FIG. 5.

p28^II-mediated inhibition directly correlates with Tax expression in the context of the native HTLV-2 promoter (LTR). (A) A total of 0.4 μg of wtHTLV-2 or 0.4 or 0.2 μg of BCHTLV-2 was transfected into 293T cells along with LTR-luciferase 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. p28^II consistently inhibits Tax-2 activity when expressed from wtHTLV-2 but not BCHTLV-2. (B) Expression of Tax-2 from cDNA with (IY620) or without (IY619) 5′UTR but under the control of the HTLV-2 promoter reverses the resistance to inhibition by p28^II that is observed with the Tax-2 CMV expression plasmid. (C) Lysates shown in panels A and B were separated on 4 to 12% SDS-PAGE gels and transferred onto nitrocellulose membranes, 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 panels A and B.