Figure 4

Single cycle infection experiments. A. ATG knockdown cells were infected with HIV-1 for 4 h, excess virus was washed away and new infections were prevented by addition of the fusion inhibitor T1249. The percentage of CA-p24 positive cells was measured at 48 post infection. The live cell population was determined by forward and side scatter (left panels). Cells were intracellularly stained for CA-p24 with a specific antibody labeled with RD1 (right panels). Mock infected SupT1, infected SupT1 and infected shAtg5 cells are shown as examples. B. FACS data for all tested cells. The percentage of CA-p24 positive cells was measured at 48 h post infection by FACS (left panel). The mean production of CA-p24 per positive cell is represented as the mean fluorescence intensity (middle panel). The concentration of CA-p24 in the culture supernatant was determined by ELISA (right panel). C. To test whether indeed early HIV-1 replication steps were inhibited, the Reverse Transcriptase 3TC drug and the Integrase inhibitor Raltegravir (RAL) were tested in single cycle infection experiments. D. SupT1 cells were either mock treated or incubated with 3-MA either starting 4 h before infection or for 48 h post infection, or both. Cells were analyzed for the percentage of CA-p24 positive cells (left panel), MFI (middle panel) and CA-p24 concentration in the culture supernatant (right panel). All single cycle infections were performed three times and per experiment every infection was performed in triplicate. A representative experiment is shown, bars represent the mean and error bars the standard deviation from the mean.