Research ArticleVIROLOGY

HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

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Science Advances  02 Oct 2019:
Vol. 5, no. 10, eaaw8651
DOI: 10.1126/sciadv.aaw8651
  • Fig. 1 sSTED-FCS measurement of lipid diffusion inside and outside HIV-1 assembly sites.

    (A) Schematic illustration of Gag and Env distribution during HIV-1 assembly, release, and maturation. (B) Representative confocal time lapse images of Jurkat T cells infected with NL4.3 Gag-iGFP HIV-1. On day 3 after infection, cells were adhered to poly-l-lysine–coated coverslips and monitored for the appearance of HIV-1 assembly sites prior to sSTED-FCS measurements. Images represent Gag-iGFP signal (blue-green-yellow) at the cell-coverslip interface at 1 (left) and 20 min (right) after adherence. Arrows indicate the appearance of the individual diffraction-limited HIV-1 assembly sites, which were subsequently analyzed by sSTED-FCS. A cell-free HIV-1 particle is indicated by an asterisk. Scale bar, 1 μm. (C) Left: Live confocal imaging was used to localize HIV-1 virus assembly sites using Gag.iGFP signal (blue-green-yellow) as a guide and to align them with the position of the circular scan orbit (red dashed line). Right: Schematic illustration of sSTED-FCS orbit over the HIV-1 assembly site. Arrows indicate the direction of the scan. Scale bar, 200 nm. (D to F) Representative confocal-mode signal intensity carpets for Gag.iGFP signal (blue-green-yellow) before (D) and after (F) acquisition of STED-mode intensity carpet for lipids (here, PE-ATTO647N) (E) inside and outside HIV-1 assembly sites. Image x and y axes correspond to the position on the scan line and signal intensity at each time point, respectively. Scale bars, 200 nm (x axis; white); 1.24 ms (y axis; gray). (G) Representative normalized autocorrelation curve of PE-ATTO647N diffusion (gray line) obtained from a single position [marked by the red asterisk in (C) and (D) to (F)] on the circular scan line within the correlation carpet. Autocorrelation curve (red) was fitted using a generic 2D diffusion model.

  • Fig. 2 Lipid mobility changes in NL4.3 Gag-iGFP HIV-1–infected Jurkat T cells.

    Median lipid diffusion coefficients (Da) as determined by sSTED-FCS measurements of 20 sites each from two independent virus preparations and infections. Box-and-whisker plots (horizontal line, median; box, 25 to 75% percentiles or IQR; whiskers, 10 to 90% measurements) showing Da values inside assembly sites (red), outside assembly sites (green), and in noninfected control cells (blue). Data were collected for (A) ATTO647N-PI(4,5)P2, (B) cholesterol (Chol-PEG-KK114), (C) SM (ATTO647N-SM), and (D) PE (ATTO647N-DPPE) analogs. Statistical significance was assessed by Wilcoxon rank sum test. ns, not significant.

  • Fig. 3 Lipid mobility changes in HIV-1 Gag.iGFP transfected Jurkat T cells.

    (A) Representative live confocal image of Jurkat T cells expressing HIV-1 Gag.iGFP. Image represents Gag.iGFP signal (blue-green-yellow) at the cell-coverslip interface at 24 hours after transfection. Scale bar, 1 μm. Zoomed-in confocal image depicts an individual HIV-1 assembly site indicated by an arrow. Scale bar, 200 nm. (B to E) Median values of the lipid diffusion coefficients (Da) were determined for (A) ATTO647N-PI(4,5)P2, (B) cholesterol (Chol-PEG-KK114), (C) SM (ATTO647N-SM), and (D) PE (ATTO647N-DPPE) analogs by sSTED-FCS on 20 sites each from two independent Gag.iGFP transfections. Box-and-whisker plots (horizontal line, median; box, 25 to 75% percentiles or IQR; whiskers 10 to 90% measurements) showing Da values inside assembly sites (red), outside assembly sites (green) and in untransfected control cells (blue). Statistical significance was assessed by Wilcoxon rank sum test.

  • Fig. 4 Lipid trapping induced by Gag during HIV-1 assembly in infected or transfected Jurkat T cells.

