Background: Gag multimerization is both necessary and sufficient for retroviral particle assembly and release. Although interactions between Gag molecules have been shown using a variety of techniques, the initial site of these interactions remains unknown. We hypothesize that initial Gag-Gag interactions occur at a specific intracellular membrane site.

Methods: Detection and analysis of intracellular Gag-Gag interactions was determined by using both Fluorescence Resonance Energy Transfer (FRET) microscopy and FRET fluorometry.

Results: Laser confocal microscopy shows Gag-GFP localized primarily in punctuate regions at the plasma membrane. In addition, Gag-YFP and Gag-CFP fusion proteins used in FRET-based scanning fluorometry experiments show Gag-Gag interactions to be present in cellular membrane fractions but absent in cytosolic fractions. Using FRET microscopy, we have shown Gag-Gag interactions not only at the plasma membrane, but also at intracellular sites.

Conclusions: We have found intracellular interactions between Gag molecules using fluorescence resonance energy transfer, a novel assay for Gag-Gag interactions. FRET microscopy, in combination with fluorescent markers that target specific membrane compartments within the cell, can be used to determine the identity of these intracellular compartments and dissect the initial steps of the retroviral assembly process. These findings will also allow the utilization of a FRET-based assay to screen for possible inhibitors of Gag-Gag interactions and the HIV assembly process.