Generation and characterization of the GGR Affinofile Cell Line. (A) Schema of the tat-rev dependent Gaussia luciferase (gLuc)-IRES-GFP reporter vector as described in the text. (B) and (C) GGR cells were maximally induced with doxycyline (Doxy, 4ng/ml) and ponasterone A (PonA, 4 μM) at the time of their seeding in 96-well plates. 16–21 hours post-seeding/induction, cells were infected with wt JR-CSF virus at varying multiplicities of infection (MOI). The titer of the virus was previously determined on stable CD4/CCR5-expressing GHOST cells where CD4/CCR5 levels are non-limiting. At 17, 24, 48, and 72 hpi, 10 μL (out of 150) of the infected cell supernatant was removed and analyzed for gLuc activity as per manufacturer’s instructions. Luciferase activity (measured as relative light units, RLU), and the corresponding signal:noise ratios at each data point are shown in (B) and (C), respectively. Mock-infected cell supernatant served as the background signal. (D) and (E) GGR cells were induced at high (3.2ng/mL Doxy, 2 μM PonA), medium (1.6ng/mL Doxy, 1μm PonA), and low (0.4ng/mL Doxy, 0.25μM PonA) levels, and infected as above with pseudotyped virus at an MOI of 0.25. Three days post-infection, supernatant was collected and analyzed for gluc expression (E), while cells from each well were individually processed for intracellular p24 staining (D) as described in methods. Data shown is representative of two independent experiments.