Thersa Sweet

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.

Developmental Expression of Wnt Signaling Factors in Mouse Brain

The Wnt signaling pathway has been implicated in a variety of biological events inducing neurogenesis. In this study, we aim to investigate the expression pattern of various components of the Wnt pathway including b-catenin and its partners LEF-1/TCF-4, GSK-3β and their nuclear target genes such as c-myc and cyclin D1 during mouse brain development. We performed a series of Western blot and immunohistochemistry of brain cortex, brainstem, and cerebellum which revealed differential accumulation of these proteins in different types of brain cells including neurons, astrocytes, and oligodendrocytes at different developmental stages. Intense cytoplasmic immunolabeling of β-catenin in 5 day old neurons throughout the cortex and brainstem significantly decreased as the brain developed, whereas the level of GSK-3β, the protein that phosphorylates β-catenin and causes its destabilization, increased during brain maturation. On the other hand, high level accumulation of LEF-1 and TCF-4 in neurons and astrocytes at the early stage of brain development diminished at the later stages. Interestingly, while the majority of LEF-1 and TCF-4 immunoreactivity was detected in the cytoplasm of neurons, it was evident that both proteins accumulated in the nuclei of astrocytes. Examination of cyclin D1, a protein that controls the cell cycle and proliferation, exhibited an intense staining in the nuclei of astrocytes throughout brain parenchyma during development. Interestingly, cyclin D was found in the cytoplasm of neurons from cortex, brainstem, and cerebellum during brain development. These data provide compelling evidence for the differential expression of the Wnt signaling pathway during brain development, and suggest that these signaling pathways may function differently in various brain regions and cell types.

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.

Molecular biology and immunoregulation of human neurotropic JC virus in CNS

The human polyomavirus, JC virus (JCV), provides an excellent model system to investigate the reciprocal interaction of the immune and nervous systems. Infection with JCV occurs during childhood and the virus remains in the latent state with no apparent clinical symptoms. However, under immunosuppressed conditions, the virus enters the lytic cycle and upon cytolytic destruction of glial cells, causes the fatal demyelinating disease of the central nervous system (CNS), named progressive multifocal leukoencephalopathy (PML). In this short review, we discuss the molecular pathogenesis of PML by highlighting the role of the immune system in modulating JCV gene activation and replication, and the latency/reactivation of this virus upon immunosuppression. Further, due to the higher incidence of PML among AIDS patients, we further elaborate on the cross‐talk between JCV and HIV‐1 by direct and indirect pathways that lead to enhanced expression of the JCV genome.

Evidence for Involvement of Transforming Growth Factor β1 Signaling Pathway in Activation of JC Virus in Human Immunodeficiency Virus 1–Associated Progressive Multifocal Leukoencephalopathy

CONTEXT Progressive multifocal leukoencephalopathy is a fatal demyelinating disease of the central nervous system frequently seen in patients with impaired immune systems, particularly acquired immunodeficiency syndrome. JC virus (JCV), a human neurotropic polyomavirus, is the etiologic infectious agent of this disease. OBJECTIVE The significantly higher incidence of progressive multifocal leukoencephalopathy in patients with acquired immunodeficiency syndrome than in patients with other immunosuppressive conditions suggests that molecular interactions between human immunodeficiency virus 1 and JCV, via the Tat protein, are responsible for the activation of the JCV enhancer/promoter and the development of progressive multifocal leukoencephalopathy. An indirect mechanism through activation of cytokines, such as transforming growth factor beta1 and Smads 3 and 4, may also be responsible for the enhancement of JCV gene expression. DESIGN Immunohistochemical analysis in progressive multifocal leukoencephalopathy samples and chloramphenicol acetyl transferase assays on cell cultures were performed to corroborate this hypothesis. RESULTS The JCV capsid protein VP-1 was found in the nuclei of oligodendrocytes and in the nuclei and cytoplasm of bizarre astrocytes. Human immunodeficiency virus proteins, including p24 and Tat, were detected in the cytoplasm of astrocytes. Tat, but not p24, was detected in oligodendrocytes, suggesting that extracellular Tat accumulates in the nuclei of oligodendrocytes, where JCV gene transcription takes place. High levels of transforming growth factor beta1 and Smads 3 and 4 were detected in JCV-infected oligodendrocytes. Results from in vitro studies confirm activation of the JCV early and late promoters by Smads 3 and 4. CONCLUSIONS These observations support our model, suggesting that the induction of transforming growth factor beta1 by human immunodeficiency virus 1 Tat can stimulate its downstream factors, including Smads 3 and 4, which in turn augment transcription of the JCV promoter in glial cells.

