The expression level of P-TEFb affects the transcriptional regulation of the α-gene promoter by ICP22 in association with VP16.

<p>(A, B) Transcriptional activity of the CMV promoter with changing levels of P-TEFb expression. CHO-k1 or Hep-2 cells were co-transfected for 36 h with the pRL-CMV reporter plasmid and P-TEFb expression plasmids (pcDNA-Cdk9 and pcDNA-CyclinT1, pcDNA3 as a control) (A) or siRNA against P-TEFb (si-Cdk9 and si-CyclinT1, si-Negative RNA as a control) (B) as indicated, then the cell lysates were analyzed with the luciferase assay. (C, D) Transcriptional activity of the viral α4 gene promoter with changing levels of P-TEFb expression. CHO-k1 or Hep-2 cells were co-transfected for 36 h with pGL-α4 reporter plasmid and P-TEFb expression plasmids (pcDNA-Cdk9 and pcDNA-CyclinT1, pcDNA3 as a control) (C) or siRNA against P-TEFb (si-Cdk9 and si-CyclinT1, si-Negative RNA as a control) (D) as indicated, then the cell lysates were analyzed by the dual luciferase assay and normalized with the renilla luciferase expression. (E) P-TEFb overcomes transcriptional inhibition of the α4 promoter by ICP22 via increasing the expression level. CHO-K1 cells were co-transfected for 36 h with pGL-α4 reporter plasmid and ICP22, P-TEFb (Cdk9 and CyclinT1) or pcDNA3 control expression plasmids, as indicated. The cell lysates were then analyzed by the dual luciferase assay and normalized with the renilla luciferase expression. (F) The expression level of P-TEFb affects transcriptional activation of the α4 promoter by VP16. Hep-2 cells were co-transfected for 36 h with pGL-α4 reporter plasmid and VP16 expression plasmid, together with P-TEFb expression plasmid (pcDNA-Cdk9 and pcDNA-CyclinT1, pcDNA3 as a control) or siRNA against P-TEFb (si-Cdk9 and si-CyclinT1, si-Negative RNA as a control), as indicated. The cell lysates were then analyzed with the dual luciferase assay and normalized with the renilla luciferase expression. (G) Decreased expression of P-TEFb influences the ability of VP16 to release transcriptional suppression of the α4 promoter by ICP22. Hep-2 cells were co-transfected for 36 h with pGL-α4 reporter plasmid, expression plasmids (ICP22 and VP16, pcDNA3 as a control) and siRNA against P-TEFb (si-Cdk9 and si-CyclinT1, si-Negative RNA as a control), as indicated. The cell lysates were then analyzed with the dual luciferase assay and normalized with the renilla luciferase expression. Error bars represent the standard deviation from triplicate samples. * P<0.05 by student's t-test.