Helene Mens , Andrea Galli  [§], Judith Gottwein , Jan Gerstoft  and Jens Bukh 
Affiliates:  Copenhagen Hepatitis C Program (CO-HEP), Department of Infectious Diseases, Hvidovre Hospital, and Department of Immunology and Microbiology, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark.  Department of Infectious Diseases, Copenhagen University Hospital, Rigshospitalet, Copenhagen, Denmark. § H.M. and A.G. contributed equally to this work.
Despite having been a cornerstone in the treatment of HCV for decades, the precise mechanism of action of ribavirin (RBV) is unknown. In other viruses, RBV has been shown to work by increasing the rate of G-to-A and C-to-T transitions. To investigate if this also holds true for HCV, we used the well-established J6/JFH1 cell-culture system to evaluate the mutagenic effect of RBV on HCV. Infected cells were treated with increasing RBV concentrations (20-150 μM). A 1245 bp piece of the NS5B was amplified, cloned and sequenced to estimate genetic distances. On average 58 sequences (range 16-81) per time point were analyzed. In addition, experiments with phosphorylated forms of ribavirin (ribavirin mono- and tri-phosphate) and a Huh7.5 cell line, stably overexpressing adenosine kinase (ADK) were carried out. We show that RBV can reduce spread and infectivity of HCV in a concentration-dependent manner, but decrease HCV RNA titers by only 1-2 logs, despite full inhibition of spread. Treatment with RBV resulted in increasing virus diversity and a right shift in hamming distances. The increase in complexity was shown to be driven by a significant increase in the frequency G-to-A transitions compared to the NTC (mean 0.09 mut per sequence (NTC) vs. 0.28 (RBV100 μM, p=0.01) and 0.69 (RBV150 μM, p<0.0001). Mutations were evenly distributed throughout the NS5B and most mutations were unique. Similar results were obtained by experiments using phosphorylated forms of RBV and by experiments using a huh7.5 cell line which stably overexpressed ADK. This study thus confirms ribavirin acting through the induction of specific transitions.