Fig. 6

HIV-1 carrying IN SIM mutants are replication defective at the early stages of viral replication, including reverse transcription, nuclear import and integration. a Schematic structure of HIV-1 RT and IN deletion provirus HIVΔRI/Gluc and the CMV-Gag-Pol-expressing plasmid harboring INwt, mutant M2, or M3. Single-cycle replicating viruses harboring INwt or mutants M2 and M3 were produced from cotransfected 293 T cells, collected by ultracentrifugation and normalized by virion-associated p24 levels. Then, equal amounts of each virus stock were used to infect C8166 T cells. At various time intervals, HIV-1 replication was determined by Gaussia luciferase assay (b) and HIV-1 p24 ELISA (c). To determine the step of viral replication that was affected, real-time quantitative PCR analysis was performed to detect the total HIV-1 DNA (d), 2-LTR circle (e) and integrated DNA levels (f) at various times post infection, as indicated