Figure 6. The mutagenic activity of amiloride is an indirect, secondary antiviral effect that correlates with increases in intracellular divalent cation (Mg²⁺ or Mn²⁺) concentrations.

(A) Dose dependence of virus titer decrease and mutation frequency increase with ribavirin treatment. The reduction in wild type virus yield (solid lines, left y axis) is shown as a function of the percentage of viruses surviving treatment with different concentrations of ribavirin. The increase in mutation frequency (dashed lines, right y-axis) of the starting population at these same drug concentrations is shown. (B) Dose dependence of amiloride treatment, as in (A). (C) Inhibition of RNA synthesis by amiloride is dose dependent. Northern blot analysis of RNA synthesis of wild type virus treated with different concentrations of amiloride. Cells were infected at MOI of 0.01 and RNA was extracted 48 hours after infection. (D–F) Treatment with Mg²⁺ and Mn²⁺, but not Na⁺ or Ca²⁺, increase the mutation frequency of wild type (D) and A372V (E), but not S299T (F). Cells were infected at MOI of 0.01 with virus in media supplemented with the indicated concentrations of salts and mutation frequencies were determined in progeny populations 48 hours after infection. * P<0.05, ** P<0.01, *** P<0.0001; ns = no statistically significant difference. (G) Passage of wild type virus in high concentrations of Mg²⁺ and Mn²⁺ selects for high fidelity A372V. Virus was passaged 20 times in 5 mM MgCl₂, 1 mM MnCl₂ or regular media. At the indicated passage numbers the virus population was sequenced. The emergence of a single point mutation resulting in a A372V change is indicated by a solid circle.