Figure 3 : Cellular cofactor affects IN oligomerization dynamics in the nucleus.

From: Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry

Figure 3

(A) The cellular cofactor TRN-SR2 has been implemented in HIV-1 nuclear import, while LEDGF/p75 tethers the PIC to the chromatin. (B) Western blot showing the depletion of TRN-SR2 and LEDGF/p75 respectively in TRN-SR2KD and LEDGF/p75KD cells. Other lanes represent LEDGF/p75 expression in the stable cell lines used in panel E. (C,D) Fluorescence intensity (C) and mean FRET ratios (D) in the cytoplasm (white) or nucleus (grey) in TRN-SR2KD, TRN-SR2SCR and HeLaP4 WT cells infected with VSV-G pseudotyped HIVIN-eGFP and HIVIN-mTFP1+IN-mVenus, respectively. (E) Representative confocal images of DAPI-stained (blue) and LEDGF/p75 immunostained (red) HeLaP4 cells. Row 1: HeLaP4 WT cells (WT), row 2: LEDGF/p75 depleted cells (LEDGF/p75KD), row 3: reversal of LEDGF/p75KD by Cre specific excision of the LEDGF/p75 knockdown cassette reconstituting wild-type LEDGF/p75 (LEDGF/p75Flox), row 4 and 5: LEDGF/p75-depleted cells complemented with wild-type (LEDGF/p75BC) or mutant LEDGF/p75D366N. (F,G) Fluorescence intensity (F) and mean FRET ratios (G) in the cytoplasm (white) or nucleus (grey) in LEDGF/p75KD, LEDGF/p75Flox and HeLaP4 WT cells infected with VSV-G pseudotyped HIVIN-eGFP and HIVIN-mTFP1+IN-mVenus, respectively. (H) Crystal structure of the IN catalytic core dimer interface and the IBD of LEDGF/p75 (PDB 2B4J). The interface mutations W131A and D366N are indicated. (I) Mean FRET ratios in the cytoplasm (white) or nucleus (grey) in HeLaP4 cells infected with VSV-G pseudotyped HIVIN-W131A-mTFP1+IN-W131A-mVenus compared to HIVIN-mTFP1+IN-mVenus. (J) Mean FRET ratios in the cytoplasm (white) or nucleus (grey) in LEDGF/p75D366N, LEDGF/p75BC and LEDGF/p75WT cells infected with HIVIN-mTFP1+IN-mVenus. SCR = scrambled; KD = knockdown; BC = back-complemented. Unless explicitly stated otherwise, the intensity and mean FRET ratios in the cytoplasm and nucleus were statistically different (p-value < 0.001). (***p-value < 0.001, **p-value < 0.01 and n.s. = not significant).