Figure 6

Y175 Mutations in Cyclin T1 impair binding to 7SK snRNA, HIV TAR and Tat. (A) CycT1-Y175 mutations abolish Tat transactivation in murine cells - 3T3 murine cells were co-transfected with the Myc-Tat, HA-CycT1 (wild type or Y175 mutant human Cyclin T1 proteins), HIV-LTR-Photinus luciferase and a Renilla luciferase reporter. 48 hr after transfection, luciferase activity was monitored. Photinus luciferase readings were normalized to Renilla expression. Fold transactivation are relative to luciferase activity in the absence of both Tat and human Cyclin T1. Rescue of Tat transactivation by wild-type Cyclin T1 was set to 100. Results are representative of the mean value of triplicate wells; error bars show ± SEM. Lower panel shows expression levels of HA-Cyclin T1 in the experiment. (B) Impaired association of Cyclin T1-Y175 mutants with HIV Tat. 293T cells were co-transfected with HA-Tagged CycT1-wild type or Y175 mutated, Flag-Tat and GFP expressing plasmids to control transfection efficiency. HA-Cyclin T1 proteins were precipitated with α-HA antibody, separated on SDS-PAGE and analyzed by Western blot with antibodies against their epitope Tags. (C) Impaired association of Cyclin T1-Y175 mutants with TAR and 7SK snRNA. 293T cells were transfected with the HIV-LTR-BFP provirus (generating TAR RNA and Tat) and with the indicated HA-Cyclin T1 wild type or mutants. 48 h post transfection RNAs were coimmunoprecipitated with α-HA (■) or non-immune (□) rabbit antibodies and analyzed by RTqPCR using primers specific to either 7SK snRNA or HIV TAR. The signal obtained from RNAs immunoprecipitated in the absence of co-transfected HA-Cyclin T1 was set to 1.