the PI3K/Akt cell survival pathway in primary human macro- phages and renders these cells resistant to cytotoxic insults [5]. In normal cells without exposure to cellular insults, this pathway remains inactivated by its negative regulator, PTEN [6]. We also demonstrated that the HIV-1 induced cytoprotection is initiated by the expression of an HIV-1 accessory protein, Tat, which lowers the PTEN level in infected macrophages and a human microglia cell line, CHME5 [5]. In the absence of negative regulation of the PI3K/Akt pathway by PTEN, HIV-1 infected macrophages may proactively react to stressful cytotoxic extracel- lular environments established by the virus-induced chemicals, ultimately elevating their chance of survival. The PI3K/Akt pathway is also commonly activated in many cancer cells and promotes their survival and outgrowth [7]. Genetic inactivation of the PTEN function, which is oncogenic, activates the PI3K/Akt pathway [8,9]. Due to the direct mechanistic connection between activation of the PI3K/Akt pathway and cancer, various attempts to screen inhibitors of key enzymes of the pathway such as PI3K and Akt kinases, have been made [10,11]. Among them, alkylphospholipds (ALPs) have been tested for their anti-cancer effect, and indeed miltefosine was recently found to be effective in the treatment of cutaneous breast cancer metastases [12]. ALPs, also termed antitumor lipids, have been found to display anti-neoplastic effects in various tissue culture and small animal models [11,13, 14,15,16]. Their mechanisms of action involve regulation of multiple cellular events related to cell proliferation, cell death and cell survival [13,15,16,17]. Our recent study reported that miltefosine is able to prevent HIV-1 infected macrophages from activating the PI3K/Akt pathway, and specifically induces death of HIV-1 infected macrophages upon exposure to insults, ultimately terminating viral production [5]. However, the mechanism by which miltefosine counteracts the PI3K/Akt pathway in HIV-1 expressing macrophages remains to be explored. In this report, we investigated whether the ALP compounds, perifosine and edelfosine, can specifically inhibit the activation of Akt kinase activity in HIV-1 expressing macrophages. Indeed, we demonstrate that perifosine and edelfosine inhibit both the activation of Akt and the kinase activity of Akt in HIV-1 expressing human microglia cell lines, and perifosine effectively induces the death of HIV-1 infected macrophages. Methods Cells, plasmids, and viruses Primary human monocyte-derived macrophages were isolated from human buffy coats (New York Blood Center) and differenti- ated as previously described [5]. M-tropic HIV-1 BaL was provided from Zeptometrix (Buffalo, NY) and VSV-G pseudotyped HIV-1 vector (DHIV-GFP) expressing EGFP and all HIV-1 proteins except Nef and Env were prepared as described [5]. Vector titers were determined with CHME5, a human fetal microglia cell line [18], and the p24 ELISA was performed for each vector or virus preparation following the manufacturer’s protocol. pTat101 is a pcDNA3.1+Hygromycin (Invitrogen) derivative expressing a 101 amino acid HIV-1 Tat cDNA derived from the YU-2 M-tropic HIV-1 strain [19]. A CHME5 subline that stably expresses full- length Tat (pTat101) and a control cell line (pCDNA3.1-Hygro) were previously selected [19]. The concentrations of cycloheximide (CHX) and E. coli serotype O26:B6 lipopolysaccharide (LPS) (Sigma) used were described in Figure legends. Cell survival assays CHME5 cells were exposed to LPS (50 mg/ml)/CHX (10 mg/ ml), and sodium nitroprusside (SNP) (Sigma) was used at 100 mM Targeted HIV-1 Reservoirs for treatment of macrophages. Two methods were employed to monitor the dead cell populations. First, the CHME5 sublines were trypsinized and mixed with 0.4% Trypan blue solution (Gibco) at a 1:1 dilution, and the number of blue (dead) cells was calculated. Second, CHME5 cells and macrophages transduced by the HIV-1 vector expressing GFP were analyzed with ethidium homodimer included in the Live/Dead-Viability/Cytotoxicity kit (Molecular Probes) to stain the dead cells as per the manufacturer’s protocol. Images were taken at a 1006 magnification at 24 hours post-treatment using a fluorescence microscope (Zeiss). Each assay was performed at least in triplicate. In each assay, three different pictures were taken and .300 transduced green cells (.100 cells per picture) were counted. The number of dead (red) cells per counted transduced (green) cells was used to determine percentage of cell death. Assays for phosphorylation and kinase activity of Akt CHME5 (16106) cells transduced with pseudotyped HIV-1 vector (,90% transduction) and CHME5 sublines were treated with LPS (50 mg/ml)/CHX (10 mg/ml) in the presence or absence of ALP compounds for 24 hours. Cells were lysed using ELB lysis buffer before performing the western blot-based Akt kinase activity assay (Cell Signaling) or ELISA-based Akt assay kit (Calbiochem) as per the manufacturers’ protocols. The same lysates were used for the western blot for total and phospho-GSK3b as described [5]. Ratios between the band intensities of phosphorylated and non-phosphorylated Akt and GSK3b levels were analyzed using ImageJ software (NIH). Virus production assay and live/dead assay with infected macrophages Primary human macrophages (86104) were infected with HIV- 1 BaL (76104 pg p24). 24 hours post-infection, cells were washed with Dulbecco’s phosphate buffered saline to eliminate the presence of excess virus. After washing, cells were cultured either in media alone, media containing SNP (100 mM), or media supplemented with 20 mM Perifosine (Cayman Chemical). Infect- ed macrophages were cultured for 12 days, and viral production at 12 days post infection was measured by p24 ELISA (Perkin Elmer) according to the manufacturer’s protocol. Cells were analyzed for the induction of cell death using the live/dead assay as described above. Merged (red + green + bright field) images are shown at 1006 magnification. Green cells represent viable cells while those with red nuclei correspond to dead or dying cells. Results Effect of ALP compounds on Akt activation and phosphorylation in HIV-1 expressing CHME5 cells We previously reported that HIV-1 expression promotes the activation of cellular signals involved in the PI3K/Akt pathway in CHME5 cells (e.g. GSK3b phosphorylation, reference 5: Figure S1) and primary human macrophages [5], which explains their elevated cell survival phenotypes. While ALP compounds, which are potential Akt inhibitors, affect cell survival signals in several cancer cell models [13,15,16,17], it has not been tested if the ALP compounds also affect the cell survival signaling in HIV-1 infected cells. Thus, first, ALP compounds were examined for the ability to inhibit the activation of Akt kinase in CHME5 cells, either transduced by the DHIV-GFP vector or expressing Tat protein. CHME5 cells were transduced by an HIV-1 vector, DHIV-GFP, expressing all HIV-1 proteins except Env and Nef; the nef gene was replaced with GFP [5], yielding .90% transduction as determined by fluorescence- activated cell sorting (FACS) for GFP expression. At two days post PLoS ONE | www.plosone.org 2 September 2010 | Volume 5 | Issue 9 | e13121