Ruma Mukerjee

Activation of the Oxidative Stress Pathway by HIV-1 Vpr Leads to Induction of Hypoxia-inducible Factor 1α Expression

The detection of biomarkers of oxidative stress in brain tissue and cerebrospinal fluid of patients with human immunodeficiency virus, type 1 (HIV)-associated dementia indicates the involvement of stress pathways in the neuropathogenesis of AIDS. Although the biological importance of oxidative stress on events involved in AIDS neuropathogenesis and the HIV-1 proteins responsible for oxidative stress remain to be elucidated, our results point to the activation of hypoxia-inducible factor 1 (HIF-1) upon HIV-1 infection and its elevation in brain cells of AIDS patients with dementia. HIF-1 is a transcription factor that is responsive to oxygen. Under hypoxic conditions, HIF-1α becomes stable and translocates to the nucleus where it dimerizes with aryl hydrocarbon receptor nuclear translocator and modulates gene transcription. Activation of HIF-1 can also be mediated by the HIV-1 accessory protein Vpr. In addition, cellular components, including reactive oxygen species, contribute to the induction of HIF-1α. Our results show that Vpr induces reactive oxygen species by increasing H2O2 production, which can contribute to HIF-1α accumulation. Interestingly, increased levels of HIF-1α stimulated HIV-1 gene transcription through HIF-1 association with HIV-1 long terminal repeat. These observations point to the existence of a positive feedback interplay between HIF-1α and Vpr and that, by inducing oxidative stress via activation of HIF-1, Vpr can induce HIV-1 gene expression and dysregulate multiple host cellular pathways.

HIV-1 Tat protein promotes neuronal dysfunction through disruption of microRNAs.

Over the last decade, small noncoding RNA molecules such as microRNAs (miRNAs) have emerged as critical regulators in the expression and function of eukaryotic genomes. It has been suggested that viral infections and neurological disease outcome may also be shaped by the influence of small RNAs. This has prompted us to suggest that HIV infection alters the endogenous miRNA expression patterns, thereby contributing to neuronal deregulation and AIDS dementia. Therefore, using primary cultures and neuronal cell lines, we examined the impact of a viral protein (HIV-1 Tat) on the expression of miRNAs due to its characteristic features such as release from the infected cells and taken up by noninfected cells. Using microRNA array assay, we demonstrated that Tat deregulates the levels of several miRNAs. Interestingly, miR-34a was among the most highly induced miRNAs in Tat-treated neurons. Tat also decreases the levels of miR-34a target genes such as CREB protein as shown by real time PCR. The effect of Tat was neutralized in the presence of anti-miR-34a. Using in situ hybridization assay, we found that the levels of miR-34a increase in Tat transgenic mice when compared with the parental mice. Therefore, we conclude that deregulation of neuronal functions by HIV-1 Tat protein is miRNA-dependent.

Role of p53 in neurodegenerative diseases.

Background: p53 plays an important role in many areas of cellular physiology and biology, ranging from cellular development and differentiation to cell cycle arrest and apoptosis. Many of its functions are attributed to its role in assuring proper cellular division. However, since the establishment of its role in cell cycle arrest, damage repair, and apoptosis (thus also establishing its importance in cancer development), numerous reports have demonstrated additional functions of p53 in various cells. In particular, p53 appears to have important functions as it relates to neurodegeneration and synaptic plasticity. Objective: In this review, we will address p53 functions as it relates to various neurodegenerative diseases, mainly its implications in the development of HIV-associated neurocognitive disorders. Conclusion: p53 plays a pivotal role in the development of neurodegenerative diseases through its interaction with cellular factors, viral factors, and/or small RNAs that have the ability to promote the development of these diseases. Hence, inhibition of p53 may present an ideal target to restore neuronal functions.

Deregulation of microRNAs by HIV-1 Vpr protein leads to the development of neurocognitive disorders.

