HIV-1 is released from infected cells as an immature particle consisting of viral RNA and Gag and Gag-Pol polyproteins. At some point during/after virus budding from the plasma membrane, the viral protease (PR) autoactivates by cleaving itself off Gag-Pol and proceeds to cleave Gag and Gag-Pol at multiple sites and in defined order. These carefully orchestrated cleavage events culminate in the formation of a mature cone-shaped viral core. Although the timely PR activation is critical for the formation of mature infectious HIV-1, the mechanism and timing of PR autoactivation remain poorly defined. We are implementing HIV-1 labeling and imaging approaches to visualize single virus assembly, PR autoactivation and substrate cleavage by total internal reflection fluorescence microscopy. These approaches rely on FRET probes in which fluorescence donor and acceptor are linked by distinct PR cleavage sites from Gag and Gag-Pol (Figure 3 and Movie 2). Knowledge of the mechanism and regulation of PR autoactivation can inform new therapeutic strategies.
