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. 2012 Oct;86(20):11194–11208. doi: 10.1128/JVI.00301-12

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.

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Fig 2 — HIV-1 Gag colocalizes with HERV-K Gag, but not MLV Gag, at the PM. HeLa cells coexpressing YFP-tagged (green) and mRFP-tagged (red) Gag proteins of different retroviruses were fixed, stained with DAPI, and examined using fluorescence microscopy at 16 h after cotransfection (A to C). Top panels, Images acquired at the midsection of the cells. Bottom panels, Images acquired at the top of the cells. Bars, 10 μm. (D) The R strength of correlation between fluorescence intensities of pairs of the indicated Gag-fluorescent protein chimeras was calculated for cells coexpressing these Gag proteins. Data from 21 to 27 cells are shown as means ± standard errors of the means (SEM). P values were determined using Student's t test. ***, P < 0.0001. (E) HeLa cells were cotransfected with plasmids encoding HERV-K Gag-YFP and HIV-1 Gag-mRFP. The supernatants of cotransfected HeLa cells were concentrated by ultracentrifugation at 16 h posttransfection. Particulate materials positive for fluorescently tagged Gag in the supernatants were examined with a fluorescence microscope as described in Materials and Methods. (F) HeLa cells were separately transfected with plasmids encoding HERV-K Gag-YFP and HIV-1 Gag-mRFP. The supernatants of transfected HeLa cells were mixed and concentrated by ultracentrifugation at 16 h posttransfection. Particulate materials positive for fluorescently tagged Gag in the supernatants were examined with a fluorescence microscope as described in Materials and Methods. Magnified views of boxed areas in the top-row images are shown in the bottom rows in panels E and F. Arrowheads indicate foci showing both green and red fluorescence signals. Bars, 10 μm. (G) The R strength of correlation between fluorescence intensities of HERV-K Gag-YFP and HIV-1 Gag-mRFP was calculated. Data from 15 images are shown as means ± SEM. P values were determined using Student's t test. ***, P < 0.0001. (H) The foci containing YFP only, mRFP only, or both YFP and mRFP were counted, and proportions relative to total Gag-positive foci were calculated. Data from three independent images are shown as means ± standard deviations. P values were determined using Student's t test. *, P < 0.01; n.s., not significant.

Fig 2 — HIV-1 Gag colocalizes with HERV-K Gag, but not MLV Gag, at the PM. HeLa cells coexpressing YFP-tagged (green) and mRFP-tagged (red) Gag proteins of different retroviruses were fixed, stained with DAPI, and examined using fluorescence microscopy at 16 h after cotransfection (A to C). Top panels, Images acquired at the midsection of the cells. Bottom panels, Images acquired at the top of the cells. Bars, 10 μm. (D) The R strength of correlation between fluorescence intensities of pairs of the indicated Gag-fluorescent protein chimeras was calculated for cells coexpressing these Gag proteins. Data from 21 to 27 cells are shown as means ± standard errors of the means (SEM). P values were determined using Student's t test. ***, P < 0.0001. (E) HeLa cells were cotransfected with plasmids encoding HERV-K Gag-YFP and HIV-1 Gag-mRFP. The supernatants of cotransfected HeLa cells were concentrated by ultracentrifugation at 16 h posttransfection. Particulate materials positive for fluorescently tagged Gag in the supernatants were examined with a fluorescence microscope as described in Materials and Methods. (F) HeLa cells were separately transfected with plasmids encoding HERV-K Gag-YFP and HIV-1 Gag-mRFP. The supernatants of transfected HeLa cells were mixed and concentrated by ultracentrifugation at 16 h posttransfection. Particulate materials positive for fluorescently tagged Gag in the supernatants were examined with a fluorescence microscope as described in Materials and Methods. Magnified views of boxed areas in the top-row images are shown in the bottom rows in panels E and F. Arrowheads indicate foci showing both green and red fluorescence signals. Bars, 10 μm. (G) The R strength of correlation between fluorescence intensities of HERV-K Gag-YFP and HIV-1 Gag-mRFP was calculated. Data from 15 images are shown as means ± SEM. P values were determined using Student's t test. ***, P < 0.0001. (H) The foci containing YFP only, mRFP only, or both YFP and mRFP were counted, and proportions relative to total Gag-positive foci were calculated. Data from three independent images are shown as means ± standard deviations. P values were determined using Student's t test. *, P < 0.01; n.s., not significant.