Nucleocapsid Promotes Localization of HIV-1 Gag to Uropods That Participate in Virological Synapses between T Cells
Figure 5
ML7 depolarizes cell morphology and Gag localization and reduces cell-to-cell transfer of Gag-YFP.
A) Transfer of Gag-YFP fluorescence from infected P2 cells to CMTMR-stained SupT1 target cells during a 3-h coculture period was measured by flow cytometry. ML7, DMSO, or antibodies, along with 10 µg/ml cycloheximide, were added at the beginning of the coculture period. Flow cytometry plots for CMTMR-labeled target cells co-cultured with uninfected cells as well as CMTMR-labeled target cells cocultured with Gag-YFP-expressing infected cells in the presence or absence of IgG or Leu3A are shown. Gate A, CMTMR-labeled target cells; gate B, double positive cells representing target cells with transferred Gag-YFP particles; and gate C, YFP-expressing cells either fused or conjugated to CMTMR-labeled target cells. B) Images of cycloheximide-treated P2 cells expressing Gag-YFP were acquired after 3-h coculture with CMTMR-stained SupT1 cells in the presence or absence of DMSO or ML7. Note that almost all the cells adopt round unpolarized morphology upon treatment with ML7 and that ML7-treated infected cells show dispersed Gag-YFP localization. The latter point is clearer in the higher magnification image (bottom panel) of a region specified in the middle row. Also note that the cell density of the cocultures in experiments shown in panel A (images shown in Figure S3 and discussed in Text S1) is 10 fold higher than in panel B. C) Relative efficiencies of cell-to-cell virus transfer were calculated as the percentage of double positive cells out of the total CMTMR-labeled cells (Virus transfer efficiency = B/(A+B+C)*100; error bars represent standard deviation). P values were determined using Student's t test. ***, P<0.001.