Fig 2.

Fluoxetine inhibits CVB3 by acting on viral protein 2C. (A) Structural formulae of fluoxetine and TBZE-029. (B) Schematic of the organization of the genome of CVB3 (top) and of protein 2C (bottom). The locations of conserved ATPase motifs A, B, and C; active-site residue K135; and the residues substituted in the TBZE-029-resistant virus (A224V, I227V, and A229V) are indicated. Amino acids are numbered according to their positions in protein 2C. (C) BGM cells were transfected with in vitro-transcribed full-length genomic RNA of WT CVB3 or the 2C_A224V,I227V,A229V or 3A_H57Y mutant (mut) virus and treated with 3 μM fluoxetine or 0.1% DMSO. At 8 h p.i., cells were lysed 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. (D) Analysis of the effect of fluoxetine on the ATPase activity of protein 2C with Pi ColorLock Gold (Innova Biosiences) in a reaction mixture containing 3 μM recombinant protein, 1 mM ATP, 10 mM HEPES (pH 7.5), 150 mM NaCl, and 10 mM MgCl₂. Where indicated, 25 μM fluoxetine was added. Phosphate release was measured through the determination of absorption at 635 nm at the indicated time points. The background of spontaneous phosphate release was subtracted. (E) Assay performed essentially as described for panel D, except that the concentration of fluoxetine was varied as indicated. Absorption was measured after 30 min.