Fig 2: Sotrastaurin-mediated PKC inhibition prevents the formation of both HIV-1-induced NEIs and the VOR complex.a–c Quiescent CD4+ T cells were pretreated with DMSO (DMSO, –) or 40 µM Sotrastaurin (Sotrast.) for 30 min before their activation with PHA and IL-2 for 48 h. Afterward, they were 6-h HIV-89.6-EGFP infected in the presence of Sotrastaurin prior to SUN2 and VAP-A immunolabeling. Nuclei were counterstained with DAPI. Samples were observed by CLSM. Single section of 10 representative cells are presented. Note the presence of NEIs in the infected cell in the absence of Sotrastaurin (left, arrows). The percentage of cells with type I or II NEIs was quantified (b 50 cells per experiment, n = 3) and the nuclear solidity was determined (c n = 10 cells). d After 6-h infection, cells were incubated for 48 h prior to FC analysis for EGFP expression. Representative FC histograms are displayed. DMSO-treated, noninfected cells were used for the gating. e Cells were treated as in a, solubilized and subjected to ORP3 IS. As a control, cells were noninfected. The input (1/50) and bound fractions were probed for ORP3, VAP-A, and Rab7 by IB. Note the absence of slower-migrating ORP3 immunoreactive species in Sotrastaurin-treated CD4+ T cells regardless of infection (red arrow). OE overexposed blot. All samples were from the same experiment. Molecular mass markers (kDa) are indicated. We used a MOI of 2. In all cases, means ± S.D. and individual values are shown. P values are indicated. Scale bars, 5 µm.

Fig 3: Silencing the VOR complex inhibits HIV-1 integrase transport into NR and productive infection.a–d HeLa cells stably transfected with plasmids carrying scrambled shRNA, shVAP-A, shVAP-B or shORP3 and expressing either ER-GFP (a, upper panels) or Rab7-RFP (a, lower panels, b–d) were 1-h HIV-Gag-iGFP infected prior to immunolabeling for either VAP-A, VAP-B, ORP3 or SUN2 and HIV-1 IN (IN-2) as indicated. Samples were observed by CLSM. Single sections are displayed. IN-2 was detected in ER-GFP+ or SUN2+ NEI of scrambled and shVAP-B cells and quantified (a arrow; b 50 NEIs per experiment, n = 3). The ratio of IN-2 in the nucleoplasm versus cytoplasm per cell was determined (c n = 150 cells from three independent experiments). The distribution of IN-2 in perinuclear region of scrambled shRNA, VAP-A-, or ORP3-silenced HeLa cells is shown in a circular histogram (d 10 cells per cell line; color code indicates an individual cell). [For Methods, see Supplementary Fig. 6d]. Cells with dual IN-2 localization are in color. e–g HeLa cells were 6-h HIV-Gag-iGFP infected, washed and incubated for 24 h prior to ICC (e) and FC (f, g). Representative fluorescent images for the indicated proteins (e) and FC histograms (f) are displayed. Scrambled shRNA-transfected, noninfected cells were used for gating. Note the reduction in Gag-iGFP expression in cells lacking VAP-A and ORP3. The percentage of GFP+ cells is plotted (g n = 3). Asterisk and arrowhead indicate Gag-iGFP in the cytoplasm (Cy) and plasma membrane (Pm), respectively. Nu nucleus. We used a MOI of 2. Means ± S.D. and individual values for each experiment (b, g) or cell (c) are presented. P values are indicated. Scale bar, 5 (a, top) or 10 (a, bottom, e) µm.

Fig 4: HIV-1 infection promotes the VOR complex formation in a microtubule-dependent manner.a PHA/IL-2-activated CD4+ T cells were noninfected (control) or 6-h HIV-89.6-EGFP infected after 30-min pretreatment with DMSO or 10 µM PRR851. The drug was present during infection. Cells were then solubilized and subjected to ORP3 IS. The bound fractions and the input (1/50) were probed for ORP3, VAP-A, and Rab7 by IB. b Cells were 5-min pretreated with DMSO or nocodazole (1 µM) and then noninfected (control) or 1-h HIV-89.6-EGFP infected, solubilized and processed for ORP3 IS and ORP3, VAP-A and Rab7 IB. All samples came from the same experiment and were run in parallel (a, b). Molecular mass markers (kDa) are indicated. Representative experiments are shown. We used a MOI of 2. Note that VOR complex formation only occurs after HIV-1 infection and is hindered by PRR851 and nocodazole. c Representation of the induction of type II NEIs by virus-laden late endosomes, a process mediated by the interaction of VOR complex proteins, namely ONM-associated VAP-A, cytoplasmic ORP3, and late endosome-associated Rab7 (left panel). Release of viral components from late endosomes into the cytoplasmic core of induced NEIs at the vicinity of the nuclear pore would facilitate their transfer to the nucleoplasm (right panel). PRR851 inhibits the interaction of the VOR complex proteins, and hence the NEI formation. ILV, intralumenal vesicles associated with late endosome/multivesicular body; INM/ONM, inner/outer nuclear membrane; MTOC, microtubule-organizing center; MTs, microtubules.

Fig 5: The VOR complex inhibition impedes the HIV-1 integrase transport into NR and productive infection.a Drug structures. b HeLa cells were 90-min incubated with DMSO or various drugs (10 µM) as indicated, detergent solubilized and ORP3 IS. Bound fractions and Rab7 input (1/50) were probed for ORP3, VAP-A, and Rab7 by IB. c HeLa cells were 30-min pretreated with DMSO or drugs (10 µM) before 1-h HIV-Gag-iGFP infection in the presence of drugs. As a control, cells were noninfected. Samples were VAP-A and HIV-1 IN (IN-2) immunolabeled. Single sections are displayed. IN-2 was detected in NEIs of DMSO/PRR846-treated cells (arrow). d–g Rab7-RFP-expressing HeLa cells were treated as in c, and IN-2 and SUN2 immunolabeled. The presence of IN-2 in SUN2+ NEI (d arrow) was quantified (e 50 NEIs per experiment, n = 3). The IN-2 nucleoplasm/cytoplasm ratio was determined (f n = 150 cells from three independent experiments). Localization of IN-2 in the perinuclear region is shown using a circular histogram (g 10 cells/condition; color code indicates an individual cell). [For Methods, Supplementary Fig. 6d]. h Drug-treated HeLa cells were 3-h HIV-Gag-iGFP infected, fractionated into cytoplasmic and nuclear fractions and probed for HIV-1 IN by IB. A quarter and the total material of the cytoplasmic and nuclear fractions were loaded. Cytoplasmic GAPDH and nuclear Lamin B1 were used as controls and run in parallel. They are derived from the same experiment (b, h). Molecular mass markers (kDa) are indicated. i–k HeLa cells were 30-min pretreated with DMSO or drugs before 6-h HIV-Gag-iGFP infection. They were 24-h incubated in the presence of drugs and then processed for ICC (i) or FC (j, k). For ICC, fixed-cells were VAP-A immunolabeled, while they were trypsinized for FC. Representative images (i) and FC histograms (j) are displayed. DMSO-treated, noninfected cells were used for gating. The GFP+ cells were quantified (k n = 3). We used a MOI of 2. Means ± S.D. and individual values for each experiment (e, k) or cell (f) are presented. P values are indicated. Scale bars, 10 (c, i) or 5 (d) µm.