Activity of ribavirin against Hantaan virus correlates with production of ribavirin-5'-triphosphate, not with inhibition of IMP dehydrogenase

Abstract

Ribavirin (RBV) is a broad-spectrum antiviral agent that inhibits the production of infectious Hantaan virus (HTNV). Although the mechanism of action of RBV against HTNV is not understood, RBV is metabolized in human cells to both RBV-5'-monophosphate, which inhibits IMP dehydrogenase, resulting in a decrease in intracellular GTP levels, and RBV-5'-triphosphate (RBV-TP), which could selectively interact with the viral RNA polymerase. To elucidate which activity of RBV was most important to its anti-HTNV activity, the mechanism of action of RBV was studied in Vero E6 cells. Incubation with 10 to 40 mug/ml RBV resulted in a small decrease in GTP levels that was not dose dependent. Increasing the RBV concentration from 10 to 40 mug/ml resulted in a decrease in viral RNA (vRNA) levels and an increase in RBV-TP formation. Mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase, also resulted in a decrease in vRNA levels; however, treatment with MPA resulted in a much greater decrease in GTP levels than that seen with RBV. Treatment with both MPA and RBV resulted in increased reduction of vRNA levels but did not result in enhanced depression of GTP levels. Although guanosine prevented the depression in GTP levels caused by RBV, guanosine only partially prevented the effect of RBV on vRNA levels. These results suggest that the inhibition of IMP dehydrogenase by RBV is of secondary importance to the inhibition of vRNA replication by RBV and that the interaction of RBV-TP with the viral polymerase is the primary action of RBV.

FIG. 1.

Inhibition of vRNA replication by RBV and MPA. Vero E6 cells infected with HTNV were treated with various concentrations of RBV or MPA for 3 days, and the amounts of HTNV S-segment vRNA or PFU were determined as described in Materials and Methods. The results are presented as percentages of untreated sample. Each data point represents the mean ± standard deviation from three independent experiments.

FIG. 2.

Effect of RBV or MPA on GTP levels. Acid-soluble extracts of cell pellets from cell cultures treated with RBV and/or MPA were analyzed by SAX HPLC to determine intracellular GTP and ATP concentrations. (A) Vero E6 cells were treated with 10 μg/ml of RBV (41 μM), 2.5 μg/ml of mycophenolic acid (7.8 μM), or no drug for 0, 1, 2, 4, 24, 48, or 72 h. (B) Vero E6 cells were treated with RBV for 4 h or MPA for 24 h. (C) Vero E6 cells were treated with RBV, MPA, or RBV plus MPA for 24 h. Each data point represents the mean ± standard deviation from three measurements. These experiments were repeated twice with similar results.

FIG. 3.

Effects of guanosine on intracellular vRNA and GTP levels. Vero E6 cells were treated with 10 μg/ml guanosine (35 μM), 40 μg/ml RBV (164 μM), 40 μg/ml RBV plus 10 μg/ml guanosine, 1 μg/ml MPA (3.1 μM), or 1 μg/ml MPA plus 10 μg/ml guanosine. After 24 h of treatment, acid-soluble extracts of the cell pellets were analyzed by SAX HPLC to determine the amounts of GTP and ATP in each sample. The effect of each treatment on HTNV S-segment vRNA was determined after 3 days of treatment. Each number represents the mean plus standard deviation from three measurements.

FIG. 4.

Metabolism of RBV in Vero cells. Vero E6 cells were treated with 10 μg/ml [³H]RBV (41 μM) for 0, 1, 2, 4, 24, 48, or 72 h. Acid-soluble extracts of the cell pellets were analyzed by SAX HPLC, and the intracellular metabolites were determined. The experiment was repeated two times with similar results. The error bars represent standard deviations.