Fig. 2: 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.