Therapeutic targeting of measles virus polymerase with ERDRP-0519 suppresses all RNA synthesis activity
Fig 3
ERDRP-0519 potently inhibits de novo RNA synthesis.
A-F) Purified recombinant WT MeV L-P complexes or complexes harboring the LT776A or LH589Y resistance mutations were incubated with either a 16-nt RNA template (A) and the represented NTPs to assess de novo RNA synthesis or a 16-nt RNA template (B), a 5’-phosphorylated 4-nt primer, and the represented NTPs to assess primer extension. Representative autoradiograms are shown for de novo initiation (C) and primer extension (D). Purified L-P complexes with an LN774A substitution in the catalytic GDNQ motif [70] served as control for specificity of the de novo initiation assay. Primer extension assays were performed in the absence of primer, template, or enzyme to control for contaminating de novo RNA synthesis driven directly by the template. Densitometry analysis was performed on elongation products of 15 to 16-nt in length (E) or 7 to 9-nt in lengths (F) and represent n = 3–4 biological repeats. EC50 values represent 4-parameter variable slope regression models, 95% confidence intervals are shown. Positions of unspecific background signals are indicated with *. G-I) Effect of ERDRP-0519 incubation on MeV primary transcription, represented in (G). Cells were infected with recMeV-Anc (MOI = 3) and incubated with vehicle (0.1% DMSO) volume control, or 0.2 μM or 0.8 μM ERDRP-0519. Infected cells were harvested 4 hours after infection. Incubation with ERDRP-0519 significantly decreased relative P-encoding mRNA amounts 4 hours post infection (H), but did not alter steepness of the MeV primary mRNA transcription gradient (I). Symbols represent individual biological repeats (n = 3), graphs show sample means. Statistical analysis through two-way ANOVA with Dunnett’s multiple comparison post-hoc test, P values are specified.