Fig 2: 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 3: Rab7+ late endosomes containing HIV-1 IN enter NEI.HeLa cells stably transfected with VAP-A-GFP were 1-h HIV-Gag-iGFP infected prior to immunolabeling for HIV-1 IN and Rab7 (a), CD63 (b) or Lamp1 (c). Single sections show transverse (a, c) and/or longitudinal (a–c) sections of a NEI. Merge and single channels (insets a1, a2, b1, c1, c2) are presented. IN-2 colocalized with Rab7 or CD63, but not Lamp1 in NEIs (yellow and magenta arrow, respectively). d Noninfected VAP-A-GFP-expressing HeLa cells were Rab7 and Lamp1 immunolabeled. Single transverse section of a NEI is shown with the merge and single channels as indicated. Insets show the absence of Lamp1 in NEI (d1, white arrow) and the colocalization of Rab7 and Lamp1 in the cytoplasmic compartment (d2, pink arrowhead). e, f HeLa cells were 1-h HIV-Gag-iGFP infected prior to immunolabeling for Rab7 and Lamp1. Nuclei were counterstained with DAPI. Single longitudinal section of a NEI is shown with the merge and single channels as indicated (e). Insets show the absence of Lamp1 in NEI (e1) and the colocalization of Rab7 and Lamp1 in cytoplasmic compartment (e2, pink arrowhead). Quantification of NEIs containing specific antigen(s) as indicated in infected cells (f, 50 cells per experiment, n = 3, means ± S.D. are shown). g VAP-A-GFP-expressing HeLa cells were 1-h infected with “bald” virus, lacking VSV-G, prior to Rab7 and HIV-1 IN immunolabeling. A single section shows transverse section of a NEI. Note that most of IN-2 remains outside the cells (asterisk) and the absence of Rab7 in NEI (double arrow). For a lower power view as well as all sections see below Supplementary Fig. 15c. We used a MOI of 2 or equivalent in the case of the “bald” virus. Scale bars, 5 µm.

Fig 4: 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 5: Native Env-pseudotyped HIV-1 infection of CD4+ T cells triggers type II NEIs biogenesis.a–f PHA/IL-2-activated CD4+ T cells were HIV-89.6-EGFP infected for 3 (a, b) or 6 (c–e left y axis, f) hours prior to immunolabeling for SUN2 and VAP-A (a–e) or Lamin B1 (f). Nuclei were counterstained with DAPI. In some experiments, cells were 30-min pretreated with PRR851 (10 µM) (c, d). Drugs were present during infection. As a control, cells were noninfected. They were observed by CLSM and serial sections of a representative cell (a) or single section of 10 cells are presented (c). Alternatively, after 6-h infection, cells were 48-h incubated prior to FC (e right y axis, n = 3). The percentage of cells with type I or II NEIs was quantified (b, d, e, 50 cells per experiment, n = 3). Note the presence of NEIs in infected cells without PRR851 (a right, c middle, f arrow). g, h The solidity was evaluated in noninfected and 6-h infected cells. After SUN2 and VAP-A immunolabeling, the surface areas were evaluated (g), and the resulting solidity presented (h, n = 10 cells). i, j Activated T cells were 3-h infected with HIV-89.6-EGFP or corresponding “bald” virus prior to VAP-A and HIV-1 IN (IN-2) immunolabeling. Single section of representative cells is shown (i). IN-2 in induced NEIs (arrows) or in the cytoplasm (asterisks) are indicated. Green dashed lines indicate IN-2 distribution around the cell surface. The percentage of cells with type II NEIs were quantified (j =50 cells per condition, n = 3). k, l The presence of IN-2 in NEIs (arrow) and late endosomes of 6 or 3 h-infected cells was observed after immunolabeling for IN-2 and SUN2 (k upper panel) or Rab7 (k lower panel), respectively. Alternatively, cells were immunolabeled for Rab7 or Lamp1 and SUN2 (l). Note that Rab7 (arrows), but not Lamp1, is present in NEIs. We used a MOI of 2 or equivalent for the “bald” virus, unless otherwise stated. In all cases, means ± S.D. and individual values are shown. P values are indicated. Scale bars, 5 µm.