Figure 1 : Single virus analysis probes IN content and conformation in functional viruses.

From: Dynamic Oligomerization of Integrase Orchestrates HIV Nuclear Entry

Figure 1

(A) Top: HeLaP4 cells are infected with VSV-G pseudotyped IN-labeled virus, produced via Vpr-transincorporation of IN-FP, for 6 hours (scissors: HIV protease recognition site). Middle: A 3D confocal image of an HIVIN-eGFP (green dots) infected HeLaP4 cell is acquired. The point spread function (PSF) of each IN-eGFP complex is fitted with a 2D Gaussian function to determine the fluorescence intensity, hence the quantity of IN-eGFP per complex. Scale bars: 5 μm. Bottom: Quasi-TIRFM imaging of HIVIN-mTFP1+IN-mVenus in infected cells enables acceptor photobleaching (AP)-FRET measurements. The intensity of the donor (mTFP1; D, pre), quenched by the proximal acceptor (mVenus; A,pre), is dequenched after photobleaching of the acceptor (mVenus; A, post) and hence its brightness increases (mTFP1; D, post). The fluorescence intensity of each viral complex in both donor images (D, pre and D, post) is used to quantify the mean FRET ratio, as a measure for the interaction between IN subunits. Scale bars: 5 μm. The 2D Gaussian function shows an increased fluorescence intensity of the donor (D, post) after acceptor photobleaching compared to the intensity before photobleaching (D, pre). (B) Single round infectivity of HIV, HIVIN-eGFP and HIVIN-mTFP1+IN-mVenus on HeLaP4 as measured by Firefly luciferase (fLuc) activity. Viruses are produced as in (A), the Vpr-transincorporated IN-FP is shown and ‘−’ represents the absence of transincorporation. The mean relative luminescence units (RLU) of two experiments performed in triplicate, normalized for protein content of cell lysates is shown, error bars represent standard deviation. (C) qPCR analysis for the kinetics of 2-LTR circle formation in HeLaP4 cells after infection with HIV in the presence of DMSO, PF74 (10 μM), RAL (0.6 μM) or EVG (0.2 μM) at 8, 24 and 48 h post infection. Error bars represent standard deviation of triplicates. (D) Percentage nuclear IN-eGFP complexes of cells treated with DMSO, PF74 (10 μM), RAL (0.6 μM) or EVG (0.2 μM) during infection with HIVIN-eGFP. Error bars represent standard error of mean (SEM); *p-value < 0.05, ***p-value < 0.001 obtained with a Mann-Whitney test.