Shohreh Amini

CNS invasion by CD14+/CD16+peripheral blood-derived monocytes in HIV dementia: perivascular accumulation and reservoir of HIV infection

Increases in circulating CD14+/CD16+ monocytes have been associated with HIV dementia; trafficking of these cells into the CNS has been proposed to play an important role in the pathogenesis of HIV-induced neurological disorders. This model suggests that events outside the CNS leading to monocyte activation initiate the process leading to HIV dementia. To investigate the role of this activated monocyte subset in the pathogenesis of HIV dementia, we examined brain specimens from patients with HIV encephalopathy (HIVE), HIV without encephalopathy, and seronegative controls. An accumulation of perivascular macrophages was observed in HIVE. The majority of these cells identified in microglial nodules and in the perivascular infiltrate were CD14+/CD16+. P24 antigen colocalized with both CD14 and CD16 suggesting that the CD14+/CD16+ macrophage is a major reservoir of HFV-1 infection in CNS. Using CD45/LCA staining, the perivascular macrophage was distinguished from resident microglia. In addition to perivascular and nodular localizations, CD16 also stained ramified cells throughout the white matter. These cells were more ramified and abundant than cells positive for CD14 in white matter. Double staining for p24 and CD16 suggests that these cells were often infected with HIV-1. The prominent distribution of CD14+ cells in HIVE prompted our analysis of soluble CD14 levels in cerebrospinal fluid. Higher levels of soluble CD14 (sCD14) were observed in patients with moderate-to-severe HIV dementia, suggesting the utility of sCD14 as a surrogate marker. CD14+/CD16+ monocytes may play a role in other neurological disorders and sCDl4 may be useful for evaluating these conditions.

HIV-1 associated dementia: symptoms and causes

Despite the use of highly active antiretroviral therapy (HAART), neuronal cell death remains a problem that is frequently found in the brains of HIV-1-infected patients. HAART has successfully prevented many of the former end-stage complications of AIDS, however, with increased survival times, the prevalence of minor HIV-1 associated cognitive impairment appears to be rising among AIDS patients. Further, HIV-1 associated dementia (HAD) is still prevalent in treated patients as well as attenuated forms of HAD and CNS opportunistic disorders. HIV-associated cognitive impairment correlates with the increased presence in the CNS of activated, though not necessarily HIV-1-infected, microglia and CNS macrophages. This suggests that indirect mechanisms of neuronal injury and loss/death occur in HIV/AIDS as a basis for dementia since neurons are not themselves productively infected by HIV-1. In this review, we discussed the symptoms and causes leading to HAD. Outcome from this review will provide new information regarding mechanisms of neuronal loss in AIDS patients.

Molecular pathway involved in HIV-1-induced CNS pathology: role of viral regulatory protein, Tat.

The broad range of histological lesions associated with HIV‐1 are somewhat subtle relative to the clinical manifestations that occur as a result of HIV infection. Although it is clear that HIV has a causative role in CNS disease, dementia appears to be a consequence of the infiltration of inflammatory cells and cytokine dysregulation rather than the amount of virus in CNS. The HIV transregulatory protein Tat plays an important intracellular as well as extracellular role in the dysregulation of cytokines. The cytokines and possibly chemokines that are induced by Tat modify the action of astrocytes such that the survival of neurons is compromised. Pathogenetic alteration induced by Tat involves a series of interactions between circulating monocyte/macrophages, endothelial cells, and astrocytes. Cytokine dysregulation induced by viral infection and extracellular Tat leads to alterations in expression of adhesion molecules and promotes migration of non‐infected inflammatory cells into the CNS compartment. We demonstrate here that recombinant HIV‐1 Tat protein introduced by stereotaxic injection into mouse brain can induce pathologically relevant alterations including macrophage invasion as well as astrocytosis. The mechanism of destruction of the CNS by Tat appears to involve autocrine and paracrine pathways that depend not only on Tat, but cytokine and chemokine signaling pathways that are altered by viral infection. In this review, we discuss various pathogenic effects of Tat in brain cells and provide experimental evidence for an increased TNF‐α level in CSF in mice injected intracerebrally with Tat protein. J. Leukoc. Biol. 65: 458–465; 1999.

