Robust signals during HIV-1 infection establish an inducible latent reservoir.

(A) Outline of experimental plan to enrich for latently infected cells following infection. Primary CD4+ T cells are infected with VSV-G pseudotyped BRU-ΔEnv-GFP and GFP-negative cells are sorted to enrich for latently infected cells. (B) Percent GFP+ HIV-expressing cells after stimulation of latent cells with various latency-reversing agents: PMA plus ionomycin, antibodies to endogenous CD3 and CD28, SBHA, and Bryostatin. Data are from seven separate donors as indicated by the color code of samples, and each LRA stimulation was performed on cells from 2–3 donors. A minimum of 250 events was required for inclusion as a data point. We had a range of 250 to 6000 events per data point depending on both donor variability and cell viability due to stimulation condition. The mean value for all data points was 1364 events and the median was 678 events. (C) Latently-infected cells were restimulated with PMA and ionomycin. HIV-1 expression was monitored by measuring Tat RNA by qRT-PCR. Values are shown as fold difference in HIV-1 transcripts over corresponding non-reactivated controls. All data in panel C are derived from 4–6 replicates and are representative of four independent experiments (see also S3 Fig). Data are presented as mean ± standard deviation. Statistical analysis performed using unpaired Student’s t test. *p<0.01, **p<0.001, ***p<0.0001.