Figure 3.

Intrabifunctional marker FRET predicts HIV-1 protease activity. A–F, SQV-treated (SQV+, A–C) and untreated (SQV−, D–F) viruses labeled with mCherry-2xCL-YFP-Vpr were immobilized on coverslips and imaged by sequentially exciting with 488 nm and 594 nm light while detecting in both YFP and mCherry emission bands. YFP and mCherry intensity detected in 488-nm/YFP and 594-nm/mCherry channels, respectively, was measured before (A and D) and after (C and F) addition of saponin. Sensitized emission FRET intensity (B and E) was detected in the 488-nm/mCherry channel and corrected for cross-talk (see “Experimental procedures” and supplemental Fig. S2). G, single-particle distributions of acceptor-normalized FRET were measured for virus labeled with Gag-imCherry and for SQV+/− virus labeled with the bifunctional marker. H, scatterplot of normalized FRET versus uncleaved mCherry content fraction (determined as the mCherry signal remaining after addition of saponin) for SQV− virus labeled with the bifunctional marker. Single-particle normalized FRET strongly correlated with single-particle fraction of uncleaved mCherry content (Pearson correlation, ρ = 0.886). More than 600 particles were analyzed for each condition.