Figure 2

Interaction of 16.4.1 with HIV-1 Rev, hCrm1 and with itself in human cells. 16.4.1 interactions in human cells were analysed with a mammalian two-hybrid assay in which the interaction of a protein fused to the Gal4 DNA-binding domain with a second protein fused to the VP16-activator domain induces transcription of a luciferase reporter gene. HEK293 cells were cotransfected with pBIND-16.4.1 plasmid for expression of Gal4-16.4.1 fusion protein, a pACT plasmid for expression of the indicated VP16-fusion protein and with the pG5luc reporter plasmid. Parallel cultures were cotransfected with pG5luc and the pBIND and pACT vectors to determine basal expression of the luciferase gene. Cells were lysed 48 hours after transfection and luciferase activities determined. Bars indicate the mean fold- induction of luciferase activity over basal expression ± SEM (standard error of the mean) and represent at least 6 independent transfection experiments. Cells coexpressing Gal4-16.4.1 and VP16 fused with Rev (grey bar), hCRM1 (black bar) or 16.4.1 (vertically striped bar) domains showed significantly stronger induction of luciferase production than cells coexpressing Gal4-16.41 and unfused VP16 (diagonally striped bar). Statistical analysis was performed by two-tailed Mann-Whitney U test.