HIV-1 Rev nuclear import mediated by importin β is selectively blocked in vitro by competitive excess of HIC. Nuclear import of GST-YFP-Rev, GST-SV40TNLS-GFP and GST-YFP-M9 was examined using in vitro nuclear import assays. Digitonin permeabilised HeLa cells were incubated with 10 μl of reaction mixtures containing 1 μg of import substrate, ATP regeneration system and nuclear import factors. Recombinant 6×His-HIC (0.5, 1 or 2 μg) was added to investigate the effect on Rev nuclear import. In all the cases, Rev nuclear signal intensities were analyzed by ImageJ for a minimum of 100 cells and illustrated by box plots (arbitrary units). Statistical significance analysis was performed with a two-tailed unpaired Student's t test *, P < 0.05; **, P < 0.01; ***, P < 0.001 (A) Recombinant HIC protein abolishes Rev nuclear import. Rabbit Reticulocyte Lysate (RRL) was employed as source of multiple import factors. (B) HIC specifically inhibits Rev nuclear import mediated by importin β but not by transportin. 2 μg of recombinant importin β or transportin was employed as the only source of import factor. (C) Schematic representation of HIV-1 Rev and deletion mutants. (D) Rev NLS domain is necessary and sufficient for Rev nuclear import inhibition by HIC. Nuclear imports of GST-YFP-Rev, GST-YFP-RevΔN1, GST-YFP-RevΔN2 and GST-YFP-RevNLS were examined using in vitro nuclear import assays. Importin β was employed as the only source of nuclear import factor.