FIG. 2.
Selection of HIV by antisense in SupT1 cells. (A) Isolation of breakthrough wt HIV from antisense selective pressure can only be measured after challenge of SupT1 cells transduced at suboptimal doses of vector. SupT1 cells were transduced with vector at an MOI of 5, 10, or 20 TU/ml and then challenged with HIVNL4-3 at an MOI of 0.1. Virus production was measured by a p24 ELISA. (B) Schematic representation of selection procedure. SupT1 cells were mock transduced or transduced with vector at an MOI of 5 and then challenged with HIVNL4-3 at an MOI of 0.1. Culture supernatants were collected twice weekly and were tested for p24 protein levels in order to determine the point of peak replication. Culture supernatants from each group were collected during peak replication, normalized for p24, and subsequently used to infect naïve and transduced SupT1 cells for a total of three rounds of selection reflecting a selection period of 56 days. (C) After three rounds of selection, naïve SupT1 cells were infected with p24-normalized supernatants collected from the final passage of the third selection during peak replication. Supernatants were collected at the indicated times postinfection and assayed for p24 production by ELISA.