Fig. 1. Enrichment and purification of HIV-latently infected cells by FACS. (A) A schematic representation of our enrichment protocol is shown. See text for details. The percentage of GFP-positive cells obtained after infection (4%) or after TNF-α treatment of GFP-negative cells (0.06%) is shown. Similar data were obtained with TPA. (B) Clonal cell lines isolated using the procedure described above were analyzed for GFP expression under basal and stimulated conditions (24 h treatment with TNF-α). (C) mRNA levels were measured for HIV and for GAPDH using TaqMan PCR in untreated and TNF-α-treated cell lines. Results are expressed as a percentage of mRNA levels measured in Jurkat cells acutely infected with HIV_NL4-3 (day 6 post-infection). Clone A72 is infected with an LTR–GFP construct (Jordan et al., 2001), clone H2 is infected with the LTR–Tat–IRES–GFP vector while clones F11 and G10 are infected with HIV-R7/E^–/GFP.

Fig. 3. Latency is associated with preferred integration in or near alphoid repeats. (A) The sequence of integration site of the HIV vector is shown for seven clonal cell lines aligned with the corresponding genomic sequence. The match in GenBank for each clone corresponded to DDBJ/EMBL/GenBank accession number M93288 for clone 82, AL591625 for clone A1, AC023948 for clone A5, M16037 for clone A7, Al354920 for clone A10, AC019063 for clone H2 and AC079801 for clone F2. The sequence corresponding to the HIV promoter is indicated by a closed box. The chromosomal location of each integration site is shown and integration sites into alphoid repeat elements are indicated. (B) A nested PCR assay designed to quantify integration in or near alphoid repeats is schematically represented. The position of primers in the two sequential PCRs are shown aligned with the HIV 5′ LTR and a putative alphoid element in the genome. (C) Four PCRs were conducted for each cell line using either primer α1, α2, α3 or α4 in combination with primer A for the first reaction and the primer pair B+C for the second PCR. PCR products amplified according to this scheme are shown for each latent cell line. (D) Real-time PCR analysis of integration in or near alphoid repeats. Primer G represents an internal fluorescent primer used for the quantification of the TaqMan reaction. (E) Quantification of PCR products after TaqMan PCR analysis as indicated in (D). Productively infected cells (GFP-positive cells in Figure 1A, panel 1; productive infection) are compared with latently infected cells (Figure 1A, panel 4; latent infection) using the Alu and alphoid PCR assay. Data is expressed as the relative signal intensity (alphoid/Alu). Numbers on top of bar indicate the ratio between latent and productive infection.

Fig. 5. Establishment of latently infected cell lines with a full-length HIV provirus. (A) Genome organization of a molecular clone of HIV encoding GFP and containing a frameshift mutation in env. (B) Schematic representation of protocol for enrichment of latently infected cells after infection of Jurkat cells with HIV-R7/E^–/GFP (see text for details). (C) Clonal cell lines isolated using the procedure described above were analyzed for GFP expression under basal and stimulated conditions (24 h treatment with TNF-α). (D) Western blot analysis of four representative Jurkat clones latently infected with HIV-R7/E^–/GFP. Clones were treated for 24 h with TNF-α (10 ng/ml) and cell lysates were analyzed by western blotting using an antiserum from an HIV-infected individual (provided by the NIH AIDS Research and Reagent Reference Program). A predominant band of 55 kDa corresponds to the Gag precursor protein. The same samples were analyzed using an antiserum specific for α-tubulin to ensure equal loading.

Fig. 2. Characterization of latently infected clones. (A) Clone 82 is competent for basal and Tat-dependent HIV promoter activity. Clone 82 was infected with viral particles containing HIV-derived vectors LTR–GFP or LTR–Tat–IRES–GFP, and GFP expression was measured by flow cytometry. As a control, Jurkat cells were infected in parallel. (B) The integrated latent HIV promoter in clone 82 is unresponsive to Tat stimulation. Clone 82 was infected with a Tat-expression retroviral vector, or treated with TPA, as a positive control. As a control of the infection process and Tat expression, a clone containing a single integration of the HIV-derived LTR–GFP vector was infected in parallel. (C) HIV promoter activity (percentage of GFP-positive cells in R2 gate) was measured in four latent clonal cell lines (82, H2, A2 and A10) 24 h after infection with retroviral particles containing the following vectors: LTR–GFP, LTR–Tat, LTR–Tat–GFP or after treatment with TNF-α. Control Jurkat cells and a cell line containing a stably integrated LTR–GFP retroviral construct (Jordan et al., 2001) were used as controls.

Fig. 4. Transcriptional activation of the HIV promoter in latently infected cells. Cells from clones 82, A1, A7 and A10 were treated as described in Materials and methods with several indicated agents and LTR expression was measured by flow cytometry. Data are expressed as percentage of cells becoming GFP-positive after a 24 h treatment.