Fidelity Variants of RNA Dependent RNA Polymerases Uncover an Indirect, Mutagenic Activity of Amiloride Compounds
Figure 5
Another polymerase variant, S299T, is resistant to both the inhibitory effect of amiloride on RNA synthesis as well as its mutagenic activity.
(A) S299T resists amiloride's antiviral activity better than wild type virus. HeLa cells were treated with different concentrations of amiloride and infected with wild type or S299T virus at an MOI of 0.01. At 48 hours, viable progeny virus was quantified by TCID50 assay. The mean virus titers (TCID50/ml) and S.E.M are shown, N = 3, * P<0.05. (B) One-step growth kinetics of wild type and S299T viruses. HeLa cells were infected at MOI of 10 and the progeny virus was quantified at different hours after infection by TCID50 assay. Mean titers (TCID50/ml) ± S.E.M are shown, N = 3, no significant difference found. (C) The relative increase in signal from time 0 to indicated time points is shown, values are means of 2 separate northern blots of samples from (B), error bars show range of values (D) One-step growth kinetics of wild type and S299T viruses in the presence of 400 µM amiloride. HeLa cells were infected at MOI of 10 and the progeny virus was quantified at different hours after infection by TCID50 assay. Mean titers (TCID50/ml) ± S.E.M are shown, N = 3, no significant differences found. (E) Northern blot analysis of RNA synthesis determined 48 hours after infection with wild type or S299T (MOI = 0.01) virus in the presence or the absence of 400 µM amiloride. Two independent treatment samples per virus are shown. (F) Average mutation frequencies of S299T untreated population and treated with ribavirin, amiloride or EIPA. *** P<0.0001; ns = no statistically significant difference. NB: All data from Figure 5 was obtained simultaneously with that for Figure 3 and separated for clarity of presentation, hence, wild type and A372V data is the same for both figures, except for (C).