The fraction of a releasable GFP-based content marker correlates with the extent of HIV-1 maturation.

<p>(A) Lysis of pseudoviruses co-labeled with Gag-iGFP (content marker, green) and the Cherry-ICAM-1 chimera (membrane marker, red). Pseudoviruses immobilized on a coverslip (left) were exposed to saponin at 37°C (0.1 mg/ml in HBSS, right). Scale bar is 8 µm. (B) The extent and kinetics of HIV-1 pseudovirus lysis. Gag-iGFP-labeled viruses produced in the presence of escalating doses of saquinavir (SQV) were immobilized onto coverslips and imaged. After the first three images, the acquisition was paused, saponin was added at the final concentration of 0.1 mg/ml, and imaging was continued for ∼5 min at 37°C. The total fluorescence of all iGFP-positive particles as a function of time was calculated for each experiment and the average fluorescence profile from 4–5 independent experiments were plotted (error bars are SEM). The fluorescence profile for 40 nM SQV-treated viruses is the mean from 2 independent experiments. (C) Pseudoviruses co-labeled with Gag-iGFP and DiD exhibit different degrees of iGFP release upon addition of saponin, from full or partial loss of content to no iGFP release. More than 200 particles were tracked for each SQV concentration. Red brackets show the total percentage of full and partial release events. Green circles show the fraction of free iGFP relative to the total amount of fluorescent protein obtained by densitometry analysis of bands shown in panel D. (D) The effect of escalating doses of SQV on HIV-1 Gag and Gag-iGFP cleavage. Pseudoviruses produced in the presence of indicated concentrations of SQV were analyzed by SDS-PAGE and blotted with antibodies to HIV-1 Gag/p24 (top panel) or with anti-GFP antibodies (bottom panel). The four intermediate bands on the GFP blot that run between Gag-iGFP and iGFP, likely correspond to MA-GFP-CA-NC, MA-GFP-CA, GFP-CA-NC and GFP-CA.