macrophages in the presence of SNP stress. Primary macrophages were infected with a high MOI of M-tropic HIV-1BaL, and monitored viral production by a p24 ELISA assay after 12 days of culture in the presence or absence of perifosine (20 mM) and SNP (100 mM). As shown in Figure 6, SNP treatment reduced viral production only by ,50%, which is consistent with the previous report that the SNP treatment usually decreases viral production 50–70% while the cell death assay showed ,5% death of the HIV infected macrophages (Figure 5) [5]. Additionally, 20 mM perifosine alone only slightly affected viral production. However, when HIV-1 infected macrophages were exposed to both perifosine and SNP, a 9-fold reduction of viral production was observed, which should be a consequence of the significant cell death in the HIV-1 infected macrophages (Figure 5). Therefore, these data observed with primary human macrophages and infectious HIV-1 support the idea that perifosine effectively reduces HIV-1 production by antagonizing the stress-induced cytoprotective phenotype of the HIV-1 infected macrophages. Discussion Targeting host factors that are utilized for the life cycle of HIV- 1 is a highly attractive anti-viral strategy. Unlike current anti-viral treatments targeting viral proteins, such as reverse transcriptase, protease and integrase, this strategy can minimize viral escape. One major obstacle in targeting host factors is that many which are utilized by HIV-1 are also essential to the host. Thus the conditionally required or non-essential host factors are better candidates as anti-HIV-1 targets. Indeed, the Akt cell survival pathway is conditionally activated only upon exposure to cytotoxic insults or in abnormal cells that require exceptional survival techniques, such as cancer cells. Figure 6. Inhibition of HIV-1 production from primary human monocyte-derived macrophages by perifosine. Primary human monocyte-derived macrophages were infected and cultured for 12 days in the presence and absence of sodium nitroprusside (SNP, 100 mM) and/or perifosine (20 mM), and the p24 levels of the supernatants on the 12th day of the culture were determined. The p24 level at day 12 for each condition was normalized by the p24 level (86 pg/ml) of the media only control (no treatment). doi:10.1371/journal.pone.0013121.g006 Targeted HIV-1 Reservoirs Importantly, it is well known that HIV-1 infected macrophages and microglia establish toxic extracellular environments by persistently secreting viral cytotoxic proteins (e.g. gp120 and Tat) and chemicals (e.g. NO), and this toxic environment induces death of nearby neurons in the CNS, leading to HIV-1 associated neurodegenerative diseases [2]. Ironically, even with the establishment of extracellular cytotoxic conditions HIV-1 infected macrophages exhibit an extended life span, (unlike infected CD4+ T cells which undergo cell death) and serve as long-term HIV-1 reservoirs [1,19,23]. Thus it is highly plausible that the pro-activation capability of the stress-induced PI3K/Akt cell survival pathway by HIV-1 infection can effectively protect the infected macrophages from the toxic local environments generated by their own proteins and chemicals, leading to long- lived viral reservoirs. In this report, we demonstrated that the ALP compounds, edelfosine, perifosine and miltefosine, were able to inhibit HIV-1 induced Akt activation, leading to the death of HIV-1 expressing macrophages and CHME5 cells. However, edelfosine treatment of primary human macrophages proved to be significantly cytotoxic at concentrations as low as 1 mM (data not shown). The precise mechanism of action of ALP compounds against Akt kinase remains unclear, though multiple reports demonstrated that the compounds reduce Akt activation in tumor tissue culture models [13,14,15,16]. While our study focused on the inhibitory function of ALP compounds against activation of the Akt cell survival pathway, we do not exclude a possibility that the inhibitory effect of these two ALP compounds on the cytoprotective phenotype of HIV-1 expressing macrophages and CHME5 cells may also result from other closely related cellular events such as the activation of the apoptotic pathway and inhibition of the anti-apoptotic pathway. In fact, whether HIV-1 infection mechanistically affects apoptosis-related events in macrophages has not been tested, presumably because unlike CD4+ T cells undergoing cell death following G2 arrest, the cell death phenotype of HIV-1 infected macrophages is absent (these cells actually live longer). Since we employed ethidium homodimer which primarily stains necrotic cells, not living or apoptotic cells, our study monitored the necrosis rate of the HIV-1 infected macrophages upon exposure to the ALP compounds. Indeed, the effect of HIV-1 infection as well as the ALP treatment on apoptosis and anti-apoptosis can be investigated in the future. In addition, while our data demonstrate that the ALP compounds effectively inhibit the HIV-1 and Tat- induced Akt activation/phosphorylation in the CHME5 model, it still remains to be confirmed that the ALP compounds also block Akt phosphorylation in HIV-1 infected macrophages. This is mainly due to technical issues: we found that the primary human macrophage lysates, particularly, after ALP treatment, were not applicable to various methods of Akt activity including western analysis. The ALP compounds investigated in this study are known to be well-tolerated in various pre-clinical trials [24] and to do little harm in normal cells in various in vitro models; this could be due to the fact that normal cells do not activate the PI3K/ Akt pathway unless they are exposed to extracellular insults. Importantly, many studies, especially from anti-Leishmania research, demonstrated that ALP compounds are effectively able to penetrate the blood-brain barrier (BBB) [25,26]. Considering that HIV-1 infected macrophages and microglia in the brain play a key role in HAND, this BBB penetration feature of ALP compounds is very important in terms of their potential clinical application. However, it is clear that further investigations and evaluations of the ALP compounds as anti-HIV agents need to be conducted. PLoS ONE | www.plosone.org 7 September 2010 | Volume 5 | Issue 9 | e13121