Figure 7.

HIC inhibits HIV-1 Rev function in a cell-specific fashion. A. HIC down-regulates Rev activity in a dose dependent manner. 293T cells were transfected with 0.1 μg of pDM128-RRE combined with 0.05 μg of HA-Rev and 1, 2 or 4 μg of FLAG-HIC. Relative CAT activity is compared with 100% for Rev activity of pDM128-RRE. Values are mean ± standard deviation. Data are representative of a minimum of three independent experiments performed in duplicate. Statistical significance analysis was performed with a two-tailed unpaired Student's t test *, P < 0.05; **, P < 0.01; ***, P < 0.001. B. The down-regulation of Rev activity by HIC is dependent on the I-mfa domain and is cell-specific. 293T or HeLa cells were transfected with 0.1 μg of pDM128-RRE combined with 0.05 μg of HA-Rev and 4 μg of FLAG HIC. Relative CAT activity was analysed as described above. C. Evaluation of siRNA knockdown of HIC. Real-time RT-PCR analysis of HIC mRNA levels for HeLa and 293T cells was performed at 72 hours following reverse transfection of three distinct HIC siRNAs or with siRNAs directed against luciferase (GL2) as negative control. These experiments were each performed in duplicates and the mean average results are shown. D. Effects of siRNA knockdown of HIC on Rev activity are cell-specific. 293T or HeLa cells were first reverse-transfected with three independent HIC siRNAs (30pmoles) or negative control siRNA (30pmoles) and after 24 hours were transfected with 0.2 μg of pDM128-RRE, 0.02 μg RL-TK and 0.02 μg of HA-Rev or parent plasmid. Relative CAT activity was analysed as described above. Values are mean ± standard deviation. Data are representative of a minimum of three independent experiments performed in duplicate.