Resting primary CD4⁺ T cells support both productive and latent HIV infection.

<p>(A) Diagram of the HIV Duo-Fluo I virus, in which eGFP has replaced the <i>nef</i> gene, and a whole transcription unit—consisting of an EF1α promoter driving the expression of an mCherry fluorescent marker—has been inserted downstream. Upon infection with the HIV Duo-Fluo I virus, cells that express GFP alone or GFP and mCherry are considered productively infected; cells that express only mCherry are considered latently infected; cells that lack expression of either fluorescent marker are considered uninfected. (B) Expression of the activation markers CD69 and CD25 in resting primary CD4^<b>+</b> T cells either left untreated or stimulated with CCL19, IL-7, or αCD3/αCD28 activating beads for 72 h. Mean Fluorescence Intensity (MFI) for CD25 expression is also shown. (C) Infection profiles of untreated or stimulated primary CD4^<b>+</b> T cells 6 days after infection via flow cytometry. Untreated resting CD4^<b>+</b> T cells were infected with either HIV Duo-Fluo I virus alone or the Vpx-containing HIV Duo-Fluo I virus. Stimulated cells were infected with HIV Duo-Fluo I alone. Productive infection (GFP+ and GFP/mCherry double-positive) and latent infection (mCherry+) were analyzed by flow cytometry. Data shown are from a single donor but are representative of three separate donors. (D) Quantified values of latent infection and productive infection from panel C. Data represents the average of three donors. (E) Ratios of latent infection to productive infection were calculated using data from panel D. Data represent the average of three donors. (F) Quantified values for reactivation of pre-integration latent virus and post-integration provirus calculated from the isolated uninfected populations (GFP/mCherry double-negative) of untreated and stimulated primary CD4^<b>+</b> T cells via flow cytometry (<a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004955#ppat.1004955.s004" target="_blank">S4 Fig</a>). Six days after infection, uninfected cells were isolated via fluorescence-activated cell sorting (FACS) and were either left unstimulated or stimulated with αCD3/αCD28 activating beads alone or αCD3/αCD28 activating beads in the presence of raltegravir for 48 h. Reactivatable pre-integration latent virus was calculated by subtracting the amount of productive infection from cells treated with αCD3/αCD28 activating beads alone and cells treated with αCD3/αCD28 activating beads in the presence of raltegravir. Reactivatable post-integration latent provirus was calculated by subtracting the amount of productive infection from unstimulated cells and cells treated with αCD3/αCD28 activating beads in the presence of raltegravir. Data represent the average of three donors. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, non-significant.