FIG 6.

Mutations in viral protein 2C confer resistance to dibucaine, pirlindole, and zuclopenthixol. (A) BGM cells were transfected with in vitro-transcribed full-length genomic RNA of wt CV-B3 or the 2C(A224V,I227V,A229V) (AVIVAV) or 3A(H57Y) mutant virus and treated with 5 μM dibucaine, 10 μM pirlindole, 10 μM fluoxetine, 2 mM GuHCl, 1 μM the PI4KIIIβ inhibitor BF738735, 3 μM zuclopenthixol, or 10 μM formoterol or mock treated. Cells were lysed at 8 h p.i., and the virus titer was determined by endpoint titration. Titers are displayed as log CCID₅₀ per milliliter, and means and standard deviations were calculated from three replicates. *, below the detection level. (B) Cells were transfected with in vitro-transcribed wt or 2C(A224V,I227V,A229V) replicon RNA and treated with the indicated amounts of dibucaine or mock treated. Firefly luciferase activity was determined at 7 h posttransfection. (C and D) Binding of dibucaine to wt (C) and mutant (D) 2C Del36 proteins was analyzed by ITC. Representative examples of the raw data are depicted at the top, and the integrated data are depicted at the bottom. Data are shown fitted to a one-site binding model.