SUMMARY 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-κB binding site previously shown to activate transcription. We now report that C/EBPβ inhibits basal and NF-κB-stimulated JCV transcription via the same site. Gel shift analysis showed C/EBPβ bound to this region in vitro and ChIP assays confirmed this binding in vivo. Further, a ternary complex of NF-κB/p65, C/EBPβ-LIP and JCV DNA could be detected in co-immunoprecipitation experiments. Mutagenesis analysis of the JCV NCCR indicated p65 and C/EBPβ-LIP bound to adjacent but distinct sites and that both sites regulate basal and p65-stimulated transcription. Thus C/EBPβ negatively regulates JCV, which together with NF-κB activation, may control the balance between JCV latency and activation leading to PML. This balance may be regulated by proinflammatory cytokines in the brain.
Mainly regulated at the transcriptional level, the cellular cyclin-dependent kinase inhibitor, CDKN1A/p21WAF1 (p21), is a major cell cycle regulator of the response to DNA damage, senescence and tumor suppression. Here, we report that COUP-TF-interacting protein 2 (CTIP2), recruited to the p21 gene promoter, silenced p21 gene transcription through interactions with histone deacetylases and methyltransferases. Importantly, treatment with the specific SUV39H1 inhibitor, chaetocin, repressed histone H3 lysine 9 trimethylation at the p21 gene promoter, stimulated p21 gene expression and induced cell cycle arrest. In addition, CTIP2 and SUV39H1 were recruited to the silenced p21 gene promoter to cooperatively inhibit p21 gene transcription. Induction of p21WAF1 gene upon human immunodeficiency virus 1 (HIV-1) infection benefits viral expression in macrophages. Here, we report that CTIP2 further abolishes Vpr-mediated stimulation of p21, thereby indirectly contributing to HIV-1 latency. Altogether, our results suggest that CTIP2 is a constitutive p21 gene suppressor that cooperates with SUV39H1 and histone methylation to silence the p21 gene transcription.
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.
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.
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.
Edited by Paul E. FraserIndividuals who are infected with HIV-1 accumulate damage to cells and tissues (e.g. neurons) that are not directly infected by the virus. These include changes known as HIV-associated neurodegenerative disorder (HAND), leading to the loss of neuronal functions, including synaptic long-term potentiation (LTP). Several mechanisms have been proposed for HAND, including direct effects of viral proteins such as the Tat protein. Searching for the mechanisms involved, we found here that HIV-1 Tat inhibits E2F transcription factor 3 (E2F3), CAMP-responsive element-binding protein (CREB), and brain-derived neurotropic factor (BDNF) by up-regulating the microRNA miR-34a. These changes rendered murine neurons dysfunctional by promoting neurite retraction, and we also demonstrate that E2F3 is a specific target of miR-34a. Interestingly, bioinformatics analysis revealed the presence of an E2F3-binding site within the CREB promoter, which we validated with ChIP and transient transfection assays. Of note, luciferase reporter assays revealed that E2F3 up-regulates CREB expression and that Tat interferes with this up-regulation. Further, we show that miR-34a inhibition or E2F3 overexpression neutralizes Tat's effects and restores normal distribution of the synaptic protein synaptophysin, confirming that Tat alters these factors, leading to neurite retraction inhibition. Our results suggest that E2F3 is a key player in neuronal functions and may represent a good target for preventing the development of HAND.
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 vsynaptic 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.Although effective in prolonging the life expectancy in patients with HIV infection (1), the highly active antiretroviral therapy (HAART) 2 in fact has resulted in an increased prevalence of HIV-associated neurocognitive disorders (HAND) (2). The molecular mechanisms leading to this phenomenon remain to be elucidated. Several reports indicated that HIVinfected macrophages and microglia produce neurotoxins (e.g. viral and cellular proteins) that have the ability to cause neuronal deregulation and to promote HAND (3, 4). The viral protein R (Vpr) is among the released HIV-1 proteins that have been considered to be deleterious to neurons; however the molecular mechanisms involved remain unclear (5-7). Vpr is packaged into the virion, and is essential for HIV-1 replication in macrophages (8, 9). Infected brain cells have been shown to release Vpr that could affects neuronal growth (10). Vpr mediates multiple functions, including nuclear import of the HIV-1 pre-integration complex, G 2 cell cycle arrest, transactivation of both viral replication, and host genes and induction of apoptosis (11,12). Vpr was also detected in a soluble form in CSF and sera of HIV-infected patients displaying neurological disorders (10). Vpr causes neuronal degeneration both in vitro and in vivo (7,10,13).A calcium channel is an ion channel, which displays selective permeability to Ca 2ϩ ions. It is sometimes synonymous as voltage-dependent Ca 2ϩ channel (VDCC) (14). Activation of particular VDCCs allows Ca 2ϩ entry into the cell, which depending on the cell type, results in muscular contraction, excitation of neurons, up-regulation of gene expression, or release of neuro...
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