Non-Metabolic Role of PKM2 in Regulation of the HIV-1 LTR.

Identification of cellular proteins, in addition to already known transcription factors such as NF-κB, Sp1, C-EBPß, NFAT, ATF/CREB, and LEF-1, which interact with the HIV-1 LTR, is critical in understanding the mechanism of HIV-1 replication in monocytes/macrophages. Our studies demonstrate upregulation of pyruvate kinase isoform M2 (PKM2) expression during HIV-1SF162 infection of monocyte/macrophages and reactivation of HIV-1 in U1 cells, a macrophage model of latency. We observed that HIV-1SF162 infection of monocyte/macrophages and reactivation of HIV-1 in U1 cells by PMA resulted in increased levels of nuclear PKM2 compared to PMA-induced U937 cells. Furthermore, there was a significant increase in the nuclear dimeric form of PKM2 in the PMA-induced U1 cells in comparison to PMA-induced U937 cells. We focused on understanding the potential role of PKM2 in HIV-1 LTR transactivation. Chromatin immunoprecipitation (ChIP) analysis in PMA-activated U1 and TZM-bl cells demonstrated the interaction of PKM2 with the HIV-1 LTR. Our studies show that overexpression of PKM2 results in transactivation of HIV-1 LTR-luciferase reporter in U937, U-87 MG, and TZM-bl cells. Using various truncated constructs of the HIV-1 LTR, we mapped the region spanning -120 bp to -80 bp to be essential for PKM2-mediated transactivation. This region contains the NF-κB binding site and deletion of this site attenuated PKM2-mediated activation of HIV-1 LTR. Immunoprecipitation experiments using U1 cell lysates demonstrated a physical interaction between PKM2 and the p65 subunit of NF-κB. These observations demonstrate for the first time that PKM2 is a transcriptional co-activator of HIV-1 LTR. J. Cell. Physiol. 232: 517-525, 2017. © 2016 Wiley Periodicals, Inc.

Regulation of the HIV-1 promoter by HIF-1α and Vpr proteins.

We previously demonstrated the ability of HIV-1 Vpr protein to activate the oxidative stress pathway, thus leading to the induction of the hypoxia inducible factor 1 alpha (HIF-1α). Therefore, we sought to examine the interplay between the two proteins and the impact of HIF-1α activation on HIV-1 transcription. Using transient transfection assays, we identified the optimal concentration of HIF-1α necessary for the activation of the HIV-1 promoter as well as the domain within HIF-1α responsible for this activation. Our findings indicated that activation of the HIV-1 LTR by Vpr is HIF-1α dependent. Furthermore, we showed that both Vpr and HIF-1α activate the HIV-1 promoter through the GC-rich binding domain within the LTR. Taken together, these data shed more light on the mechanisms used by Vpr to activate the HIV-1 promoter and placed HIF-1α as a major participant in this activation.

Role of hexokinase-1 in the survival of HIV-1-infected macrophages.

Viruses have developed various strategies to protect infected cells from apoptosis. HIV-1 infected macrophages are long-lived and considered reservoirs for HIV-1. One significant deciding factor between cell survival and cell death is glucose metabolism. We hypothesized that HIV-1 protects infected macrophages from apoptosis in part by modulating the host glycolytic pathway specifically by regulating hexokinase-1 (HK-1) an enzyme that converts glucose to glucose-6-phosphate. Therefore, we analyzed the regulation of HK-1 in HIV-1 infected PBMCs, and in a chronically HIV-1 infected monocyte-like cell line, U1. Our results demonstrate that HIV-1 induces a robust increase in HK-1 expression. Surprisingly, hexokinase enzymatic activity was significantly inhibited in HIV-1 infected PBMCs and in PMA differentiated U1 cells. Interestingly, we observed increased levels of mitochondria-bound HK-1 in PMA induced U1 cells and in the HIV-1 accessory protein, viral protein R (Vpr) transduced U937 cell derived macrophages. Dissociation of HK-1 from mitochondria in U1 cells using a pharmacological agent, clotrimazole (CTZ) induced mitochondrial membrane depolarization and caspase-3/7 mediated apoptosis. Dissociation of HK-1 from mitochondria in Vpr transduced U937 also activated caspase-3/7 activity. These observations indicate that HK-1 plays a non-metabolic role in HIV-1 infected macrophages by binding to mitochondria thereby maintaining mitochondrial integrity. These results suggest that targeting the interaction of HK-1 with the mitochondria to induce apoptosis in persistently infected macrophages may prove beneficial in purging the macrophage HIV reservoir.

HIV-1 Vpr modulates macrophage metabolic pathways: a SILAC-based quantitative analysis.

Human immunodeficiency virus type 1 encoded viral protein Vpr is essential for infection of macrophages by HIV-1. Furthermore, these macrophages are resistant to cell death and are viral reservoir. However, the impact of Vpr on the macrophage proteome is yet to be comprehended. The goal of the present study was to use a stable-isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry-based proteomics approach to characterize the Vpr response in macrophages. Cultured human monocytic cells, U937, were differentiated into macrophages and transduced with adenovirus construct harboring the Vpr gene. More than 600 proteins were quantified in SILAC coupled with LC-MS/MS approach, among which 136 were significantly altered upon Vpr overexpression in macrophages. Quantified proteins were selected and clustered by biological functions, pathway and network analysis using Ingenuity computational pathway analysis. The proteomic data illustrating increase in abundance of enzymes in the glycolytic pathway (pentose phosphate and pyruvate metabolism) was further validated by western blot analysis. In addition, the proteomic data demonstrate down regulation of some key mitochondrial enzymes such as glutamate dehydrogenase 2 (GLUD2), adenylate kinase 2 (AK2) and transketolase (TKT). Based on these observations we postulate that HIV-1 hijacks the macrophage glucose metabolism pathway via the Vpr-hypoxia inducible factor 1 alpha (HIF-1 alpha) axis to induce expression of hexokinase (HK), glucose-6-phosphate dehyrogenase (G6PD) and pyruvate kinase muscle type 2 (PKM2) that facilitates viral replication and biogenesis, and long-term survival of macrophages. Furthermore, dysregulation of mitochondrial glutamate metabolism in macrophages can contribute to neurodegeneration via neuroexcitotoxic mechanisms in the context of NeuroAIDS.