In the course of experiments aimed at deciphering the inhibition mechanism of mycophenolic acid and ribavirin in hepatitis C virus (HCV) infection, we observed an inhibitory effect of the nucleoside guanosine (Gua). Here, we report that Gua and not the other standard nucleosides inhibits HCV replication in human hepatoma cells. Gua did not directly inhibit the in vitro polymerase activity of NS5B, but it modified the intracellular levels of nucleoside di-and triphosphate (NDPs and NTPs), leading to deficient HCV RNA replication and reduction of infectious progeny virus production. Changes in the concentrations of NTP or NDP modified NS5B RNA polymerase activity in vitro, in particular de novo RNA synthesis and template switching. Furthermore, the Gua-mediated changes were associated with a significant increase in the number of indels in viral RNA, which may account for the reduction of the specific infectivity of the viral progeny, suggesting the presence of defective genomes. Thus, a proper NTP:NDP balance appears to be critical to ensure HCV polymerase fidelity and minimal production of defective genomes. 3 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 Author summaryRibonucleoside metabolism is essential for replication of RNA viruses. In this article we describe the antiviral activity of the natural ribonucleoside guanosine (Gua). We demonstrate that hepatitis C virus (HCV) replication is inhibited in the presence of increasing concentrations of this ribonucleoside and that this inhibition does not occur as a consequence of a direct inhibition of HCV polymerase. Cells exposed to increasing concentrations of Gua show imbalances in the intracellular concentrations of nucleoside-diphosphates and triphosphates and as the virus is passaged in these cells, it accumulates mutations that reduce its infectivity and decimate its normal spreading capacity.imbalance of the concentrations of NDP and NTP results in the inhibition of HCV polymerase activity in vitro, and iv) Gua treatment is associated with an increase of indel frequency in progeny HCV RNA. The results provide evidence of a metabolism-dependent mechanism of generation of defective HCV genomes.including viral entry, primary translation and genome replication, overall efficiency of which is proportional to reporter gene activity [12]. The results (Fig 2A) show that a selective HCV entry inhibitor, hydroxyzine, strongly interferes with reporter gene accumulation, as previously documented [13] (Figure 2A). Of the four natural nucleosides, only Gua exerted a significant inhibitory role, as shown by reduced luciferase levels in these cells (Figure 2A), and suggesting that an early step of the infection preceding viral assembly is significantly inhibited by Gua. To further dissect the impact of Gua on HCV replication, we analyzed the effect of Gua, Ade, Cyt and Uri treatment at different times in the replication of a dicistronic subgenomic genotype 2a (JFH-1) replicon bearing a luciferase reporter gene [12]. The objective was to analyze if the effect took place at t...