Detection of HIV-1 Tat and JCV capsid protein, VP1, in AIDS brain with progressive multifocal leukoencephalopathy

HIV-1 infection can lead to severe central nervous system (CNS) clinical syndromes in more than 50% of HIV-1 positive individuals. Progressive multifocal leukoencephalopathy (PML) is the frequent opportunistic infection of the CNS which is seen in as high as 5% of AIDS patients. Results from previous cell culture studies showed that the HIV-1 regulatory protein, Tat can potentiate transcription of the human neurotropic virus, JCV, the causative agent for PML in cells derived from the human CNS. In this communication we examine the presence of the HIV-1 regulatory protein, Tat, as well as the HIV-1 and JCV structural proteins, p24 and VP1, respectively in AIDS/PML clinical samples. We demonstrate high level expression of the JCV capsid protein, VP1, in oligodendrocytes and to some degree in astrocytes of AIDS with PML. In HIV-1+ samples expression of HIV-1 core protein, p24 was detected in perivascular monocytic cells and to a lesser extent in astrocytes and endothelial cells. A lack of p24 expression in oligodendrocytes suggested no infection of these cells with HIV-1. Interestingly, HIV-1 Tat was detected in various infected cells as well as in uninfected oligodendrocytes from HIV-1+ tissue, supporting the earlier in vitro findings that secreted Tat from the infected cells can be localized in the neighboring uninfected cells. The presence of Tat in oligodendrocytes was particularly interesting as this protein can up-modulate JCV gene transcription and several key cell cycle regulatory proteins including cyclin E, Cdk2, and pRb. The data presented here provide in vivo evidence for a role of HIV-1 Tat in the pathogenesis of AIDS/PML by acting as a positive regulatory protein that affects the expression of JCV and other cell regulatory proteins in the CNS.

Role of HIV-1 Vpr in AIDS pathogenesis: relevance and implications of intravirion, intracellular and free Vpr

Vpr, a 14-kDa, 96 amino acid protein, is conserved among the primate lentiviruses HIV-1, HIV-2 and Simian Immunodeficiency virus supporting the notion that it plays an important role in virus life cycle in vivo. Vpr appears to have several functions including cell cycle arrest at G2 stage, apoptosis, nuclear localization, nuclear import of the pre-integration complex, cation selective channel activity and transcriptionally activate HIV-1 LTR and other heterologous promoters. Over the years, we have addressed several issues pertaining to Vpr including the amount of Vpr present in the virus particles and structure-function relationship of Vpr. Here, we have reviewed the sources of Vpr that may potentially contribute to the cytopathic features observed in the context of HIV-1 infection. There are three different sources of Vpr available in the infected individuals to initiate the pathogenic effects. These include cell-associated, virion-associated (infectious, infectious-non productive, and non-infectious defective viruses) and free Vpr (cell-free and virus-free). A potential role of Vpr in neuropathogenesis of HIV infection in CNS was also suggested by early studies demonstrating neurotoxicity of recombinant Vpr protein. Interestingly, free Vpr (cell-free and virus-free) has been demonstrated in the serum of HIV-1 infected individuals and in the CSF of AIDS patients with neurological dysfunctions. Based on the toxic effects of extra-cellular Vpr on cells noted in several studies, it is likely that free Vpr could contribute to the bystander cell depletion in lymphoid tissues, peripheral blood, and the CNS. These results led us to propose a model for the role of Vpr in AIDS pathogenesis.

Evidence for BAG3 modulation of HIV-1 gene transcription†

A family of co‐chaperone proteins that share the Bcl‐2‐associated athanogene (BAG) domain are involved in a number of cellular processes, including proliferation and apoptosis. Among these proteins, BAG3 has received increased attention due to its high levels in several disease models and ability to associate with Hsp70 and a number of other molecular partners. BAG3 expression is stimulated during cell response to stressful conditions, such as exposure to high temperature, heavy metals, and certain drugs. Here, we demonstrate that BAG3 expression is elevated upon HIV‐1 infection of human lymphocytes and fetal microglial cells. Furthermore, BAG3 protein was detectable in the cytoplasm of reactive astrocytes in HIV‐1‐associated encephalopathy biopsies, suggesting that induction of BAG3 is part of the host cell response to viral infection. To assess the impact of BAG3 upregulation on HIV‐1 gene expression, we performed transcription assays and demonstrated that BAG3 can suppress transcription of the HIV‐1 long terminal repeat (LTR) in microglial cells. This activity was mapped to the κB motif of the HIV‐1 LTR. Results from in vitro and in vivo binding assays revealed that BAG3 suppresses interaction of the p65 subunit of NF‐κB with the κB DNA motif of the LTR. Results from binding and transcriptional assay identified the C‐terminus of BAG3 as a potential domain involved in the observed inhibitory effect of BAG3 on p65 activity. These observations reveal a previously unrecognized cell response, that is, an increase in BAG3, elicited by HIV‐1 infection, and may provide a new avenue for the suppression of HIV‐1 gene expression. J. Cell. Physiol. 210: 676–683, 2007.

