Figure 3

Lentiviral IN affinity for imp7 correlates with infection phenotype in imp7 KD cells. (A) GST-imp7 (top left), GST (middle), or GST-LEDGF^326–530 (bottom) immobilized on glutathione sepharose beads were incubated in the absence (lane 1), or presence (lanes 2–6) of untagged recombinant HIV-1, HIV-2, SIVmac, EIAV, or BIV IN in the pull-down buffer containing 130 mM NaCl. Proteins bound to glutathione sepharose beads were resolved in an SDS PAGE gel and detected by staining with Coomassie Blue. Input quantities of each soluble protein used are shown to the right. Migration positions of GST-Imp7, INs, GST, and GST-LEDGF^326–530, and the molecular weight markers are indicated. (B) Non-tagged Imp7 was incubated in the absence (lane 3) or presence (lanes 4–9) of C-terminally hexahistidine-tagged INs from HIV-1, HIV-2, SIVmac, EIAV, BIV and Ni-NTA agarose beads in a pull down buffer containing 150 mM NaCl. Proteins captured on the resin were separated in a tricine SDS PAGE gel and detected with Coomassie Blue. Lanes 1 and 2 show 100% and 20% Imp7 input, respectively. (C) GST (lanes 3, 6, 9, 12, 15), GST-LEDGF^326–530 (lanes 4, 7, 10, 13, 16), or GST-imp7 (lanes 5, 8, 11, 14, 17) were incubated without (lanes 3–5), or with non-tagged HIV-1 (lanes 6–8, 12–14) or HIV-2 (lanes 9–11, 15–16) INs. The pull-down buffer contained 150 mM (lanes 3–11) or 400 mM (lanes 12–17) NaCl. Lanes 1 and 2 contained input quantities of HIV-1 and HIV-2 INs, respectively. (D) DxR KD and imp7 KD cell populations were infected with VSV-G pseudotyped HIV-1, (pHR'), SIVmac, HIV-2 and EIAV vectors expressing GFP at an MOI of 0.03 and the percentage of GFP+ cells counted 24 hours after infection by flow cytometry. Data are expressed as average percentage of infection relative to control (shDxR) ± SD of two independent experiments performed in duplicate.