Inhibition of HIV-1 replication in U937 cells by lentivirus-mediated shRNA. (A) The structure of the shRNA lentiviral expression vector. The HIV-1–based lentivirus vector for expressing shRNA was constructed using Gateway technology. pCS-EG/shRNA consisted of U6-shRNA upstream of an EF1α promoter–driven EGFP expression cassette, which allowed simultaneous expression of shRNA and EGFP. (B) U937 cells were infected with lentivirus expressing either shNef366 (Lenti shNef366) or shLacZ (Lenti control) at an MOI of 1. After 2 hours of infection, cells were washed and maintained in culture. Cells expressing EGFP were analyzed by FACS, and EGFP⁺ cells were collected. EGFP⁺ cells were analyzed by FACSaria 1 week later (designated as U937/Lenti control and U937/Lenti shNef366). (C) U937/Lenti cont or U937/Lenti shNef366 cells (1 × 10⁵/well) were infected with HIV-1_NL432, and the culture supernatants of these cells were collected at 3- or 4-day intervals after infection. The level of p24 antigen in the culture supernatants was measured by ELISA. (D) HIV-1–infected cells were collected and total DNA was prepared 12 hours after infection. Total HIV-1 and 2LTR DNA was analyzed by qRT-PCR. The amount of HIV-1–specific DNA per cell was normalized to β-globin gene expression. The data represent the average ± SD of 3 independent experiments.