Studies have shown that HIV-infected patients develop neurocognitive disorders characterized by neuronal dysfunction. The lack of productive infection of neurons by HIV suggests that viral and cellular proteins, with neurotoxic activities, released from HIV-1-infected target cells can cause this neuronal deregulation. The viral protein R (Vpr), a protein encoded by HIV-1, has been shown to alter the expression of various important cytokines and inflammatory proteins in infected and uninfected cells; however the mechanisms involved remain unclear. Using a human neuronal cell line, we found that Vpr can be taken up by neurons causing: (i) deregulation of calcium homeostasis, (ii) endoplasmic reticulum-calcium release, (iii) activation of the oxidative stress pathway, (iv) mitochondrial dysfunction and v- synaptic retraction. In search for the cellular factors involved, we performed microRNAs and gene array assays using human neurons (primary cultures or cell line, SH-SY5Y) that we treated with recombinant Vpr proteins. Interestingly, Vpr deregulates the levels of several microRNAs (e.g. miR-34a) and their target genes (e.g. CREB), which could lead to neuronal dysfunctions. Therefore, we conclude that Vpr plays a major role in neuronal dysfunction through deregulating microRNAs and their target genes, a phenomenon that could lead to the development of neurocognitive disorders.

Modulation of JC virus transcription by C/EBPβ

The polyomavirus JC (JCV) causes the demyelinating disease progressive multifocal leukoencephalopathy (PML). Infection by JCV is very common in childhood after which the virus enters a latent state, which is poorly understood. Under conditions of severe immunosuppression, especially AIDS, JCV may reactivate to cause PML. Expression of JC viral proteins is regulated by the JCV non-coding control region (NCCR), which contains an NF-kappaB binding site previously shown to activate transcription. We now report that C/EBPbeta inhibits basal and NF-kappaB-stimulated JCV transcription via the same site. Gel shift analysis showed C/EBPbeta bound to this region in vitro and ChIP assays confirmed this binding in vivo. Further, a ternary complex of NF-kappaB/p65, C/EBPbeta-LIP and JCV DNA could be detected in co-immunoprecipitation experiments. Mutagenesis analysis of the JCV NCCR indicated p65 and C/EBPbeta-LIP bound to adjacent but distinct sites and that both sites regulate basal and p65-stimulated transcription. Thus C/EBPbeta negatively regulates JCV, which together with NF-kappaB activation, may control the balance between JCV latency and activation leading to PML. This balance may be regulated by proinflammatory cytokines in the brain.

Association of p65 and C/EBPβ with HIV-1 LTR modulates transcription of the viral promoter

In human immunodeficiency virus type 1 (HIV‐1) latently infected cells, NF‐kappaB (NF‐κB) plays a critical role in the transcriptional induction of the HIV‐1 promoter. The trans‐activating ability of NF‐κB can be modified by another nuclear factor C/EBPβ that can physically bind to NF‐κB and regulate its activity. Because the HIV‐1 promoter also contains a C/EBPβ site adjacent to the NF‐κB site, the present study examined cooperative functional in vivo interaction of the p65 subunit of NF‐κB and C/EBPβ, and the impact of Tat in this event. We demonstrated that ectopic expression of p65 along with Tat increases p65 binding to HIV‐1 LTR, and that this increase correlates with enhanced HIV‐1 promoter activity. Further, co‐expression of C/EBPβ and Tat leads to a decrease in p65 binding, which allows C/EBPβ to bind more efficiently to the LTR. Inhibition of p65 expression by siRNA significantly decreases C/EBPβ‐binding and LTR expression. Using ChIP assay, we confirmed the existence of an interchange between p65 and C/EBPβ and their abilities to bind to the LTR in vivo. These observations demonstrate that a delicate balance of interaction between p65, C/EBPβ, and Tat can dictate the level of HIV‐1 LTR transcription. J. Cell. Biochem. 100: 1210–1216, 2007.

HIV-1 Vpr deregulates calcium secretion in neural cells.

The lack of productive infection of neurons by HIV-1 suggests that the neuronal damage seen in AIDS patients with cognitive disorders is caused indirectly via viral and cellular proteins with neurotoxic activity. Among HIV-1 proteins, Vpr has been shown to deregulate expression of various important cytokines and inflammatory proteins in infected and uninfected cells. However, the mechanisms underlying these changes remain unclear. Here, we demonstrate that neurons can take up Vpr that is released into the supernatant of HIV-infected microglia. We also found that administration of recombinant Vpr (rVpr) to human neurons resulted in a slow but sustained elevation of intracellular calcium [Ca(2+)]i. Interestingly, our data also show that [Ca(2+)]i elevation by Vpr leads to ROS production and impairs glutamate signaling in neuronal cells. Vpr disturbs calcium homeostasis through downregulation of endogenous PMCA. Finally, we found that the permeability of the plasma membrane increases in neurons treated with Vpr. Therefore, we conclude that soluble Vpr is a major viral factor that causes a disturbance in neuronal communication leading to neuronal dysfunction. The outcome of these studies will advance the understanding of HIV-1 pathogenesis and will help in the development of new therapeutic approaches.

