M117 activates E2F-dependent transcription.

(A) NIH-3T3 cells were first co-transfected with the reporter plasmid pGL2-Firefly-E2F or pGL2-Firefly-E2Fmut and the control plasmid pRL-Renilla. After 24 hours, cells were infected at an MOI of 3 TCID₅₀/cell with viruses expressing full-length (FL) or mutant M117. Cell lysates were harvested 24 hpi and luciferase activities were measured. Mean ±SEM of 3 independent experiments are shown. Firefly luciferase activity was normalized to Renilla luciferase activity and values relative to mock are shown. M117 mutant viruses were compared to M117-FL. (B) NIH-3T3 cells were synchronized by serum starvation for 24 hours, infected at an MOI of 3 TCID₅₀/cell (without serum) and harvested at 24 hpi for RNA extraction and qRT-PCR with primers specific for the indicated genes. Mean ±SEM of 4 independent experiments are shown. Transcripts were quantified using the ΔΔCt method and normalized to a housekeeping gene (Gapdh). Values relative to mock-infected cells are shown. M117 mutant viruses were compared to M117-FL. (C) Cells were infected as in (B) and protein lysates were harvested at the indicated times and subjected to Western Blot analysis. (D) Cell lysates of two E2F3-positive (g0 cl1 and cl2) and two E2F3-negative NIH-3T3 cells (g2 and g3) were harvested and subjected to Western Blot analysis. (E) E2F3-positive and negative cells were transfected, infected, and analyzed as in (A). Means ±SEM of 4 independent experiments are shown. ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001.