Physical and in silico approaches identify DNA-PK in a Tax DNA-damage response interactome

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.     

Infection of human immunodeficiency virus and intracellular viral Tat protein exert a pro-survival effect in a human microglial cell line

The interaction of human immunodeficiency virus type 1 (HIV-1) with CD4+ T lymphocytes is well studied and typically results in virally induced cytolysis. In contrast, relatively little is known concerning the interplay between HIV-1 and microglia. Recent findings suggest that, counter-intuitively, HIV-1 infection may extend the lifespan of microglia. We developed a novel cell line model system to confirm and mechanistically study this phenomenon. We found that transduction of a human microglial cell line with an HIV-1 vector results in a powerful cytoprotective effect following apoptotic challenge. This effect was reproduced by ectopic expression of a single virus-encoded protein, Tat. Subsequent studies showed that the pro-survival effects of intracellular Tat could be attributed to activation of the PI-3-kinase (PI3K)/Akt pathway in the microglial cell line. Furthermore, we found that expression of Tat led to decreased expression of PTEN, a negative regulator of the PI-3-K pathway. Consistent with this, decreased p53 activity and increased E2F activity were observed. Based on these findings, a model of possible regulatory circuits that intracellular Tat and HIV-1 infection engage during the cytoprotective event in microglia has been suggested. We propose that the expression of Tat may enable HIV-1 infected microglia to survive throughout the course of infection, leading to persistent HIV-1 production and infection in the central nervous system.     

Analysis of p300 acetyltransferase substrate specificity by MALDI TOF mass spectrometry

Acetyltransferase enzymes target specific lysine residues in substrate proteins. While the list of histone and nonhistone substrates is growing, the mechanisms of substrate selection remain unclear. Here, we describe a mass spectrometric approach to examine the site selection of the acetyltransferase p300 in the HIV-1 protein Tat. Tat is acetylated by p300 at a single lysine (K50) within its basic RNA-binding domain. To determine the sequence requirements for K50 recognition within this domain, we synthesized mixtures of "degenerated" Tat peptides, in which one of the surrounding residues was substituted by all proteinogenic amino acids. Peptide mixtures were assembled based on nonoverlapping peptide masses and acetylated by p300 in a standard in vitro acetylation reaction. Analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry identified amino acid substitutions that prevented acetylation by p300. This approach represents a fast and comprehensive screening method that was applied to the six surrounding residues of K50 in Tat. It can be applied to any known acetyltransferase substrate and might help to define consensus recognition sequences for individual acetyltransferase enzymes.     

HIV-1 Tat inhibits NGF-induced Egr-1 transcriptional activity and consequent p35 expression in neural cells

Infection with HIV-1 causes degeneration of neurons leading to motor and cognitive dysfunction in AIDS patients. One of the key viral regulatory proteins, Tat, which is released by infected cells, can be taken up by various uninfected cells including neurons and by dysregulating several biological events induces cell injury and death. In earlier studies, we demonstrated that treatment of neuronal cells with Tat affects the nerve growth factor (NGF) signaling pathway involving MAPK/ERK. Here we demonstrate that a decrease in the level of Egr-1, one of the targets for MAPK, by Tat has a negative impact on the level of p35 expression in NGF-treated neural cells. Further, we demonstrate a reduced level of Egr-1 association with the p35 promoter sequence in NGF-treated cells expressing Tat. As p35, by associating with Cdk5, phosphorylates several neuronal proteins including neurofilaments and plays a role in neuronal differentiation and survival, we examined kinase activity of p35 complexes obtained from cells expressing Tat. Results from H1 kinase assays showed reduced activity of the p35 complex from Tat-expressing cells in comparison to that from control cells. Accordingly, the level of phosphorylated neurofilaments was diminished in Tat-expressing cells. Similarly, treatment of PC12 cells with Tat protein or supernatant from HIV-1 infected cells decreased kinase activity of p35 in these cells. These observations ascribe a role for Tat in altering p35 expression and its activity that affects phosphorylation of proteins involved in neuronal cell survival.     

Functional cross-talk of HIV-1 Tat with p53 through its C-terminal domain

Human immunodeficiency virus type 1 (HIV-1) Tat repressed the p53-dependent gene expression through its C-terminal domain of Tat (amino acid residues 73-86) independent of the involvement of NF-kappaB and coactivator CBP/p300. Although Tat did not directly bind to p53, this repression required the N-terminal domain of p53. In contrast, Tat and p53 cooperated in the activation of HIV-1 gene expression. Thus, the cross-talk between Tat and p53 may be linked with cellular transformation by HIV-1 infection or activation of HIV-1 replication.     

HIV-1 Tat protein-induced VCAM-1 expression in human pulmonary artery endothelial cells and its signaling

Expression of cell adhesion molecule in endothelial cells upon activation by human immunodeficiency virus (HIV) infection is associated with the development of atherosclerotic vasculopathy. We postulated that induction of vascular cell adhesion molecule-1 (VCAM-1) by HIV-1 Tat protein in endothelial cells might represent an early event that could culminate in inflammatory cell recruitment and vascular injury. We determined the role of HIV-1 Tat protein in VCAM-1 expression in human pulmonary artery endothelial cells (HPAEC). HIV-1 Tat protein treatment significantly increased cell-surface expression of VCAM-1 in HPAEC. Consistently, mRNA expression of VCAM-1 was also increased by HIV-1 Tat protein as measured by RT-PCR. HIV-1 Tat protein-induced VCAM-1 expression was abolished by the NF-kappaB inhibitor pyrrolidine dithiocarbamate (PDTC) and the p38 MAPK inhibitor SB-203580. Furthermore, HIV-1 Tat protein enhanced DNA binding activity of NF-kappaB, facilitated nuclear translocation of NF-kappaB subunit p65, and increased production of reactive oxygen species (ROS). Similarly to VCAM-1 expression, HIV-1 Tat protein-induced NF-kappaB activation and ROS generation were abrogated by PDTC and SB-203580. These data indicate that HIV-1 Tat protein is able to induce VCAM-1 expression in HPAEC, which may represent a pivotal early molecular event in HIV-induced vascular/pulmonary injury. These data also suggest that the molecular mechanism underlying the HIV-1 Tat protein-induced VCAM-1 expression may involve ROS generation, p38 MAPK activation, and NF-kappaB translocation, which are the characteristics of pulmonary endothelial cell activation.     

