Fig. 1.
Isolation of single-cell clones from the H9MN cell line. (A) Schematic diagrams of the strategies used to isolate H9MN single-cell clones harboring defective proviruses. Flow cytometry plot of HIV-1 MN cells showing the expression of intracellular Gag p24 protein (clone: KC-57) and cell surface Env gp120 (clone: 447–52D) protein. The H9MN cells were flow-sorted into three populations based on cell surface expression of Env gp120 protein and CD3: gp120hi, gp120int, and gp120lo. The flow-sorted cells were serially diluted in 96-well plates and left to grow to a confluent status (2 weeks). Cells from the wells exhibiting growth (visually inspected by microscopy) were transferred to 48-well plates. A small aliquot of cells from the wells in the 48-well plates was taken to detect the presence of HIV-DNA by 5′LTR-to-3′LTR PCR. Cells from wells positive for HIV-DNA were further expanded in 24-well plates for an additional week. Upon completion of the expansion culture, one portion (1 × 106) of the cells was used for simultaneous extraction of DNA and RNA (after cDNA synthesis), followed by 5′LTR-to-3′LTR single-genome amplification and direct amplicon sequencing; the other portion (2 × 106) of the cells was used for Western blot and confocal microscopy (1 × 106 cells) for detection of HIV-1 proteins. (B) Proviral sequences present in the three sorted fractions of H9MN cells.