothenburg

Chalmers, Mathematical Sciences, Applied Mathematics and Statistics

Megan Wind-Rotolo

Bristol-Myers Squibb

Kristoffer Hellstrand

University of Gothenburg

Helene Norder

University of Gothenburg

Ka Wei Tang

University of Gothenburg

Wallenberg Lab.

Martin Lagging

University of Gothenburg

Journal of Virology

0022-538X (ISSN) 1098-5514 (eISSN)

Vol. 92 19 e01087

Subject Categories

Microbiology

Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)

Microbiology in the medical area

DOI

10.1128/JVI.01087-18

PubMed

30045981

More information

Latest update

12/10/2018