    Confinement indices are based on the relative normalized cumulative frequency distribution of the diffusion coefficients Da (for Da = 0.001 to 1 μm2/s, normalized to 100 for Da = 1 μm2/s), which were arbitrarily divided into three distinct diffusion regimes, slow (Da < 0.01 μm2/s), intermediate (0.01 ≤ Da < 0.1 μm2/s), and fast (Da ≥ 0.1 μm2/s). The sum of cumulative frequencies within each regime resulted in values of the confinement index for each regime as ratio of the latter values for lipid diffusion within and outside the assembly sites or within the assembly sites and noninfected (or nontransfected) cells. Values of the confinement indices found for PI(4,5)P2, Chol, SM, and PE analogs for HIV-1 Gag.eGFP-transfected (black bars) and HIV-1-iGFP–infected (gray bars) Jurkat T cells. Chol and PI(4,5)P2 analogs show strong confinement (indices >1 for intermediate and especially slow regimes) inside assembly sites for both transfected-only or fully infected Jurkat T cells, while neither SM nor PE analogs exhibited confinement inside assembly sites (confinement index ~1 for all regimes). *, no diffusion coefficients below 0.01 μm2/s were observed for PE analogs.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/10/eaaw8651/DC1

    Sections S1 and S2. Characterization of fluorescent lipid analogs

    Section S3. MHC-I mobility at HIV-1 assembly sites in NL4.3 Gag-iGFP HIV-1–infected Jurkat T cells

    Section S4. STED microscope calibration

    Section S5. PI(4,5)P2 clustering and trapping by full-length HIV-1 Gag on biomimetic membranes

    Section S6. Budding efficiency comparison in Jurkat T cells transfected with Gag.eGFP or with a mixture of Gag.eGFP/Gag (ratio, 1:3)

    Section S7. Cumulative frequency distributions observed in infected cells for the different lipids

    Section S8. Cumulative frequency distributions observed in transfected cells for the different lipids

    Fig. S1. Fluorescent lipid analog structures.

    Fig. S2. Fluorescent lipid analog mobility in Jurkat T cells.

    Fig. S3. MHC-I mobility at HIV-1 assembly sites in NL4.3 Gag-iGFP HIV-1–infected Jurkat T cells.

    Fig. S4. STED microscope calibration results.

    Fig. S5. Changes in the lateral mobility of ATTO647N-PI(4,5)P2 upon addition of Gag on SLBs.

    Fig. S6. Jurkat T cell coelectroporation with Gag.eGFP and Gag plasmids.

    Fig. S7. Cumulative frequency distributions of diffusion coefficient observed in HIV-1–infected T cells.

    Fig. S8. Cumulative frequency distributions of diffusion coefficient observed in HIV-1 Gag-transfected T cells.

    Movie S1. Drift stabilized time lapse movie of a representative NL4.3 Gag-iGFP HIV-1–infected Jurkat T-cell showing already present and newly developing virus assembly sites.

    References (62, 63)

  • Supplementary Materials

    The PDF file includes:

    • Sections S1 and S2. Characterization of fluorescent lipid analogs
    • Section S3. MHC-I mobility at HIV-1 assembly sites in NL4.3 Gag-iGFP HIV-1–infected Jurkat T cells
    • Section S4. STED microscope calibration
    • Section S5. PI(4,5)P2 clustering and trapping by full-length HIV-1 Gag on biomimetic membranes
    • Section S6. Budding efficiency comparison in Jurkat T cells transfected with Gag.eGFP or with a mixture of Gag.eGFP/Gag (ratio, 1:3)
    • Section S7. Cumulative frequency distributions observed in infected cells for the different lipids
    • Section S8. Cumulative frequency distributions observed in transfected cells for the different lipids
    • Fig. S1. Fluorescent lipid analog structures.
    • Fig. S2. Fluorescent lipid analog mobility in Jurkat T cells.
    • Fig. S3. MHC-I mobility at HIV-1 assembly sites in NL4.3 Gag-iGFP HIV-1–infected Jurkat T cells.
    • Fig. S4. STED microscope calibration results.
    • Fig. S5. Changes in the lateral mobility of ATTO647N-PI(4,5)P2 upon addition of Gag on SLBs.
    • Fig. S6. Jurkat T cell coelectroporation with Gag.eGFP and Gag plasmids.
    • Fig. S7. Cumulative frequency distributions of diffusion coefficient observed in HIV-1–infected T cells.
    • Fig. S8. Cumulative frequency distributions of diffusion coefficient observed in HIV-1 Gag-transfected T cells.
    • Legend for movie S1
    • References (62, 63)

    Download PDF

    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.avi format). Drift stabilized time lapse movie of a representative NL4.3 Gag-iGFP HIV-1–infected Jurkat T-cell showing already present and newly developing virus assembly sites.

    Files in this Data Supplement:

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