Activation of p90 RSKs and the HIV-1 LTR is specific to KSHV ORF45. (A) The empty vector, KSHV FLAG-ORF45, EBV FLAG-ORF45, or MHV68 FLAG-ORF45 was transfected into HEK293T cells. At 24 h posttransfection, the medium was replaced with serum-free medium and the cells were maintained for an additional 24 h. Lysates of serum-starved cells were prepared and analyzed by Western blotting with RSK phosphorylation-specific antibodies to pT359/S363 or pS380, total RSK, and FLAG. (B) The empty vector, KSHV FLAG-ORF45, EBV FLAG-ORF45, or MHV68 FLAG-ORF45 was transfected into NH1 or NH2 cells, and luciferase levels were determined at 48 h posttransfection. Western blot analysis of β-tubulin served as a loading control. Statistical significance was determined by Student t test (***, P < 0.0001).