FIG. 4.

Intron sequences-splicing and 5′UTR are not required for p28^II-mediated inhibition of Tax activity. (A) A total of 0.4 μg of wtHTLV-2 and IY587 plasmids or 0.1 μg of the indicated Tax-2 cDNA plasmids was transfected into 293T cells, along with the LTR-luciferase reporter in the absence or presence of a 4× molar ratio of p28^II expression plasmid. After 48 h of culture, cells were lysed, and luciferase activity was measured. Experiments were done in triplicate, and error bars reflect standard deviations. CMV-βgal was used to adjust for transfection efficiency. Negative control (NC), cells that are transfected with empty vector. p28^II inhibited Tax-2 activity that was expressed from both wtHTLV-2 and IY587, indicating that truncating intron-1 does not influence p28^II-mediated inhibition or Tax-2 expression. All cDNA expression plasmids showed minimal or no inhibition by p28^II. (B) The same lysates shown in panel A were separated on 4 to 12% SDS-PAGE gels and transferred onto a nitrocellulose membrane, followed by Western blotting with antibodies specific for Tax-2, p28^II, or actin (LC). Reduction of Tax protein correlates with loss of Tax activity shown in panel A.