HIV-1 Tat potentiates TNF-induced NF-kappa B activation and cytotoxicity by altering the cellular redox state.

This study demonstrates that human immunodeficiency virus type 1 (HIV-1) Tat protein amplifies the activity of tumor necrosis factor (TNF), a cytokine that stimulates HIV-1 replication through activation of NF-kappa B. In HeLa cells stably transfected with the HIV-1 tat gene (HeLa-tat cells), expression of the Tat protein enhanced both TNF-induced activation of NF-kappa B and TNF-mediated cytotoxicity. A similar potentiation of TNF effects was observed in Jurkat T cells and HeLa cells treated with soluble Tat protein. TNF-mediated activation of NF-kappa B and cytotoxicity involves the intracellular formation of reactive oxygen intermediates. Therefore, Tat-mediated effects on the cellular redox state were analyzed. In both T cells and HeLa cells HIV-1 Tat suppressed the expression of Mn-dependent superoxide dismutase (Mn-SOD), a mitochondrial enzyme that is part of the cellular defense system against oxidative stress. Thus, Mn-SOD RNA protein levels and activity were markedly reduced in the presence of Tat. Decreased Mn-SOD expression was associated with decreased levels of glutathione and a lower ratio of reduced:oxidized glutathione. A truncated Tat protein (Tat1-72), known to transactivate the HIV-1 long terminal repeat (LTR), no longer affected Mn-SOD expression, the cellular redox state or TNF-mediated cytotoxicity. Thus, our experiments demonstrate that the C-terminal region of HIV-1 Tat is required to suppress Mn-SOD expression and to induce pro-oxidative conditions reflected by a drop in reduced glutathione (GSH) and the GSH:oxidized GSH (GSSG) ratio.(ABSTRACT TRUNCATED AT 250 WORDS)     

Novel PI3K/Akt Inhibitors Screened by the Cytoprotective Function of Human Immunodeficiency Virus Type 1 Tat

The PI3K/Akt pathway regulates various stress-related cellular responses such as cell survival, cell proliferation, metabolism and protein synthesis. Many cancer cell types display the activation of this pathway, and compounds inhibiting this cell survival pathway have been extensively evaluated as anti-cancer agents. In addition to cancers, several human viruses, such as HTLV, HPV, HCV and HIV-1, also modulate this pathway, presumably in order to extend the life span of the infected target cells for productive viral replication. The expression of HIV-1 Tat protein exhibited the cytoprotective effect in macrophages and a human microglial cell line by inhibiting the negative regulator of this pathway, PTEN. This cytoprotective effect of HIV-1 appears to contribute to the long-term survival and persistent HIV-1 production in human macrophage reservoirs. In this study we exploited the PI3K/Akt dependent cytoprotective effect of Tat-expressing CHME5 cells. We screened a collection of compounds known to modulate inflammation, and identified three novel compounds: Lancemaside A, Compound K and Arctigenin that abolished the cytoprotective phenotype of Tat-expressing CHME5 cells. All three compounds antagonized the kinase activity of Akt. Further detailed signaling studies revealed that each of these three compounds targeted different steps of the PI3K/Akt pathway. Arctigenin regulates the upstream PI3K enzyme from converting PIP2 to PIP3. Lancemaside A1 inhibited the movement of Akt to the plasma membrane, a critical step for Akt activation. Compound K inhibited Akt phosphorylation. This study supports that Tat-expressing CHME5 cells are an effective model system for screening novel PI3K/Akt inhibitors.     

MiR-217 is involved in Tat-induced HIV-1 long terminal repeat (LTR) transactivation by down-regulation of SIRT1

MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression and may contribute to the development and progression of many infective diseases including human immunodeficiency virus 1 (HIV-1) infection. The Tat protein is fundamental to viral gene expression. In this study, our goal was to investigate the regulation of a specific miRNA (known as miR-217) in multinuclear activation of galactosidase indicator (MAGI) cells and explore the mechanisms by which miR-217 influenced Tat-induced HIV-1 transactivation through down-regulation of SIRT1 expression. We showed that miR-217 was up-regulated when Tat was expressed in multinuclear activation of galactosidase indicator cells. Forced expression of "miR-217 mimics" increased Tat-induced LTR transactivation. In addition, miR-217 significantly inhibited SIRT1 protein expression by acting on the 3'-UTR of the SIRT1 mRNA. In turn, the decrease in SIRT1 protein abundance provoked by miR-217 affected two important types of downstream signaling molecules that were regulated by Tat. Lower expression of SIRT1 caused by miR-217 enhanced Tat-induced phosphorylation of IKK and p65-NFkB and also exacerbated the loss of AMPK phosphorylation triggered by Tat. Our results uncover previously unknown links between Tat and a specific host cell miRNA that targets SIRT1. We also demonstrate that this regulatory mechanism impinges on p65-NFkB and AMPK signaling: two important host cell pathways that influence HIV-1 pathogenesis. Our results also suggest that strategies to augment SIRT1 protein expression by down-regulation of miR-217 may have therapeutic benefits to prevent HIV-1 replication.