TNF Alpha Production in Morphine-Treated Human Neural Cells Is NF-κB-Dependent

The cytokine tumor necrosis factor alpha (TNFα) is a key factor in several inflammatory diseases and its levels increase in response to a variety of internal or external stimuli. The regulation of the TNFα promoter is mediated by several transcription factors including the nuclear factor kappa B protein (NF-κB). This study examines the role of NF-κB in the regulation of TNFα production by morphine in microglia. Using reverse transcriptase polymerase chain reaction, we demonstrated the presence of morphine receptors in these cells. We next demonstrated the ability of morphine to promote TNFα production and secretion by these cells using a cytokine array assay. Transient transfection experiments led to the identification of the region located between nucleotides −751 and −615 within the TNFα promoter as being responsive to morphine treatment. The DNA sequence of this region contains a motif indicative of a potential NF-κB binding site. The use of a small interfering RNA directed against p65, a subunit of NF-κB, demonstrated that TNFα induction by morphine is NF-κB-dependent. All of the effects of morphine were reversed by the morphine inhibitor, naloxone. These data provide important insights into the effects of morphine on microglia.

Involvement of the p53 and p73 transcription factors in neuroAIDS.

HIV-associated dementia (HAD) is the most common AIDS-associated neurological disorder and is characterized by the development of synaptodendritic injury to neurons. To advance HAD therapy, it is crucial to identify the mechanisms and factors involved. The viral protein HIV-1 Tat is among those factors and is released by HIV-1-infected cells and can be taken up by adjacent neuronal cells leading to neurotoxic effects. Multiple cellular host proteins have been identified as Tat cofactors in causing neuronal injury. Interestingly, most of these factors function through activation of the p53 pathway. We have now examined the ability of Tat to activate the p53 pathway leading to the induction of endogenous p53 and p73 in neuronal cells. We found that Tat induced p53 and p73 levels in SH-SY5Y cells and that this induction caused retraction of neurites. In the absence of either p53 or p73, Tat failed to induce dendritic retraction or to activate the proapoptotic proteins, such as Bax. Further, we found that p53-accumulation in Tat-treated cells depends on the presence of p73. Therefore, we conclude that Tat contributes to neuronal degeneration through activation of a pathway involving p53 and p73. This information will be valuable for the development of therapeutic agents that affect these pathways to protect CNS neurons and prevent HAD.

HIV-1 Vpr: a closer look at the multifunctional protein from the structural perspective.

The human immunodeficiency virus-1 (HIV-1) Vpr protein plays multiple roles in HIV-1 replication. In early infection, Vpr provides help in the nuclear localization of pre-integration complex. Subsequently, Vpr induces cell cycle arrest of infected cells at G2/M phase. Cell cycle arrest facilitates higher rate of viral gene transcription. Vpr is also capable of activating transcription of viral and heterologous genes. Vpr induces apoptosis in infected cells leading to loss of immune cells and onset of clinical AIDS. Interestingly, Vpr is also considered as a passenger protein in the virus particles as it is incorporated into the virus particles through interaction with Gag. The structure of full length Vpr has been resolved recently through NMR. In this review, we have analysed the functions of Vpr using the available data from structural perspective. Packing of the three helices of Vpr around a core formed by hydrophobic side chains and integrity of helical domains are critical for Vpr functions. The distinct functions of Vpr have been attributed to structural integrity of different domains. The unique distribution of acidic and basic residues in Vpr is an interesting feature. Two hydrophobic pockets on the structure of Vpr are proposed to be important targets for modulating Vpr functions. The inter-relationship between different functions of Vpr is discussed in the context of structure. Based on bioinformatics analysis, we propose new targets for modulating Vpr functions, which need to be validated experimentally.