Ribavirin can be mutagenic for arenaviruses

Abstract

Arenaviruses include several important human pathogens, and there are very limited options of preventive or therapeutic interventions to combat these viruses. An off-label use of the purine nucleoside analogue ribavirin (1-β-d-ribofuranosyl-1-H-1,2,4-triazole-3-carboxamide) is the only antiviral treatment currently available for arenavirus infections. However, the ribavirin antiviral mechanism action against arenaviruses remains unknown. Here we document that ribavirin is mutagenic for the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) in cell culture. The mutagenic activity of ribavirin on LCMV was observed under single- and multiple-passage regimes and could not be accounted for by a decrease of the intracellular GTP pool promoted by ribavirin-mediated inhibition of inosine monophosphate dehydrogenase (IMPDH). Our findings suggest that the antiviral activity of ribavirin on arenaviruses might be exerted, at least partially, by lethal mutagenesis. Implications for antiarenavirus therapy are discussed.

Fig. 1.

Inhibition of LCMV replication by ribavirin. BHK-21 cells were infected with LCMV Armstrong (Arm) 53b at an MOI of either 0.01 PFU/cell or 10 PFU/cell. Viral titers were determined at 48 h postinfection (p.i.) in triplicate, and standard deviations (error bars) are given. The horizontal and vertical lines indicate the viral titer and ribavirin concentration that yield the IC₉₉ values (concentration of ribavirin that produces a 99% inhibition of LCMV infectious progeny production), given in the text as the average of triplicate determinations. The broken line indicates the limit of detection of LCMV infectivity. Note the different scale of the abscissa in the two plots. Procedures for LCMV infection in the presence or absence of ribavirin and for the determination of infectivity by plaque assays are detailed in Materials and Methods.

Fig. 2.

LCMV progeny production in the presence or absence of ribavirin (R) and guanosine (GU). BHK-21 cells were infected with LCMV Arm 53b at an MOI of 10 PFU/cell either in the absence of drugs or in the presence of 100 μM guanosine (+GU) or 20 μM or 100 μM ribavirin (+R20 and +R100, respectively) (−R, no ribavirin), as indicated for each column. LCMV infectivity (top panel) and number of LCMV RNA molecules measured by quantitative RT-PCR (bottom panel) were determined in the cell culture supernatant at 48 h p.i. Background values (PFU/ml and RNA/ml measured at time zero) have been subtracted from each titer and number of RNA molecules, respectively. The broken lines indicate the limit of detection for LCMV infectivity and the number of RNA molecules. Values are means of triplicate determinations, and standard deviations are given. Procedures for infections, titration by plaque assay and LCMV RNA quantification are described in Materials and Methods.

Fig. 3.

Inhibition of LCMV progeny production by ribavirin. BHK-21 cells were infected with LCMV Arm 53b at an MOI of either 0.01 PFU/cell (left panels) or 10 PFU/cell (right panels) in the presence of the indicated concentrations of ribavirin (R) (0 to 100 μM). The broken lines indicate the limit of detection of LCMV infectivity and number of RNA molecules. Viral titers obtained in the presence of 100 μM ribavirin at an MOI of 0.01 PFU/cell and at 48 h at an MOI of 10 PFU/cell were below the limit of detection (indicated by a short black bar next to the abscissa). Background values (PFU/ml and RNA/ml measured at time zero) have been subtracted from each titer and number of RNA molecules, respectively. (A to F) Virus titers (A and B) and number of LCMV RNA molecules measured by quantitative RT-PCR (C and D) in the supernatants of the infected cultures at 24 and 48 h postinfection were used to calculate the corresponding specific infectivities (E and F). The asterisks in parentheses indicate that a specific infectivity was calculated assuming 1 PFU/ml, since the virus titer was below the limit of detection. Procedures for infections, titration by plaque assay, and LCMV RNA quantification are described in Materials and Methods.

Fig. 4.

Infectious progeny production during serial passages of LCMV in the presence of ribavirin and guanosine. BHK-21 cells were infected with LCMV Arm 53b at an MOI of 0.01 PFU/cell either in the presence or absence of 10 μM ribavirin (R) or 100 μM guanosine (GU), or both, as indicated. The progeny virus was used to infect a fresh BHK-21 cell monolayer under the same conditions, and infection was repeated for a total of nine passages. Virus titers were determined in triplicate in the supernatant of the infected cultures at 48 h p.i. The broken line indicates the limit of detection of LCMV infectivity. Procedures for infections in the presence or absence of drugs and titration by plaque assay are described in Materials and Methods.