High level of nuclear MS RNA in CCL19-treated latently infected CD4+ T-cells. (A) Schematic diagram of the broad classes of HIV mRNA, including multiply spliced (MS) singly spliced (SS) and unspliced (US) HIV RNA (adapted from ). Location of primers and probes used for real-time PCR quantification of US, SS and MS RNA are shown. (B) Resting CD4+ T cells were activated with IL-2/PHA, CCL19 or unactivated and infected with NL4.3 or NL4.3Δenv. The fold increase in US RNA at day 4 post infection (relative to day 0; left panels) and the absolute copies per million cells of MS RNA (right panels) is shown for (B) total RNA following infection with WT NL4.3 (n = 5) and (C and D) nuclear (open) and cytoplasmic (grey) fractions following infection with (C) NL4.3 and (D) NL4.3Δenv. The mean (column) and individual data (open symbols) are shown. The ratio of MS RNA to integrated DNA for each donor is also shown (C and D, far right panel). The detection limit for US RNA and MS RNA was 200 copies/106 cells and is shown as dashed line for MS RNA. * = P < 0.05.