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Fig. 1 | Retrovirology

Fig. 1

From: HIV-1 latency and virus production from unintegrated genomes following direct infection of resting CD4 T cells

Fig. 1

Kinetics of responsiveness to activating agents following initial infection of resting CD4 T cells. a Experimental design. After infection, samples of the IL-4-treated resting CD4 T cells were maintained in culture with or without raltegravir (RAL) for the indicated time period and were then stimulated by αCD3/CD28 activation beads or prostratin plus trichostatin (Pro/TSA) for 2 days before analysis by flow cytometry. IL-4 was replenished every 7 days. Raltegravir was added on the day of infection and on days 3, 7, 14 and 21. b Emergence of GFP+ productively infected cells. Pro/TSA or αCD3/CD28 beads were added 2 days prior to analysis. Only the GFP+ cells are displayed, and the areas under the curves represent the number of GFP+ cells present in each sample. Red arrows indicate subpopulations which we have previously shown to contain predominantly unintegrated HIV-1 (uDNA) or to contain at least one copy of integrated HIV-1 DNA (iDNA) [6]. One representative of 5 experiments is shown. Additional file 1: Fig. S1A in presents a similar experiment performed using an Int-D116N integrase active site mutant. c Percentage of cells that were GFP+. Experiment was performed in triplicate. To account for proliferation of GFP-negative cells, %GFP+ was calculated as the number of GFP+ cells divided by the number of total live cells, which was adjusted for proliferation using the Expansion Index in the Proliferation Platform of FlowJo 9. Cell divisions were measured by eFluor670 dilution. Typically ≤16 % of cells underwent division through day 14, with essentially all of them GFP-negative (Additional file 1: Fig. S4A). d Cell populations predominantly expressing HIV-1 from unintegrated (GFPlow) or integrated HIV-1 (GFPhi) can be parsed in αCD3/CD28 activated T cells by accounting for cellular heterogeneity (side scatter [SSC] profile). Equivalent results were obtained utilizing the Int-D116N active site mutant (Additional file 1: Fig S1B)

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