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. 2023 Jul 20;14:4368. doi: 10.1038/s41467-023-39262-2

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© The Author(s) 2023

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. 4 — a HIV-1_NL4-3ΔNef particles were produced in the presence of indicated plasmids and titered on TZM-Bl indicator cells by luciferase assay. Results represent the mean of n = 7 independent experiments ± SEM. b HIV-1_NL4-3ΔRTΔNef virus particles containing Gag-GFP and CD63-mRFP were produced in the presence of indicated hSERINC5 or mTMEM16F plasmid, bound to anti-CD63 magnetic beads, stained with Alexa647-annexin V, and imaged by flow cytometry. Histograms depict annexin V intensity for the GFP-positive population. Results reflect one representative experiment of n = 3 biological replicates. c PS exposure was assessed as in (b) and plotted in relation to virus infectivity as in (a) for the indicated hSERINC5 (S5) or mTMEM16F proteins. Values represent mean ± SD from n = 3 independent experiments. d HIV-1_NL4-3ΔNef or NefC-expressing virus particles were produced in the presence of indicated hSERINC5 or mTMEM16F plasmid and analyzed as in (b). Histograms depict annexin V intensity for the GFP-positive population from one representative of n = 3 independent experiments. e Annexin V mean fluorescence intensity is shown for HIV-1 particles ± Nef as in (d). Values represent the mean ± SEM from n = 3 independent experiments. Percent annexin V-positive MLV particles imaged by confocal microscopy as in (Supplementary Fig. 9). Values represent the mean ± SEM from five independent experiments. f HIV-1_NL4-3ΔRTΔNef virus particles were produced in the presence of WT hSERINC5 or the F397L mutant and the conformational state of HIV-1 Env was analyzed by single-molecule FRET (see also Supplementary Fig. 10). N is the number of individual dynamic molecule traces complied into a population FRET histogram (gray lines) and fitted into a three-state Gaussian distribution (solid) centered at ~0.15-FRET, ~0.35-FRET, and ~0.6-FRET. Histogram error bars represent the mean FRET probabilities ± SEM. g HIV-1_NL4-3ΔRTΔNef virus particles were produced in the presence of mTMEM16F GY (blue) or mTMEM16F DW (red) and the conformational state of HIV-1 Env was analyzed by single-molecule FRET as in (f). Source data are provided as a Source data file.