Figure 3. Transfection of a Transportin-3 allele insensitive to TNPO3 si4 restores protein expression, HIV infectivity, and partially restores wild-type HIV integration site distributions.

(A) Western blot showing Transportin-3 levels in cells treated with TNPO3 si4 in the presence or absence of the Transportin-3 rescue plasmid. Cells were cotransfected with siRNA and either empty vector plasmid or rescue plasmid encoding siRNA-resistant alleles of Transportin-3 expressed from the CMV promoter and harvested at 48 hr post-transfection for analysis. Transportin-3 is reduced after co-transfection with siRNA and empty vector, and overexpressed after co-transfection with siRNA and rescue plasmid. Endogenous levels of Transportin-3 are shown in cells transfected with the control siRNA targeting GL2 and an empty vector. (B) HIV infection in cells treated with TNPO3 si4 in the presence or absence of the Transportin-3 rescue plasmid. Cells were co-transfected as above. 48 hr after transfection cells were infected with a VSVG-pseudotyped HIV-1 vector carrying a GFP reporter. At 48 hpi cells were harvested and the percent of cells expressing GFP was determined by flow cytometry. The Y-axis shows relative infection compared to infection in the control (GL2 siRNA + empty vector-transfected) cells. (C) Average gene density in 1 Mb windows surrounding HIV integration sites in cells depleted or rescued for Transportin-3 expression. Asterisks denote significant differences as determined by the Mann–Whitney test (*P<0.05; **P<0.01; ***P<0.001).