FIG 5.

RSK activation is necessary but not sufficient to transcriptionally activate the HIV-1 LTR. (A) Cell lysates from HEK293T cells transfected with the plasmids indicated were analyzed by Western blotting with RSK phosphorylation-specific antibodies to pT359/S363, pS380, total RSK, and FLAG. (B) NH1 and NH2 cells were transfected with the plasmids indicated. Luciferase levels were determined at 48 h posttransfection. (C) Whole-cell extracts from HEK293T cells transfected with either WT or F66A mutant ORF45 were subjected to either anti-FLAG or anti-HA immunoprecipitation. Inputs and immunoprecipitated material were analyzed by Western blotting with antibodies against FLAG, RSK, and histone H3. (D) Lysates of HEK293T cells transfected with the plasmids indicated were analyzed by Western blotting with phosphorylation-specific antibodies to pT359/S363 or pS380, total RSK, and FLAG. (E) NH1 and NH2 cells were transfected with either WT or F66A mutant ORF45. At 48 h posttransfection, luciferase levels were determined. (F) NH1 and NH2 cells were transfected with either WT or F66A mutant ORF45. At 48 h posttransfection, RNAPII ChIP analysis was performed. Immunoprecipitated DNA was detected by qPCR with primers located within the HIV-1 LTR or luciferase gene body (ORF). (G) Jurkat 1G5 cells were transfected with the plasmids indicated, and luciferase levels were determined at 24 h posttransfection. Western blot analysis of β-tubulin served as a loading control. (H) RT-qPCR analysis of RNA extracted from panel G. Statistical significance was determined by Student t test (**, P < 0.001; ***, P < 0.0001).