Cooperative interaction of C/EBPβ and Tat modulates MCP-1 gene transcription in astrocytes

The chemoattractant protein 1 (MCP-1) is one of the most potent monocyte chemoattractants whose level is elevated during the course of AIDS dementia. Earlier studies showed that HIV-1 Tat protein is able to induce transcription of the MCP-1 promoter in astrocytic cells. Furthermore, the TGFbeta-1 signaling pathway through its regulatory proteins, Smads, modulates Tat activation of MCP-1. Here, we demonstrate that C/EBPbeta, whose activity is enhanced by a variety of cytokines during the course of viral infection, can stimulate basal- and Tat-mediated transcription of MCP-1 in human astrocytic cells. Results using promoter deletion mutants suggested the importance of multiple C/EBPbeta binding sites scattered within -200 to +1 of the MCP-1 promoter in the observed activity. Results from DNA binding studies have shown that the interaction of C/EBPbeta with its DNA motif is diminished by the C/EBPbeta homologous protein, CHOP, which possesses the ability to suppress the stimulatory effect of C/EBPbeta on MCP-1 transcription. Tat, which possesses the ability to interact with C/EBPbeta, alleviates the negative effect of CHOP and restores C/EBPbeta interaction with the DNA. Furthermore, Smad3 and its C-terminal regulatory motif, MH2, interact with C/EBPbeta and modulate its DNA binding and transcriptional activity on the MCP-1 promoter. Our results show that the physical and functional interactions of C/EBPbeta and Tat are severely affected by the presence of Smad3 and MH2. Altogether, these observations identify C/EBPbeta as a new partner for Tat in stimulating MCP-1 transcription in astrocytes and suggest that the delicate balance among the downstream regulatory proteins of several cytokines and immunomodulators can dictate the level of expression of chemoattractants, including MCP-1. Hence, inappropriate expression and function of regulatory proteins such as C/EBPbeta and Smads by Tat may induce MCP-1 production in astrocytes and contribute to the neuropathogenesis of AIDS through stimulation of inflammation in the CNS.

Role of HIV-1 Tat and CC Chemokine MIP-1α in the pathogenesis of HIV associated central nervous system disorders

Two syndromes affecting cognitive and motor function in the setting of AIDS have been described as HIV encephalopathy (HIVE) and progressive multifocal leukoencephalopathy (PML). HIVE is characterized by the presence of microglial nodules with accompanying astrocytosis. PML is a fatal demyelinating disease of the white matter induced by the human papovavirus JCV which causes cytolytic destruction of glial cells. In addition to the effect of HIV-1 induced immune suppression, HIV may act directly as a co-factor for stimulation of JCV replication in AIDS patients, in part due to Tat-induced activation of JCV gene transcription. Since Tat has been implicated in CNS pathogenesis, we examined its localization in CNS specimens from HIV infected patients with HIVE and PML as well as controls. Based on the observation of CC chemokine induction in monocytes by Tat, we also examined the cellular localization of the CC chemokine Macrophage Inflammatory Protein-1alpha (MIP-1alpha) and its cognate receptor CCR-5 in these samples. In HIVE, Tat was primarily localized in astrocytes and microglia, within the nodular lesions. In PML, a marked increase in the number of Tat positive astrocytes was observed. In both HIVE and PML, prominent expression of MIP-1alpha and CCR-5 was found within areas containing histopathological lesions. CCR-5 positivity of microglia was localized primarily to nodular lesions in HIVE. In PML, increased numbers of cells with monocyte/microglial morphology were observed relative to HIVE. The increased MIP-1 alpha positivity, and potentially other chemokines, may contribute to the pathogenesis of PML in the setting of HIV infection. Tat may play an important role in the pathogenesis of both HIV associated CNS disease states, acting indirectly through cytokine and chemokine dysregulation.

Cdk9 phosphorylates p53 on serine 392 independently of CKII

The tumor suppressor p53 is an important cellular protein, which controls cell cycle progression. Phosphorylation is one of the mechanisms by which p53 is regulated. Here we report the interaction of p53 with another key regulator, cdk9, which together with cyclin T1 forms the positive transcription elongation complex, p‐TEFb. This complex cooperates with the HIV‐1 Tat protein to cause the phosphorylation of the carboxyl terminal domain (CTD) of RNA polymerase II and this facilitates the elongation of HIV‐1 transcription. We demonstrate that cdk9 phosphorylates p53 on serine 392 through their direct physical interaction. Results from protein–protein interaction assays revealed that cdk9 interacts with the C‐terminal domain (aa 361–393) of p53, while p53 interacts with the N‐terminal domain of cdk9. Transfection and protein binding assays (EMSA and ChIP) demonstrated the ability of p53 to bind and activate the cdk9 promoter. Interestingly, cdk9 phosphorylates serine 392 of p53, which could be also phosphorylated by casein kinase II. Kinase assays demonstrated that cdk9 phosphorylates p53 independently of CKII. These studies demonstrate the existence of a feedback‐loop between p53 and cdk9, pinpointing a novel mechanism by which p53 regulates the basal transcriptional machinery. J. Cell. Physiol. 208: 602–612, 2006.

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.