Limits...
Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy.

Chugh P, Bradel-Tretheway B, Monteiro-Filho CM, Planelles V, Maggirwar SB, Dewhurst S, Kim B - Retrovirology (2008)

Bottom Line: Interestingly, the expression of HIV-1 or SIV Tat is sufficient to mediate this cytoprotective effect, which is dependent on the basic domain of Tat - a region that has previously been shown to bind p53.Next, we observed that this interaction appears to contribute to the downregulation of PTEN expression, since HIV-1 Tat was found to compete with PTEN for p53 binding; this is known to result in p53 destabilization, with a consequent reduction in PTEN protein production.Since HIV-1 infected macrophages display highly elevated Akt activity, our results collectively show that PI3K/Akt inhibitors may be a novel therapy for interfering with the establishment of long-living HIV-1 infected reservoirs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, School of Medicine, University of Rochester Medical Center 601 Elmwood Avenue Box 672 Rochester, New York 14742 USA. Pauline_Chugh@urmc.rochester.edu

ABSTRACT

Background: Unlike CD4+ T cells, HIV-1 infected macrophages exhibit extended life span even upon stress, consistent with their in vivo role as long-lived HIV-1 reservoirs.

Results: Here, we demonstrate that PI3K/Akt inhibitors, including clinically available Miltefosine, dramatically reduced HIV-1 production from long-living virus-infected macrophages. These PI3K/Akt inhibitors hyper-sensitize infected macrophages to extracellular stresses that they are normally exposed to, and eventually lead to cell death of infected macrophages without harming uninfected cells. Based on the data from these Akt inhibitors, we were able to further investigate how HIV-1 infection utilizes the PI3K/Akt pathway to establish the cytoprotective effect of HIV-1 infection, which extends the lifespan of infected macrophages, a key viral reservoir. First, we found that HIV-1 infection activates the well characterized pro-survival PI3K/Akt pathway in primary human macrophages, as reflected by decreased PTEN protein expression and increased Akt kinase activity. Interestingly, the expression of HIV-1 or SIV Tat is sufficient to mediate this cytoprotective effect, which is dependent on the basic domain of Tat - a region that has previously been shown to bind p53. Next, we observed that this interaction appears to contribute to the downregulation of PTEN expression, since HIV-1 Tat was found to compete with PTEN for p53 binding; this is known to result in p53 destabilization, with a consequent reduction in PTEN protein production.

Conclusion: Since HIV-1 infected macrophages display highly elevated Akt activity, our results collectively show that PI3K/Akt inhibitors may be a novel therapy for interfering with the establishment of long-living HIV-1 infected reservoirs.

Show MeSH

Related in: MedlinePlus

HIV-1 infection promotes membrane recruitment of Akt's PH domain, resulting in increased Akt activity. (A) Primary human macrophages were sequentially infected with M-tropic HIV-1 YU-2 and Ad.CMV-EGFP-PHAkt expressing the PH domain of Akt, and localization of the PH domain of Akt was assessed by fluorescence microscopy. Heat inactivated YU-2 was used as a negative control, and treatment with epidermal growth factor (EGF) was used as a positive control for Akt activation. HIV-1 infected macrophages were treated with 10 μM Miltefosine (Milt.) for inhibition of Akt. BF: bright field. GFP: green fluorescent protein. Inset: High magnification images of representative cells. The percentage of membrane localized PH-Akt is shown with the SD from three independent experiments. (B) Assay for Akt kinase activity. Macrophage and CHME5 cells were transduced with HIV vector and lysed. Using these lysates, an Akt kinase activity assay was performed using GSK3β as a substrate. Western blots of phospho-GSK3β (GSK3β-P) and α-Tubulin (loading control) are shown along with the fold induction of Akt kinase activity relative to control. Fold increase of Akt kinase activity is also shown. The error bars denote the SD from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2265748&req=5

Figure 3: HIV-1 infection promotes membrane recruitment of Akt's PH domain, resulting in increased Akt activity. (A) Primary human macrophages were sequentially infected with M-tropic HIV-1 YU-2 and Ad.CMV-EGFP-PHAkt expressing the PH domain of Akt, and localization of the PH domain of Akt was assessed by fluorescence microscopy. Heat inactivated YU-2 was used as a negative control, and treatment with epidermal growth factor (EGF) was used as a positive control for Akt activation. HIV-1 infected macrophages were treated with 10 μM Miltefosine (Milt.) for inhibition of Akt. BF: bright field. GFP: green fluorescent protein. Inset: High magnification images of representative cells. The percentage of membrane localized PH-Akt is shown with the SD from three independent experiments. (B) Assay for Akt kinase activity. Macrophage and CHME5 cells were transduced with HIV vector and lysed. Using these lysates, an Akt kinase activity assay was performed using GSK3β as a substrate. Western blots of phospho-GSK3β (GSK3β-P) and α-Tubulin (loading control) are shown along with the fold induction of Akt kinase activity relative to control. Fold increase of Akt kinase activity is also shown. The error bars denote the SD from three independent experiments.

