Fidelity Variants of RNA Dependent RNA Polymerases Uncover an Indirect, Mutagenic Activity of Amiloride Compounds

Abstract

In a screen for RNA mutagen resistance, we isolated a high fidelity RNA dependent RNA polymerase (RdRp) variant of Coxsackie virus B3 (CVB3). Curiously, this variant A372V is also resistant to amiloride. We hypothesize that amiloride has a previously undescribed mutagenic activity. Indeed, amiloride compounds increase the mutation frequencies of CVB3 and poliovirus and high fidelity variants of both viruses are more resistant to this effect. We hypothesize that this mutagenic activity is mediated through alterations in intracellular ions such as Mg ²⁺ and Mn ²⁺, which in turn increase virus mutation frequency by affecting RdRp fidelity. Furthermore, we show that another amiloride-resistant RdRp variant, S299T, is completely resistant to this mutagenic activity and unaffected by changes in ion concentrations. We show that RdRp variants resist the mutagenic activity of amiloride via two different mechanisms: 1) increased fidelity that generates virus populations presenting lower basal mutation frequencies or 2) resisting changes in divalent cation concentrations that affect polymerase fidelity. Our results uncover a new antiviral approach based on mutagenesis.

Author Summary

RNA viruses have extreme mutation frequencies, due in large part to the erroneous nature of the viral RNA dependent RNA polymerases (RdRp) that replicate their genomes. Since RdRp lack proofreading and repair mechanisms, the use of base analogs as RNA mutagens to increase lethal mutations and extinguish the virus population is a promising antiviral strategy. Recently, a screen for resistance to this antiviral treatment identified a higher fidelity RdRp variant of poliovirus, indicating that RdRp fidelity can be modulated by single amino acid substitutions. To extend these observations to other viruses, we performed a similar screen using Coxsackie virus B3 (CVB3). We identified a new high fidelity RdRp variant which was also resistant to amiloride compounds that have no known mutagenic activity. Using wild type and RdRp fidelity variants of poliovirus and CVB3, we show that amiloride compounds do have mutagenic activity and act on RNA virus populations indirectly, by altering intracellular ion concentrations that affect polymerase fidelity. Our results identify a new means of targeting viruses through increases in mutation frequency using non-nucleoside compounds that alter the cellular environment in which the virus replicates.