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

• Published in: Journal of virology Vol. 92; no. 19
• Main Authors: Nyström, Kristina, Wanrooij, Paulina H, Waldenström, Jesper, Adamek, Ludmila, Brunet, Sofia, Said, Joanna, Nilsson, Staffan, Wind-Rotolo, Megan, Hellstrand, Kristoffer, Norder, Helene, Tang, Ka-Wei, Lagging, Martin
• Format: Journal Article
• Language: English
• Published: United States American Society for Microbiology 01.10.2018
• Subjects: antiviral activity; Antiviral Agents - metabolism; Antiviral Agents - pharmacology; Cell Line, Tumor; Dose-Response Relationship, Drug; Gene Expression Regulation; gene-expression; Guanosine Triphosphate - metabolism; Hepacivirus - drug effects; Hepacivirus - genetics; Hepacivirus - growth & development; Hepacivirus - metabolism; Hepatitis B Core Antigens - genetics; Hepatitis B Core Antigens - metabolism; hepatitis C virus; Hepatocytes - drug effects; Hepatocytes - enzymology; Hepatocytes - virology; High-Throughput Nucleotide Sequencing; Host-Pathogen Interactions; Humans; infection; inosine triphosphate; Inosine triphosphate pyrophosphatase; interferon; ITP pyrophosphatase; ITPA; ITPase; Microbiology in the medical area; Mikrobiologi inom det medicinska området; Mutagenesis; mycophenolic-acid; Nucleotides - metabolism; population; Pyrophosphatases - antagonists & inhibitors; Pyrophosphatases - genetics; Pyrophosphatases - metabolism; pyrophosphohydrolase deficiency; real-time pcr; Ribavirin; Ribavirin - metabolism; Ribavirin - pharmacology; RNA, Small Interfering - genetics; RNA, Small Interfering - metabolism; RNA, Viral - genetics; RNA, Viral - metabolism; Signal Transduction; therapy; treatment response; Virus-Cell Interactions; ribavirin; ITP pyrophosphatase; inosine triphosphate pyrophosphatase; hepatitis C virus; ITPase; mutagenesis; ITPA
• ISSN: 0022-538X; 1098-5514; 1098-5514
• DOI: 10.1128/jvi.01087-18

A third of humans carry genetic variants of the ITP pyrophosphatase (ITPase) gene ( ) 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 effect of reduced ITPase activity was assessed by means of transfection of hepatocytes (Huh7.5 cells) with a small interfering RNA (siRNA) directed against 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 by recombinant ITPase to a similar extent as ITP, a naturally occurring substrate of ITPase, and reducing 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 genotype in addition to novel antiviral strategies. 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 by ITPase.