Figure 3.

CVB3 2A^S35G confers resistance to DENSpm. (A) Vero cells were left untreated and were infected with CVB3 and 2A mutant protease at an MOI of 0.1. Samples were collected every 24 h and titered via plaque assay. (B) Vero cells were treated with increasing doses of DENSpm, from 10 μM to 100 μM, for 16 h prior to infection with wildtype CVB3 or 2A protease mutant. Viral titers were determined via plaque assay at 48 hpi. Viral titers were used to calculate the percent replication in DENSpm, by dividing the titer of the virus after infection of DENSpm-treated cells by the titer of the virus after infection of untreated cells at 48 hpi. (C) Thin layer chromatograms resolving the polyamines putrescine (Put), spermidine (Spm), and spermine (Spm) after treatment with DENSpm. Arrows indicate the band corresponding to each polyamine. (D) Vero cells were treated with increasing doses of DFMO, from 100 μM to 1 mM, for 4 days prior to infection with wildtype CVB3 or 2A protease mutant. Viral titers were determined via plaque assay at 48 hpi. Viral titers were used to calculate the percent replication in DFMO, by dividing the titer of the virus after infection of DFMO-treated cells by the titer of the virus after infection of untreated cells at 48 hpi. (E) Thin layer chromatograms resolving the polyamines putrescine (Put), spermidine (Spm), and spermine (Spm) after treatment with DFMO. (F) Huh7 cells and (G) HeLa cells were treated with increasing doses of DENSpm and infected with WT or 2A protease mutant as in (B). * p ≤ 0.05, ** p ≤ 0.01 using Student’s t-test (n ≥ 2), comparing treated samples to untreated controls. Error bars represent ± 1 SEM.