Mentions: The PTEN phosphatase normally converts PIP3 to PIP2. During the activation of the cell survival pathway, high levels of PIP3 lead to the recruitment of the Akt kinase to the plasma membrane by binding to the PH domain of Akt. Therefore, we investigated the effect of HIV-1 infection on the membrane recruitment of Akt. For this, we employed an adenoviral vector that expresses an EGFP-PH fusion protein, in which the PH domain of Akt was fused to the C-terminus of EGFP (Ad.CMV-EGFP-PHAkt). In order to detect the localization of PH Akt during HIV-1 infection, we first infected primary human macrophages with HIV-1 YU-2 and transduced these infected cells 48 hours later with Ad.CMV-EGFP-PHAkt. As shown in Figure 3A, macrophages treated with heat-inactivated HIV-1 displayed diffuse localization of the PH domain throughout the cell. In contrast, HIV-1 YU-2 infection resulted in a distinct localization of EGFP-PHAkt to the plasma membrane. This membrane localization is typically observed following treatment with epidermal growth factor (EGF), which is known to activate the PI3K/Akt pathway [26,27]. Interestingly, we also found that treatment of HIV-1 infected macrophages with the Akt inhibitor Miltefosine inhibited the recruitment of PH-AktGFP to the plasma membrane (Figure 3A). Since Miltefosine inhibits Akt through mimicry of the PH domain, it is likely that Miltefosine binds to PIP3, blocking the recruitment of PH-Akt to the membrane. The percentage of macrophages in which PH domain membrane recruitment was observed is shown below panel 3A. These results suggest that HIV-1 infection in macrophages induces plasma membrane recruitment of Akt which can be reversed using Miltefosine, and our results above suggest that this is likely due to the reduced levels of PTEN expression.


Akt inhibitors as an HIV-1 infected macrophage-specific anti-viral therapy.

Chugh P, Bradel-Tretheway B, Monteiro-Filho CM, Planelles V, Maggirwar SB, Dewhurst S, Kim B - Retrovirology (2008)

HIV-1 infection promotes membrane recruitment of Akt's PH domain, resulting in increased Akt activity. (A) Primary human macrophages were sequentially infected with M-tropic HIV-1 YU-2 and Ad.CMV-EGFP-PHAkt expressing the PH domain of Akt, and localization of the PH domain of Akt was assessed by fluorescence microscopy. Heat inactivated YU-2 was used as a negative control, and treatment with epidermal growth factor (EGF) was used as a positive control for Akt activation. HIV-1 infected macrophages were treated with 10 μM Miltefosine (Milt.) for inhibition of Akt. BF: bright field. GFP: green fluorescent protein. Inset: High magnification images of representative cells. The percentage of membrane localized PH-Akt is shown with the SD from three independent experiments. (B) Assay for Akt kinase activity. Macrophage and CHME5 cells were transduced with HIV vector and lysed. Using these lysates, an Akt kinase activity assay was performed using GSK3β as a substrate. Western blots of phospho-GSK3β (GSK3β-P) and α-Tubulin (loading control) are shown along with the fold induction of Akt kinase activity relative to control. Fold increase of Akt kinase activity is also shown. The error bars denote the SD from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2265748&req=5

