Fig. 4

Viral gene expression is responsive to the NAD⁺/NADH ratio. a) Uninfected cells were treated with 10 mM NAM for 24 h, and total cellular NAD⁺ and NADH concentrations determined as described previously. N = 3 for all cell lines. Fold-increase in treated over untreated is shown in bold in the gray bar (mean of the treated/mean of the untreated). Error bars show standard deviations of the NAD⁺/NADH ratios. P-values were determined using a student’s t-test. b) Impact of NAM on viral gene expression in persistently-infected cells. Persistently-infected BJAB and KE37 (> 50 dpi) were treated with 10 mM NAM for 24 h. Following RT-qPCR, fold-change in viral is calculated using 2^−∆∆Ct as described in the Material and Methods, with untreated sample as reference (set to 1) and cellular gene EIF1 as the housekeeping gene. Dashed gray line shows fold change = 1. Error bars show standard deviation in replicate wells. One representative experiment for each cell line is shown. This experiment was repeated three times with similar results using BJAB cells. This experiment was repeated four times for hexon in KE37 cells with similar results and twice for E1A and E3 with similar results