Figure 1.

Transactivation of the MYC promoter through the PRF element by KSHV vIRF. (A) Western blot for cMYC (Upper) and vIRF protein (Lower) in NIH 3T3 cells expressing vIRF (clones C2 and C7) or empty vector (clone C0). Cells were harvested during exponential growth to prevent contact inhibition effects on cMYC expression, and equal amounts (100 μg) of total protein were loaded in each lane. (B) Dominant-negative cMYC (DNMYC) inhibits soft agar colony formation of vIRF-expressing NIH 3T3 clones after 18 days in culture. C7 cells expressing vIRF or C0 cells containing empty vector were plated on soft agar in the presence (100 nM) or absence of 4HT. C0 cells (1 and 2) do not form colonies in soft agar, whereas large C7 soft-agar colonies are present in the absence of 4HT (3). 4HT treatment of C7 cells markedly reduces the number and size of C7 cell colonies. (C) Luciferase promoter-reporter assays showing that increasing amounts of pvIRF expression plasmid transactivates pBB-Luc but not pΔPRFBB-Luc, in which the PRF ISRE sequence is deleted. This effect occurs in murine pre-B 18-81 cells and in IRF1/2−/− MEF (2 μg of reporter plasmid used for all conditions). The reverse expression construct, pvFRI (5 μg), is inactive, whereas YY1 (2 μg) activates both pBB-Luc and the pΔPRFBB-Luc reporters. vIRF does not transactivate the irrelevant pGL3-control-Luc plasmid. (D) vIRF transactivation of MYC is resistant to cycloheximide. Northern blot for luciferase expression from the pBB-Luc plasmid in IRF1/2−/− cells 6 hr after 100 nM 4HT treatment, with cycloheximide pretreatment (100 μg/ml) 1.5 hr before 4HT treatment. Lanes 1, 3, and 5 have empty vector, and lanes 2, 4, and 6 have an expression plasmid encoding a 4HT-responsive vIRF-ER fusion protein. (E) DN-IRF expression plasmid does not activate pBB-Luc but instead inhibits vIRF MYC transactivation in IRF1/2−/− cells, consistent with model 1.