HIV-1 Nef and Env expression in individual cells at various phases of the cell cycle. A) HIV-1 protein expression was monitored by flow cytometric analysis. OM10.1 cells were washed in Hanks' balanced salt solution, fixed in 70% ice-cold ethanol, and stained with mouse anti-human monoclonal antibodies to cyclin E, cyclin A, Nef, Env, or a non-specific immunoglobulin (DAKO) overnight. Cells in G1 and G2 were identified by DNA content and divided the intervening region into 10 to 12 equal increments to allow the calculation of mean protein-associated fluorescence and mean DNA content. The exact same analysis was performed on the appropriate aliquot stained with non-specific immunoglobulin. Protein fluorescence was determined by subtracting the mean from the nonspecific samples. Samples were washed in PBS-BSA and stained with a fluorescein isothiocyanate-labeled goat anti-mouse secondary antibody (1:30; DAKO) for 30 min in the dark. The bottom of panel A shows HIV-1 gene expression in G0, early G1, G1/S, S, and G2 phases of cell cycle by RT-PCR with primers for Env and Nef [58, 59]. B) Cell cycle analysis of cells after G0 release. Cells were synchronized at G0 by serum starvation and samples were removed from the medium at each time point, washed with PBS without Mg2+ or Ca2+, fixed with 70% ethanol, and stained with propidium iodide followed by cell-sorting analysis on a Coulter EPICS cell analyzer. The FACS profile for each time point is presented along with the percentage of cells in G1, S, and G2 (upper right side boxes of each histogram).