Evidence for Activation of the TGF-β1 Promoter by C/EBPβ and Its Modulation by Smads

The transforming growth factor-beta1 (TGF-beta1) is a cytokine involved in many biological events inlcuding immunosuppression, angiogenesis, cell growth, and apoptosis. Expression of TGF-beta1 at the transcriptional level is controlled by a series of ubiquitous and specialized factors whose activities can be modulated by a variety of signaling events. Here we demonstrate that activity of the TGF-beta1 promoter is increased by C/EBPbeta, a DNA-binding transcription factor whose activity can be influenced by several immunomodulators, in astrocytes and microglial cells. Interestingly, expression of Smad3 and Smad4, the downstream regulators of the TGF-beta1-signaling pathway, impairs the activity of C/EBPbeta on the TGF-beta1 promoter. Further, we demonstrate that MH2, a common domain among Smads that has protein-binding activities, interacts with C/EBPbeta and decreases its association with a region of the TGF-beta1 promoter that is responsive to C/EBPbeta activation. Interestingly, the p65 subunit of nuclear factor-kappaB (NF-kappaB), which also interacts with C/EBPbeta, cooperates with MH2 and decreased DNA-binding and transcriptional activities of C/EBPbeta on the TGF-beta1 promoter. These observations indicate that an autoregulatory mechanism, involving the MH2 domain of Smads, modulates activation of the TGF-beta1 promoter by C/EBPbeta. Further, our results show that the interplay between NF-kappaB and C/EBPbeta has an impact on the ability of C/EBPbeta to stimulate TGF-beta1 transcription, hence, suggesting that the cross-communication of signaling pathways that modulate NF-kappaB and C/EBPbeta may dictate the level of TGF-beta1 promoter activity.

IGF-IR dependent expression of Survivin is required for T-Antigen mediated Protection from Apoptosis and Proliferation of Neural Progenitors

The insulin-like growth factor-1 receptor (IGF-IR) and the human polyomavirus JCV protein, T-antigen cooperate in the transformation of neuronal precursors in the cerebellum, which may be a contributing factor in the development of brain tumors. Because it is not clear why T-antigen requires IGF-IR for transformation, we investigated this process in neural progenitors from IGF-IR knockout embryos (ko-IGF-IR) and from their wild-type nontransgenic littermates (wt-IGF-IR). In contrast to wt-IGF-IR, the brain and dorsal root ganglia of ko-IGF-IR embryos showed low levels of the antiapoptotic protein Survivin, accompanied by elevated numbers of apoptotic neurons and an earlier differentiation phenotype. In wt-IGF-IR neural progenitors in vitro, induction of T-antigen expression tripled the expression of Survivin and accelerated cell proliferation. In ko-IGF-IR progenitors induction of T-antigen failed to increase Survivin, resulting in massive apoptosis. Importantly, ectopic expression of Survivin protected ko-IGF-IR progenitor cells from apoptosis and siRNA inhibition of Survivin activated apoptosis in wt-IGF-IR progenitors expressing T-antigen. Our results indicate that reactivation of the antiapoptotic Survivin may be a critical step in JCV T-antigen-induced transformation, which in neural progenitors requires IGF-IR.

Interplay between NFBP and NF‐κB modulates tat activation of the LTR

Interplay of the HIV‐1 regulatory protein, Tat, with several cellular factors plays an important role in transcriptional regulation of the viral promoter, the long terminal repeat (LTR). Special attention has been paid to NF‐κB, a family of inducible transcription factors, which interact with a specific DNA motif within the LTR. Here, we report on the physical and functional interaction of NFBP, a recently identified protein that interacts with the P65 subunit of NF‐κB, with HIV‐1 Tat. NFBP colocalizes with Tat in the nucleus and nucleoli, recognizes the amino acid residues 37 to 48 of Tat, and its interaction is modulated by RNA molecules. The interaction of NFBP with Tat modulates the synergism between Tat and P65 in activating LTR transcription. In the absence of the κB‐binding sites, NFBP augments the TAR‐dependent activation by Tat, yet it interferes with the synergistic effect of P65 and Tat on LTR transcription.

Evidence for involvement of NFBP in processing of ribosomal RNA.

Ribosomal RNA (rRNA) in vertebrates is initially transcribed as a single 47S precursor which is modified by the addition of 2′‐O‐methyl ribose moieties, pseudouridines, and methyl groups, followed by cleavage at several sites to produce the mature 28S, 18S, and 5.8S rRNAs. Cleavage of the rRNA precursor to generate the 18S rRNA is mediated by a ribonucleoprotein (RNP) complex termed the processome containing U3, a box C/D small nucleolar RNA (snoRNA), and at least 28 cellular proteins. We previously identified a novel human RNA binding protein, NF‐κB binding protein (NFBP), which is the human homolog of Rrp5p, a protein component of the yeast U3 processome. Here, we show that NFBP colocalizes with and coprecipitates U3 in the nucleolus. We also demonstrate that NFBP is essential for the generation of 18S rRNA as maturation of the 18S rRNA is repressed in the absence of NFBP. Using Northern blot analyses, we further show that NFBP is specifically necessary for cleavages at sites A0, 1, and 2, as unprocessed intermediate forms of rRNA accumulated in the absence of NFBP. J. Cell. Physiol. 214: 381–388, 2008.