FIG. 3.

Identification of mutations causal for resistance. (A) Sequence analysis of the noncoding genomic termini (LS and TS), intergenic regions, and viral N, P/C/V, and L proteins of the 13 resistant MeV clones in comparison with those of the respective sensitive input strains. Envelope components located between the P and L open reading frames were not examined. Coding changes in viral structural proteins are marked in black, silent mutations are shown in gray, and changes in the nonstructural C protein are displayed in parentheses. Three mutation cluster zones were identified (changes are shown in bold). Noncoding regions were unchanged in all cases. Shaded areas for MeV-Edm variants were not sequenced. (B) With the exception of Edm-1*, a transient MeV replicon reporter assay identified for each adapted clone a single mutation in L that reconstitutes complete resistance. Selected candidate mutations were rebuilt individually in an MeV-Edm L expression plasmid, and RdRp-driven reporter activity was assessed upon transfection of replicon-encoding plasmids into BHK-T cells and incubation in the absence or presence of 30 μM AS-136A. Values are normalized for those obtained with the unmodified replicon system in the absence of AS-136A and represent averages from at least three independent experiments ± standard errors. Results are based on a CAT (constructs marked “1”) or an identical firefly luciferase (constructs marked “2”) reporter. (C) Dose-response curves demonstrate higher base RdRp activity in the replicon assay when the L(R1233Q) mutation (found in Edm-1*) is present, resulting in intermediate-level resistance. The luciferase-based MeV reporter was used for all experiments; values were normalized as described for panel B and represent averages from seven independent experiments ± standard errors of the means. Asterisks represent P values derived from t test analysis (***, P < 0.001; **, P < 0.01; *, P < 0.05).