Generation of, and virus replication in, an H9 T-cell population stably expressing HIV-Env-TMD-CT. A. schematic representation of the SIN lentiviral vector pWPI-Env-TMD-CT employed. An internal human EF1-α promoter drives expression of a transcriptional unit consisting of the Env-TMD-CT gene, an IRES element and the gene for GFP. The composition of the Env-TMD-CT gene is described in the text. SP; signal peptide sequence of tissue plasminogen activator, Env-TMD; membrane anchor of HIV-Env, CT; cytoplasmic domain of HIV-Env. B. FACS for GFP expression (left panels) and indirect immunofluorescence analyses for Env-TMD-CT expression (right panels) of H9 T-cell populations stably transduced with pWPI (H9-GFP) and pWPI-Env-TMD-CT vector particles (H9-CT). Immunofluorescence of paraformaldehyde-fixed permeabilised H9 cells was performed with rabbit anti-gp160 serum shown to contain antibodies against the Env CT, followed by biotinylated goat anti-rabbit IgG and streptavidin phycoerythrin. Identical exposure times were used to generate the images from the H9-GFP and the H9-CT cells. C. Western blot analysis of H9 cells stably transduced with pWPI (H9-GFP) and pWPI-Env-TMD-CT (H9-CT) (left panel) and of cytosolic (C) and membrane (M) fractions of H9-CT cells (right panel) with gp41 Mab Chessie 8  as indicated. After stripping, the right blot was reprobed with rabbit antibodies specific for the cytoplasmic protein 14-3-3 γ (C-16) (Santa Cruz Biotechnology). D. Replication kinetics of Wt-pNL-4-3 (Wt) (filled-in symbols) and pNL-Tr712 virions (Tr712) (empty symbols) in H9 cells (circles), H9-GFP cells (triangles) and H9-CT cells (squares). Infections were initiated with 100 ng virus per 106 cells, produced by transfection of the respective plasmids in 293T cells. 5 h p.i., the cells were thoroughly washed and the course of infections followed by measurement of newly released HIV-CA in the supernatant by ELISA. E. Western blot analyses of equalised amounts (by CA-ELISA of culture supernatants) of lysates of H9-GFP and H9-CT cells infected with pNL-Δ Env (Δ), pNL-Wt (Wt) and pNL-Tr712 (712) (left) and of equalised amounts (by CA-ELISA of ultracentrifuged particles) of the respective virions released into the media (right). The top portions of the filters have been probed with anti-gp120 serum and the bottom portion with anti-CT antibodies (Chessie 8).