During human immunodeficiency virus (HIV)-1 infection, perturbations in neuron-glia interactions may culminate in neuronal damage. Recently, purinergic receptors have been implicated in the promotion of virus-induced neurotoxicity and supporting the viral life cycle at multiple stages. The astrocytes robustly express purinergic receptors. We therefore sought to examine if P2X7R, a P2X receptor subtype, can mediate HIV-1 Tat-induced neuronal apoptosis. Tat augmented the expression of P2X7R in astrocytes. Our data reveal the involvement of P2X7R in Tat-mediated release of monocyte chemoattractant protein (MCP-1) /chemokine (C-C motif) ligand 2 (CCL2) from the astrocytes. P2X7R antagonists, such as the oxidized ATP, A438079, brilliant blue G, and broad spectrum P2 receptor antagonist suramin, attenuated Tat-induced CCL2 release in a calcium-and extracellular signal-regulated kinase (ERK)1/2-dependent manner. Calcium chelators, (1,2-bis(oaminophenoxy) ethane-N,N,N',N'-tetraacetic acid) acetoxymethyl ester and EGTA, and ERK1/2 inhibitor U0126 abolished chemokine (C-C motif) ligand 2 release from astrocytes. Furthermore, in human neuronal cultures, we demonstrated P2X7R involvement in Tat-mediated neuronal death. Importantly, in the TUNEL assay, the application of P2X7R-specific antagonists or the knockdown of P2X7R in human astrocytes reduced HIV-Tat-induced neuronal death significantly, underlining the critical role of P2X7R in Tat-mediated neurotoxicity. Our study provides novel insights into astrocyte-mediated neuropathogenesis in HIV-1 infection and a novel target for therapeutic management of neuroAIDS.
Cashew nut shell liquid (CNSL) has been used in traditional medicine for the treatment of a wide variety of pathophysiological conditions. To further define the mechanism of CNSL action, we investigated the effect of cashew nut shell extract (CNSE) on two matrix metalloproteinases, MMP-2/gelatinase A and MMP-9/gelatinase B, which are known to have critical roles in several disease states. We observed that the major constituent of CNSE, anacardic acid, markedly inhibited the gelatinase activity of 3T3-L1 cells. Our gelatin zymography studies on these two secreted gelatinases, present in the conditioned media from 3T3-L1 cells, established that anacardic acid directly inhibited the catalytic activities of both MMP-2 and MMP-9. Our docking studies suggested that anacardic acid binds into the MMP-2/9 active site, with the carboxylate group of anacardic acid chelating the catalytic zinc ion and forming a hydrogen bond to a key catalytic glutamate side chain and the C15 aliphatic group being accommodated within the relatively large S1Ј pocket of these gelatinases. In agreement with the docking results, our fluorescence-based studies on the recombinant MMP-2 catalytic core domain demonstrated that anacardic acid directly inhibits substrate peptide cleavage in a dosedependent manner, with an IC 50 of 11.11 M. In addition, our gelatinase zymography and fluorescence data confirmed that the cardol-cardanol mixture, salicylic acid, and aspirin, all of which lack key functional groups present in anacardic acid, are much weaker MMP-2/MMP-9 inhibitors. Our results provide the first evidence for inhibition of gelatinase catalytic activity by anacardic acid, providing a novel template for drug discovery and a molecular mechanism potentially involved in CNSL therapeutic action.
