Fig. 1: VSV-G-pseudotyped HIV-1 is endocytosed and induces type II NEIs. | Nature Communications

Fig. 1: VSV-G-pseudotyped HIV-1 is endocytosed and induces type II NEIs.

From: HIV-1-induced nuclear invaginations mediated by VAP-A, ORP3, and Rab7 complex explain infection of activated T cells

Fig. 1: VSV-G-pseudotyped HIV-1 is endocytosed and induces type II NEIs.The alternative text for this image may have been generated using AI.

a HeLa cells were 1-h HIV-Gag-iGFP infected prior to VAP-A and HIV-1 IN (IN-2) immunolabeling. As controls, noninfected cells or no HIV-1 IN antibody were used (Supplementary Fig. 2a). Samples were observed by CLSM and single sections are displayed. Longitudinal (top and bottom panels) or transverse (middle) sections of NEI (arrow) are shown. b–d Cells were 1-, 3-, or 5-h infected prior to VAP-A and IN-2 immunolabeling. Nucleoplasmic (Nu) and cytoplasmic (Cy) IN-2 were quantified as defined by regions of interest (ROI, b) and their ratio per cell from one representative experiment (c) or total IN-2 intensity per cell are indicated (d 50 cells per experiment, n = 3). e, f Cells were 3-h infected (or not) prior to VAP-A and SUN2 immunolabeling. Type I (VAP-A–SUN2+) and II (VAP-A+SUN2+) NEIs (e green and yellow/red arrows, respectively) were quantified (f 100 cells per experiment, n = 3). g, h Cells were 30-min pretreated with DMSO or DNS (80 µM) prior to 3-h HIV-Gag-iGFP infection followed by VAP-A and HIV-1 IN immunolabeling (g). Note that IN-2 accumulated in NEI (yellow arrow) and/or dispersed into cytoplasm and nucleoplasm in the control, whereas it remained outside the DNS-treated cells (asterisk). Total IN-2 intensity is presented (h, >50 cells per experiment, n = 3). i–m Colocalization of IN-2 with Rab proteins. Experimental methodology (i). Cells were baculovirus infected to express Rab5/Rab7-RFP and then HIV-Gag-iGFP infected. At different times, cells were processed for ICC for HIV-1 IN and counterstained with DAPI. Noninfected cells expressing Rab5/7-RFP were analyzed (control). Composite images revealed colocalizations of IN-2 and Rab5-RFP (j white arrow) or Rab7-RFP (k green arrow) at early and late time points, which are rendered in a 3D image (l) and quantified using Pearson’s R correlation coefficient (m n = 25 cells per time point). We used a MOI of 2. In all cases, the means ± S.D. and, where appropriate, the individual values for each experiment are shown. P values are indicated. Scale bars, 2 (l), 5 (a, b), 10 (e, g, j, k) µm.

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