Figure 3: HIV-1 infection promotes membrane recruitment of Akt's PH domain, resulting in increased Akt activity. (A) Primary human macrophages were sequentially infected with M-tropic HIV-1 YU-2 and Ad.CMV-EGFP-PHAkt expressing the PH domain of Akt, and localization of the PH domain of Akt was assessed by fluorescence microscopy. Heat inactivated YU-2 was used as a negative control, and treatment with epidermal growth factor (EGF) was used as a positive control for Akt activation. HIV-1 infected macrophages were treated with 10 μM Miltefosine (Milt.) for inhibition of Akt. BF: bright field. GFP: green fluorescent protein. Inset: High magnification images of representative cells. The percentage of membrane localized PH-Akt is shown with the SD from three independent experiments. (B) Assay for Akt kinase activity. Macrophage and CHME5 cells were transduced with HIV vector and lysed. Using these lysates, an Akt kinase activity assay was performed using GSK3β as a substrate. Western blots of phospho-GSK3β (GSK3β-P) and α-Tubulin (loading control) are shown along with the fold induction of Akt kinase activity relative to control. Fold increase of Akt kinase activity is also shown. The error bars denote the SD from three independent experiments.
Mentions: The PTEN phosphatase normally converts PIP3 to PIP2. During the activation of the cell survival pathway, high levels of PIP3 lead to the recruitment of the Akt kinase to the plasma membrane by binding to the PH domain of Akt. Therefore, we investigated the effect of HIV-1 infection on the membrane recruitment of Akt. For this, we employed an adenoviral vector that expresses an EGFP-PH fusion protein, in which the PH domain of Akt was fused to the C-terminus of EGFP (Ad.CMV-EGFP-PHAkt). In order to detect the localization of PH Akt during HIV-1 infection, we first infected primary human macrophages with HIV-1 YU-2 and transduced these infected cells 48 hours later with Ad.CMV-EGFP-PHAkt. As shown in Figure 3A, macrophages treated with heat-inactivated HIV-1 displayed diffuse localization of the PH domain throughout the cell. In contrast, HIV-1 YU-2 infection resulted in a distinct localization of EGFP-PHAkt to the plasma membrane. This membrane localization is typically observed following treatment with epidermal growth factor (EGF), which is known to activate the PI3K/Akt pathway [26,27]. Interestingly, we also found that treatment of HIV-1 infected macrophages with the Akt inhibitor Miltefosine inhibited the recruitment of PH-AktGFP to the plasma membrane (Figure 3A). Since Miltefosine inhibits Akt through mimicry of the PH domain, it is likely that Miltefosine binds to PIP3, blocking the recruitment of PH-Akt to the membrane. The percentage of macrophages in which PH domain membrane recruitment was observed is shown below panel 3A. These results suggest that HIV-1 infection in macrophages induces plasma membrane recruitment of Akt which can be reversed using Miltefosine, and our results above suggest that this is likely due to the reduced levels of PTEN expression.

Bottom Line: Interestingly, the expression of HIV-1 or SIV Tat is sufficient to mediate this cytoprotective effect, which is dependent on the basic domain of Tat - a region that has previously been shown to bind p53.Next, we observed that this interaction appears to contribute to the downregulation of PTEN expression, since HIV-1 Tat was found to compete with PTEN for p53 binding; this is known to result in p53 destabilization, with a consequent reduction in PTEN protein production.Since HIV-1 infected macrophages display highly elevated Akt activity, our results collectively show that PI3K/Akt inhibitors may be a novel therapy for interfering with the establishment of long-living HIV-1 infected reservoirs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Microbiology and Immunology, School of Medicine, University of Rochester Medical Center 601 Elmwood Avenue Box 672 Rochester, New York 14742 USA. Pauline_Chugh@urmc.rochester.edu

ABSTRACT

Background: Unlike CD4+ T cells, HIV-1 infected macrophages exhibit extended life span even upon stress, consistent with their in vivo role as long-lived HIV-1 reservoirs.

Results: Here, we demonstrate that PI3K/Akt inhibitors, including clinically available Miltefosine, dramatically reduced HIV-1 production from long-living virus-infected macrophages. These PI3K/Akt inhibitors hyper-sensitize infected macrophages to extracellular stresses that they are normally exposed to, and eventually lead to cell death of infected macrophages without harming uninfected cells. Based on the data from these Akt inhibitors, we were able to further investigate how HIV-1 infection utilizes the PI3K/Akt pathway to establish the cytoprotective effect of HIV-1 infection, which extends the lifespan of infected macrophages, a key viral reservoir. First, we found that HIV-1 infection activates the well characterized pro-survival PI3K/Akt pathway in primary human macrophages, as reflected by decreased PTEN protein expression and increased Akt kinase activity. Interestingly, the expression of HIV-1 or SIV Tat is sufficient to mediate this cytoprotective effect, which is dependent on the basic domain of Tat - a region that has previously been shown to bind p53. Next, we observed that this interaction appears to contribute to the downregulation of PTEN expression, since HIV-1 Tat was found to compete with PTEN for p53 binding; this is known to result in p53 destabilization, with a consequent reduction in PTEN protein production.

Conclusion: Since HIV-1 infected macrophages display highly elevated Akt activity, our results collectively show that PI3K/Akt inhibitors may be a novel therapy for interfering with the establishment of long-living HIV-1 infected reservoirs.

Show MeSH
Related in: MedlinePlus