FRET analysis of HIV‐1 Gag and GagPol interactions

The Gag protein of HIV multimerizes to form viral particles. The GagPol protein encoding virus‐specific enzymes, such as protease, reverse transcriptase, and integrase, is incorporated into HIV particles via interactions with Gag. The catalytically active forms of these enzymes are dimeric or tetrameric. We employed Förster resonance energy transfer (FRET) assays to evaluate Gag–Gag, Gag–GagPol, and GagPol–GagPol interactions and investigated Gag and Pol interdomains tolerant to fluorescent protein insertion for FRET assays. Our data indicated that the matrix (MA)–capsid (CA) domain junction in the Gag region and the Gag C terminus were equally available for Gag–Gag and Gag–GagPol interaction assays. For GagPol dimerization assays, insertion at the MA–CA domain junction was most favorable.