Targeted HIV-1 Reservoirs Primary human monocyte-derived macrophages were infected with 76104 pg p24 of M-tropic HIV-1 BaL, which is MOI .5 as previously reported [5], and the infected macrophages were cultured for 12 days in various conditions shown in Figure 5. Under these culture conditions, more than 95% of the macrophages were infected at 12 days post infection as determined by the p24 intracellular staining (data not shown). The cells were then analyzed for live and dead phenotypes by staining with live (green) and dead (red) cell specific dyes. In this study, three concentrations of perifosine were used, 1, 5 and 20 mM, and 100 mM sodium nitroprusside (SNP) which is a nitric oxide producer, well known to be an HIV-1 related cellular stress [22]. As shown in Figure 5A and 5B, HIV-1 infected macrophages showed effective cell survival (green cells) for 12 days in the absence of SNP and perifosine (media alone), which is contrasted to the HIV-1 infected CD4+ T cells known to undergo cell death within a few days post infection. Under this culture condition, the basal level of death of HIV-1 infected macrophages was ,10%. Furthermore, HIV-1 infected macrophages displayed strong survival (green) with only a slight increase (,2-fold) of the basal cell death (Figure 5A and 5B) even in the presence of the 100 mM SNP stress which normally induces .90% cell death in the uninfected macrophages [19], confirming the cytoprotective phenotype of the HIV-1 infected macrophages. When the infected macrophages were treated with the three different concentrations of perifosine without SNP, the cells still exhibited only background cell death (Figure 5A and 5B), suggesting that perifosine does not impose nonspecific cytotoxicity on the infected macrophages at these concentrations. However, simultaneous treatment of perifo- sine and SNP to the culture revealed elevated death compared to the perifosine or SNP singly treated control cells (Figure 5A). Moreover, cell death increased in a dose-dependent manner with a rise in perifosine concentration in the presence of SNP (Figure 5B). Thus, the data in Figure 5 suggest that perifosine induces the death of HIV-1 infected macrophages, specifically when the cells were exposed to cellular stress which does not normally induce the death of the HIV-1 infected macrophages. Effect of ALPs on HIV-1 production in macrophages The data presented in Figure 5 demonstrate that perifosine induces death of HIV-1 infected macrophages when they are exposed to a level of cellular stress which alone does not kill the HIV-1 infected macrophages. Thus, finally, we tested if perifosine is able to reduce viral production from HIV-1 infected Figure 5. Anti-cytoprotective activity of perifosine in HIV-1 infected primary human macrophages. Primary human monocyte-derived macrophages isolated from blood donors were infected with HIV-1 Bal at MOI 5, and cultured for 12 days in the presence and absence of sodium nitroprusside (SNP, 100 mM) and/or several concentrations of perifosine. Note that 100 mM SNP induces nearly 95% death of uninfected human macrophages [5]. (A) Cells were stained with ethidium homodimer and calcein to distinguish between dead (red) and live (green) cells, respectively, on day 12. Images were taken with a fluorescence microscope, and the images shown (merged bright, red, and green fields) are representatives of 3 experiments conducted in duplicate. (B) Percent cell death was determined by counting dead and live cells. The values are averages of the duplicates for all conditions performed with macrophages isolated from three separate healthy blood donors. C: infection with no treatment, PF: perifosine. doi:10.1371/journal.pone.0013121.g005 PLoS ONE | www.plosone.org 6 September 2010 | Volume 5 | Issue 9 | e13121