FIG. 7.

The I domain mediates Gag-Gag interactions. (A) Gag fusion constructs were expressed in 293T cells and harvested by Dounce homogenization. Whole-cell lysates were analyzed by fluorometry. The intensities of curves were normalized based on the amount of YFP expressed. The YFP-alone emission curve for each pair of constructs was subtracted from the curve of the CFP-YFP combination (i.e., MA-CFP-MA-YFP curve − MA-YFP-alone curve), resulting in an emission peak at 527 nm representing FRET with the background subtracted for each construct. (B) Gag384-CFP and Gag384-YFP were cotransfected in Mel JuSo cells, and images were obtained with a Zeiss LSM 510-Meta confocal microscope. The image represents YFP excitation-emission before photobleaching. (C) The same cell as that shown in panel B following photobleaching of the indicated regions at 514 nm. (D) Emission spectra were obtained from the selected regions of interest before (diamonds) and after (squares) photobleaching. The excitation wavelength for the scan was 458 nm. (E) Gag384(R380,384A)-CFP cotransfected with Gag384(R380,384A)-YFP. The image represents YFP excitation-emission. (F) The same cell as that shown in panel E following photobleaching at 514 nm. (G) Spectral data obtained from the indicated regions of panels E and F before bleaching (diamonds) and after bleaching (squares).