HIV-1 Vpr Modulates Macrophage Metabolic Pathways: A SILAC-Based Quantitative Analysis

Abstract

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.

Glutamate metabolism in HIV-1 infected macrophages: Role of HIV-1 Vpr

“…3,13 Our earlier studies using SILAC-based proteomics analysis demonstrated that numerous enzymes in the glycolytic pathway and TCA cycle were altered in response to HIV-1 Vpr in macrophages. 16 In this study, we evaluated alteration in metabolic profile in response to HIV-1 Vpr in macrophages to correlate with the observed changes in expression profile of critical enzymes in the glycolytic pathway and TCA cycle. In fact, metabolomics has provided sensitive, fast, and robust tools for analyzing metabolite levels during viral infections.…”

“…16 To corroborate changes in the intracellular and extracellular metabolite levels we assessed the expression levels of selected enzymes in the TCA cycle in response to HIV-1 Vpr overexpression in U937 macrophages in comparison to the control Ad-null transduced macrophages on day 1, 3 and 7 (Fig. 4).…”

“…Our recent SILAC-based proteomics study showed that HIV-1 Vpr overexpression in macrophages induces changes at the protein level in many of the enzymes involved in cellular metabolism. 16 HAD has been linked to macrophage toxin production including glutamate. [17][18][19][20] Increased glutamate levels are also seen in the plasma, and cerebrospinal fluid (CSF) of HIV-1 infected patients.…”

“…16 The development of a metabolomics platform to evaluate the final products of the metabolic pathways induced in macrophages overexpressing Vpr can offer a better understanding of mechanisms responsible not only for viral reservoir persistence in macrophages but also for the pathogenesis of HAD. Such biomarker(s) will help in early detection and classification of neurocognitive impairment.…”

ATP5B (ATP synthase, H+ transporting, mitochondrial F1 complex, beta polypeptide)

“…ATP5B was shown to be upregulated by Tat protein of HIV-1 virus (López-Huertas et al, 2013). ATP5B protein was also shown to be physically interacted with Vpr, a HIV-1 virus protein in HEK293 cells (Barrero et al, 2013). Metabolic changes are common characteristic features of cancer tissues.…”