Of the various genetic subtypes of human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2) and simian immunodeficiency virus (SIV), only in subtype C of HIV-1 is a genetically variant NF-B binding site found at the core of the viral promoter in association with a subtype-specific Sp1III motif. How the subtype-associated variations in the core transcription factor binding sites (TFBS) influence gene expression from the viral promoter has not been examined previously. Using panels of infectious viral molecular clones, we demonstrate that subtype-specific NF-B and Sp1III motifs have evolved for optimal gene expression, and neither of the motifs can be replaced by a corresponding TFBS variant. The variant NF-B motif binds NF-B with an affinity 2-fold higher than that of the generic NF-B site. Importantly, in the context of an infectious virus, the subtype-specific Based on genetic variation, human immunodeficiency virus type 1 (HIV-1) is classified into four distinct groups (M, N, O, and P), and group M is subclassified into nine molecular subtypes (A, B, C, D, F, G, H, J, and K) and numerous circulating recombinant forms, including A/E (1). The global distribution of HIV-1 subtypes is uneven, with C, A, and B being the most widespread subtypes. Subtype C is predominant in southern and eastern African countries, India, and Nepal and in recombinant forms in China, and it is currently emerging in southern Brazil. Subtype C is responsible for approximately half of the global HIV-1 infections and more than 95% of the infections in India (2). The factors that contribute to the widespread expansion of subtype C are not well understood. Diverse viral subtypes differ from one another up to 30 to 35% in certain gene segments, such as the envelope (3). A genetic variation to this large an extent is expected to have a significant impact on the biological properties of the subtypes influencing their relative fitness properties. Subtype-specific genetic variations in elements such as the viral promoter (enhancer and other regulatory elements), regulatory proteins, and structural proteins may underlie the biological differences, although drawing such a correlation between these factors may not always be possible.The individual components of the viral promoter, including the modulator region, the enhancer, and the core promoter, are characterized by several subtype-specific molecular variations. Such differences have been mapped to many transcription factor binding sites (TFBS), including USF, c-Myb, NF-AT, Ap-1, NF-B, and Sp1, and regulatory elements such as the TATA box and
Extracellular Tat (eTat) plays an important role in HIV-1 pathogenesis. The presence of anti-Tat antibodies is negatively correlated with disease progression, hence making Tat a potential vaccine candidate. The cytotoxicity and moderate immunogenicity of Tat however remain impediments for developing Tat-based vaccines. Here, we report a novel strategy to concurrently enhance the immunogenicity and safety profile of Tat. The grafting of universal helper T-lymphocyte (HTL) epitopes, Pan DR Epitope (PADRE) and Pol711 into the cysteine rich domain (CRD) and the basic domain (BD) abolished the transactivation potential of the Tat protein. The HTL-Tat proteins elicited a significantly higher titer of antibodies as compared to the wild-type Tat in BALB/c mice. While the N-terminal epitope remained immunodominant in HTL-Tat immunizations, an additional epitope in exon-2 was recognized with comparable magnitude suggesting a broader immune recognition. Additionally, the HTL-Tat proteins induced cross-reactive antibodies of high avidity that efficiently neutralized exogenous Tat, thus blocking the activation of a Tat-defective provirus. With advantages such as presentation of multiple B-cell epitopes, enhanced antibody response and importantly, transactivation-deficient Tat protein, this approach has potential application for the generation of Tat-based HIV/AIDS vaccines.
In India, the low prevalence of HIV-associated dementia (HAD) in the Human immunodeficiency virus type 1 (HIV-1) subtype C infection is quite paradoxical given the high-rate of macrophage infiltration into the brain. Whether the direct viral burden in individual brain compartments could be associated with the variability of the neurologic manifestations is controversial. To understand this paradox, we examined the proviral DNA load in nine different brain regions and three different peripheral tissues derived from ten human subjects at autopsy. Using a highly sensitive TaqMan probe-based real-time PCR, we determined the proviral load in multiple samples processed in parallel from each site. Unlike previously published reports, the present analysis identified uniform proviral distribution among the brain compartments examined without preferential accumulation of the DNA in any one of them. The overall viral DNA burden in the brain tissues was very low, approximately 1 viral integration per 1000 cells or less. In a subset of the tissue samples tested, the HIV DNA mostly existed in a free unintegrated form. The V3-V5 envelope sequences, demonstrated a brain-specific compartmentalization in four of the ten subjects and a phylogenetic overlap between the neural and non-neural compartments in three other subjects. The envelope sequences phylogenetically belonged to subtype C and the majority of them were R5 tropic. To the best of our knowledge, the present study represents the first analysis of the proviral burden in subtype C postmortem human brain tissues. Future studies should determine the presence of the viral antigens, the viral transcripts, and the proviral DNA, in parallel, in different brain compartments to shed more light on the significance of the viral burden on neurologic consequences of HIV infection.
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