FIG 4.

The binary latency trajectory of the GFP^High population delineates the viral promoter into strong (3- and 4-κB) and weak (2-, 1-, and 0-κB) LTRs. (A) Experimental schemes to study the kinetics of latency establishment in EGFP^High and d2EGFP^High cells. (B to E) Comparative kinetic profiles of EGFP MFIs, percentages of EGFP⁺ cells, d2EGFP MFIs, and percentages of d2EGFP⁺ cells, respectively. Mean values from experimental triplicates ± SD are shown and are representative of results from two independent experiments. Two-way ANOVA with Bonferroni posttest correction was used for the statistical evaluation (***, P < 0.001; ns, nonsignificant). (F) Stacked histogram profiles of EGFP^High cells during latency establishment. The sorted EGFP^High cells (GFP^H) (MFI, >10⁴ RFU), comprising a homogeneous population of Tat-mediated transactivated cells, transitioned to the EGFP⁻ phenotype (GFP⁻) through an EGFP^Low cluster (GFP^L) (MFI, ∼10² to 10⁴ RFU), representing cells with a basal level of transcription without an intermediate phenotype. No Inf, Jurkat cells not infected. (G) Stacked histogram profiles of d2EGFP^High cells during latency establishment identify regions of d2EGFP⁻ (GFP⁻) (MFI, <10³ RFU), d2EGFP^Low (GFP^L) (MFI, ∼10³ to 10⁴ RFU), and d2EGFP^High (GFP^H) (MFI, >10⁴ RFU) phenotypes. Of note, given the shorter half-life of d2EGFP, the stability of the d2EGFP^Low phenotype was extremely transient; hence, the present system lacked a distinct d2EGFP^Low cluster at any time point, unlike in the EGFP system (Fig. 3C).