Expression and subcellular localization of precursor Gal-Pol polyprotein were detected by immunofluorescence microscopy as described previously with some modifications [24] . Briefly, HeLa cells cultured on coverslip in 24-well culture plate were transfected with 600 ng pNL4-3-DE-EGFP and 200 ng pHEF-VSV-G. Two days post-transfection, the cells were fixed with 500 ml 4% paraformaldehyde (PFA) at room temperature for 15 min. After washing 3 times with PBS, 500 ml of 50 mM ammonium chloride was incubated with the cells for 10 min to neutralize residual PFA. The cells were washed 3 times with PBS and treated with 0.05% Triton X-100 for 3 min. After washing 3 times with PBS, 500 ml 10% normal rabbit serum (NRS) in PBS was added to block the slip overnight at 4uC. Primary antibody (mouse monoclonal antiintegrase, 1:100, Santa Cruz) and secondary antibody (Texas Red dye-conjugated Rabbit anti-mouse IgG, 1:100, Jackson Immu-noResearch) were incubated with samples in dark at room temperature. Following each incubation, samples were subjected to 3 washes with 1% NRS in PBS. With 3 additional PBS washes, the coverslip was mounted using fluorescence mounting medium (Dako) and observed under LSM510 Meta confocal microscope (Carl Zeiss).