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. 2021 Mar 18;65(4):e02605-20. doi: 10.1128/AAC.02605-20

FIG 6.

FIG 6

Enisamium is metabolized in humans and metabolite VR17-04 inhibits the viral RNA polymerase in vitro. (A) Effect of enisamium on the steady-state IAV vRNA, 5S rRNA, and GFP levels, with quantification shown in the graph. Levels of 5S rRNA and IAV vRNA were analyzed by primer extension (middle panel). PA and tubulin expression were analyzed by Western blotting (bottom panel). A mutant IAV RNA polymerase containing two amino acid substitutions in the PB1 active site (PB1a) was used as a negative control. Data points represent means and standard deviations of three independent enisamium titrations and matching GFP measurements or primer extensions. M, marker. (B) Effect of extracts from A549 cells treated with enisamium on IAV RNA polymerase activity in vitro. Five hundred or 2,500 μg of enisamium was added to A549 cells for 24 h. Next, cells were lysed and 1/10 of the lysate was added to in vitro polymerase assays. RNA polymerase products were analyzed by 20% denaturing PAGE. (C) Quantification of the activity of the IAV RNA polymerase in vitro in the presence of enisamium, VR17-04, or T-705 triphosphate (T-705-TP). Data points represent means and standard deviations of three independent titrations in RNA polymerase assays. (D) Phase I metabolites identified in human plasma samples. (E) Activity of the IAV RNA polymerase in the presence of different VR17-04 concentrations.