SIV Nef decreases cellular iron levels and attenuates lentiviral replication in macrophages. (A) THP-1 infected with HIV-1 expressing the indicated Nef proteins and eCFP via an IRES were stained with the green fluorescent dye CA-AM as described in the Methods Section. CA-AM is quenched upon the availability of chelatable iron within the cell. This decrease in fluorescence emission was assessed by FACS specifically in the HIV-1 infected and therefore eCFP positive population. Depicted is the increase in cellular chelatable iron relative to the 239 Nef expressing cells. Each symbol represents a single measurement from eight experiments with two to three independent virus stocks. (B) Primary macrophages from two donors were infected with 50 ng of normalized HIV-1 expressing the indicated Nef proteins. Aliquots of cell culture supernatants were taken in two to three day intervals and the amount of p24 production was measured by ELISA. The graphs show replication curves with mean values of four independent infections. (C) The cumulative p24 production is the absolute amount of released p24 during the culture period. Mean values and SEM for the two donors shown are calculated from the four independent infections. Similar results were obtained with macrophages from two other donors.