Inosine triphosphate pyrophosphatase dephosphorylates ribavirin triphosphate and reduced enzymatic activity potentiates mutagenesis in hepatitis C virus
Artikel i vetenskaplig tidskrift, 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.

Inosine triphosphate pyrophosphatase

ITPase

hepatitis C virus

ITPA

Mutagenesis

Mutagenesis

ITP pyrophosphatase

Ribavirin

Författare

Kristina Nyström

Göteborgs universitet

Paulina Wanrooij

Umeå universitet

Jesper Waldenström

Göteborgs universitet

Ludmila Adamek

Göteborgs universitet

Sofia Brunet

Göteborgs universitet

Joanna Said

Göteborgs universitet

Staffan Nilsson

Göteborgs universitet

Chalmers, Matematiska vetenskaper, Tillämpad matematik och statistik

Megan Wind-Rotolo

Bristol-Myers Squibb

Kristoffer Hellstrand

Göteborgs universitet

Helene Norder

Göteborgs universitet

Ka Wei Tang

Göteborgs universitet

Wallenberg Lab.

Martin Lagging

Göteborgs universitet

Journal of Virology

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

Vol. 92 19 e01087

Ämneskategorier

Mikrobiologi

Medicinsk bioteknologi (med inriktning mot cellbiologi (inklusive stamcellsbiologi), molekylärbiologi, mikrobiologi, biokemi eller biofarmaci)

Mikrobiologi inom det medicinska området

DOI

10.1128/JVI.01087-18

PubMed

30045981

Mer information

Senast uppdaterat

2018-10-18