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. 2020 Jul 14;11:3505. doi: 10.1038/s41467-020-17256-8

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a, b MDMs were co-infected with two VSV-G/HIVeGFP pseudoviruses labeled with INmNG or INmCherry (MOI 2), fixed at 6 hpi and immunostained for NSs (SC35). a A central section of MDM nucleus showing merger of INmNG and INmCherry VRCs in NSs (double positive IN clusters residing in NSs, yellow arrows). Single-labeled INmNG and INmCherry VRCs are marked with dashed green and red circles, respectively. NS contours are marked with semi-transparent gray dashed lines. b Fluorescence intensities associated with INmNG puncta inside (SC35(+)) or outside (SC35(−)) of NSs are plotted. c–f HIV-1 CA determines the nuclear penetration and speckle association of VRCs. c, d MDMs were co-infected with VSV-G-pseudotyped INmNG-labeled HIVeGFP virus bearing wild-type CA (green) and INmCherry-labeled pseudoviruses (MOI 2) containing either wild-type CA or the A77V CA (red). At 24 hpi, cells were fixed, immunostained for lamin, and colocalization of INmNG and INmCherry in the nucleus was quantified. c A central section of MDM nucleus shows nuclear INmNG spot for wild-type CA (white box) and nuclear membrane-associated INmCherry puncta of the A77V CA mutant (white arrows). The yellow arrows point to A77V CA INmCherry puncta on the nuclear side of Lamin. Inset shows the intranuclear WT CA INmNG spot (green) lacking A77V CA INmCherry signal. d The fraction of nuclear INmNG colocalizing with INmCherry puncta is shown. e, f MDMs were infected with INmNG-labeled VSV-G pseudotyped A77V CA mutant virus, fixed at 24 hpi, and immunostained for Lamin (blue) and SC35 (red). e Images show the A77V mutant INmNG puncta localized to the nuclear membrane failed to penetrate the nucleoplasm and reach SC35 compartments. f Quantification of the A77V CA mutant INmNG puncta colocalization with lamin or SC35(+) NS compartment. Scale bars in (a, c, e) are 5 µm and 0.5 µm in the inset (c). Error bars in (d, f) are SEM from n > 60 nuclei analyzed from three independent experiments. Source data are provided as a Source Data file.