Effect of ATA on viral replication. Huh7 (A), HeLa (B), and AD293 (C) cells were infected with vaccinia virus (WR) at an MOI of 5. AD293 cells (D) were infected with Ad-enhanced green fluorescent protein at an MOI of 0.1. Cells were treated with the indicated concentrations of ATA. Plaque assays for vaccinia virus were performed in triplicate on BSC-1 cells 24 hpi. Plaque assays for Ad were performed in triplicate on AD293 cells and were counted by immunostaining 24 hpi. (E) The CC₅₀ values in Huh7 (▪), HeLa (⧫), and AD293 (▴) cells were determined. Cells were treated for 24 h with the indicated concentrations of ATA (μg/ml) and then washed three times with PBS, followed by the addition of WST-1 reagent, and the absorbance was measured at 450 nm. All experiments were performed in triplicate. Error bars represent the standard errors of the means.

ATA inhibits the early phase of replication. (A) HeLa cells were infected with vaccinia virus (Cop) at an MOI of 5 and treated with ATA (400 μg/ml) at different times postinfection. Bar 1, cell control (no ATA); bar 2, 0 hpi; bar 3, 1 hpi; bar 4, 3 hpi; bar 5, 6 hpi. Infected cells were also treated with ATA for 1 h (bar 6), 3 h (bar 7), and 6 h (bar 8), after which cells were washed three times with PBS and fresh growth medium was added. In each experiment, the virus yield was determined 24 hpi by using plaque assays on BSC-1 cells. All experiments were performed in triplicate. Error bars represent the standard errors of the means. (B) HeLa cells were treated with ATA (400 μg/ml) and collected at 0 h (control) (no ATA) (lane 1), 10 min (lane 2), 30 min (lane 3), 1 h (lane 4), 3 h (lane 5), 6 h (lane 6), 8 h (lane 7), 8 h (control) (no ATA) (lane 8), and 24 h (control) (no ATA) (lane 9). The levels of phospho-ERK, total ERK, and β-actin were determined. Cells were also treated with ATA for 1 h (lane 10), 3 h (lane 11), and 6 h (12 h), after which cells were washed three times with PBS and fresh growth medium was added. The level of phospho-ERK, total ERK, and β-actin were determined 24 h posttreatment.

ATA inhibits vaccinia virus gene transcription. HeLa cells were infected with vaccinia virus (WR) at an MOI of 5 and treated with (+) or without (−) 400 μg/ml of ATA. RNA was extracted from cells collected at the indicated times for RT-PCR. The lanes at 0 hpi represent uninfected cell controls.

ATA inhibits H1L tyrosine phosphatase activity. Reactions were performed at a substrate concentration equal to the K_m value (1 mM), at 37°C, with 2.5 μg of H1L in the presence of serial dilutions of ATA (0 to 53 μg/ml). Reactions were performed in triplicate. Error bars represent the standard errors of the means.