HIV-1-infected T cell lines.

The following reagents were obtained through the NIH AIDS Reagent Program, Division of AIDS, NIAID, NIH: J-Lat full-length clones (clones A1, A7, 82, 6.3, and 8.4) from Eric Verdin (38). J-Lat cell line 11.1 was kindly provided by Emilie Besnard (from Eric Verdin’s laboratory at the Gladstone Institute, San Francisco, CA). These clonal cell lines were derived from the CD4⁺ T lymphoblastoid Jurkat parental cell line, and each of them contains a latent HIV-1 provirus in a single integration site (79). The lines A1, A7, and 82 harbor a mini-HIV-1 cassette that contains only the Tat and green fluorescent protein (GFP) open reading frames (ORF) under the transcriptional control of the 5′ long terminal repeat (5′ LTR). In contrast, J-Lat 6.3, 8.4, and 11.1 cells carry a full-length integrated HIV-1 genome with a nonfunctional Env due to a frameshift, and the nef gene was replaced by the GFP ORF. In all the J-Lat clones, HIV-1 is replication incompetent and the genomic site of integration is associated with their susceptibility to HIV-1 reactivation (38).

All J-Lat lines were grown in 5% CO₂ in 1640 RPMI (Gibco) supplemented with 25 mM HEPES, 10% heat-inactivated fetal bovine serum (FBS; Nucleus Biologics), 2 mM l-glutamine (Gibco), and 100 U/ml penicillin and 100 μg/ml streptomycin (Gibco). The cells were maintained at 37°C in a humidified incubator with 5% CO₂ and split every other day (generally at a 1:4 or 1:5 ratio) to an approximate density of 2.5 × 10⁵ cells/ml. Under these basal culture conditions, little (A1, A7, and 11.1 cells) or no (82, 6.3, and 8.4 cells) GFP can be detected by flow cytometry (Fig. 1C, open squares).