Fig 2: LEDGF D366N mutant is uncoupled from HIV-IN but interacts with other binding partners. (a–c) In vivo co-localization of the LEDGF D366N and cellular binding partners. Wild-type HEK293T, LEDGF KO and LEDGF D366N cells were transfected with flag-tagged IWS1, JPO2 and HIV-IN (panels a, b and c, respectively) and fixed 30 hrs later. Fluorescence microscopy to assess localization of proteins: LEDGF/p75 and LEDGF D366N were detected using αLEDGF480–530 (shown in green), whereas the transfected flag-tagged proteins were detected with αFlag Ab (shown in red). (a,b) LEDGF WT (top panel) and LEDGF D366N (lower panel) co-localizes with IWS1 and JPO2. (c) LEDGF D366N mutant did not co-localize with HIV-IN (lower panel), while in HEK293T WT cells HIV-IN is retained in the nucleus by binding to LEDGF/p75 (top panel). Scale Bar: 10 µm. (d) Co-immunoprecipitation of LEDGF/p75 and LEDGF D366N by cellular binding partners. HEK293T_LEDGF KO cells were transfected with either WT or D366N mutant LEDGF plasmid along with either one of the plasmids encoding Flag-tagged binding partners: IWS1, JPO2 or HIV IN. Cells were lysed 24 h later and lysates were incubated with anti-FLAG® M2-agarose affinity resin to capture the binding partner protein, which was then visualized by Western blot using αFlag Ab and αLEDGF480–530.

Fig 3: Increase of intensity of Tpr at the nuclear periphery by LEDGF/p75 overexpression and vice versa.The 293 T cells were cotransfected with GFP-Tpr and (a) HA alone or (b) HA-Nup153 or (c) HA-LEDGF/p75. (d) As control we cotransfected GFP-Nup98 with HA-LEDGF/p75. HA fused proteins were detected using a monoclonal anti-HA antibody and a secondary antibody conjugated with Cy3. (e) RT–PCR, mean of ratio between relative mRNA of PSIP1 and of Tpr genes by actin in Tpr depleted versus control HeLa and Jurkat cells is shown in the histogram. (f) Flow cytometry analysis show the percentage of GFP-Tpr-positive cells when cotransfected with HA, HA-LEDGF/p75 and HA-Nup153. (g) Relative increase of Nup GFP-positive cells on overexpression of LEDGF/p75 relative to the endogenous level of LEDGF/p75 based on flow cytometry data. S.d. of three independent experiments are shown.

Fig 4: Dynamic subpopulations of LEDGF/p75 revealed by sFRAP, CP and FCS. (A) sFRAP experiment of eGFP and eGFP-LEDGF/p75 in living HeLa cells. The diffusion coefficient we calculated for eGFP after fitting with a pure diffusion model was only 10.28 µm2/s (Table 1). For eGFP the observed recovery was lower than expected (solid orange curve) because of unavoidable recovery-while-photobleaching, which occurred because the diffusion time of eGFP (tdiff = 97 ms) was too small with respect to the acquisitioning time of the microscope (65 ms per iteration on our setup) (40). For eGFP-LEDGF/p75, the arrow indicates the decrease in mobility of eGFP-LEDGF/p75 due to chromatin interactions. DeGFP = 33 µm2/s is the D we measured with FCS, DeGFP-LEDGF/p75 = 22 µm2/s is calculated from DeGFP with Equation 8. See also Supplementary Figure S1. (B) CP experiment of eGFP and eGFP-LEDGF/p75. Upper panel is a 80–100-s zoom. (C) Concentration dependence of the amount of photobleaching of eGFP-LEDGF/p75 during the first 20 s. Solid line = fit with Equation 6. (D) FCS experiment of eGFP and eGFP-LEDGF/p75. Solid lines = two-component normal diffusion model. See also Supplementary Figure S2. Error bars = SD.

Fig 5: Characterization of SupT1_LEDGF D366N. (a) Sanger sequencing results showing consensus between HDR template and allele 1. The HDR template is shown with red characters indicating the sites of nucleotide substitutions, generating a VspI restriction site at D366N and deleting the PAM site. The sequence for allele 2 shows a 22-nucleotide deletion gRNA1 (“−” indicates deletion), resulting in KO of the PSIP1 allele. (b) Western blot analysis for the SupT1_LEDGF D366N clone using C-terminal specific LEDGF antibody, αLEDGF480–530. SupT1_WT and KO cells are included as controls. (c) Immunocytochemical analysis of LEDGF D366N protein (green) in SupT1_LEDGF D366N cells (bottom panel) recapitulates that of LEDGF/p75 in SupT1_WT cells (top panel). Phalloidin (white) was used to stain F-actin. Scale Bar: 10 µm.