UMLS:C0019693 - FACTA Search

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Query: UMLS:C0019693 (HIV)
170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The HIV-1-specific virus protein U (Vpu) is an amphipathic membrane-associated phosphoprotein that probably possesses an N-terminal hydrophobic membrane anchor and a hydrophilic moiety. Because of an as yet undefined cytotoxic effect and the low concentration of Vpu in host cells, it has not been possible to obtain sufficient quantities of this protein for biochemical and structural investigations. We describe the synthesis, in two forms, of 50-residue peptides representing the polar cytoplasmic domain of Vpu: pVpu, comprising the wild-type Vpu sequence of HIV-1, strain HTLVIIIB, from position Ile32 to Leu81, and a mutant, pVpum2/6, with replacement of Ser52-56 with Asn. This mutation affects the two phosphorylation sites of Vpu for casein kinase-2 (CK-2), the enzyme that phosphorylates Vpu in HIV-1-infected cells. The peptides were purified to homogeneity and characterized by N-terminal sequencing, mass spectrometry and SDS-PAGE. Furthermore, both peptides were immunologically characterized by Western blot and ELISA using Vpu-specific monoclonal and polyclonal antibodies. Recombinant human CK-2 caused in vitro phosphorylation of pVpu, but had no effect on the mutant pVpum2/6. Investigation of the peptides by circular dichroism showed that addition of trifluoroethanol stabilized the alpha-helical secondary structure. Preliminary 1H NMR spectroscopic data indicated that, in the presence of trifluoroethanol, both peptides in solution have stable secondary structures with considerable alpha-helical content.
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PMID:Synthesis and characterization of the hydrophilic C-terminal domain of the human immunodeficiency virus type 1-encoded virus protein U (Vpu). 838 59

The electrophoretic mobility shift assays (EMSA) with the use of the synthetic HIV-1 NF-kappa B motif as a probe, showed that LPS-treatment of J774 cells (a mouse macrophage cell line) leads to the activation of the fast-moving (denoted as B1) and the slow-moving NF-kappa B (denoted as B2). The binding of both B1 and B2 to the NF-kappa B probe was inhibited specifically by either unlabelled NF-kappa B, or competitor probes, but not by unrelated probes. LPS-induced activation of NF-kappa B was inhibited by a protein kinase A (PKA) inhibitor (H-89), but not by a protein kinase C (PKC) inhibitor (H-7). PMA itself failed to activate NF-kappa B and the depletion of PKC did not prevent LPS-induced activation of NF-kappa B. The pre-treatment of J774 cells with dibutyric cAMP, forskolin, prostaglandin E2 or cholera toxin resulted in NF-kappa B activation. Thus, these data suggested a probable involvement of PKA in LPS-induced NF-kappa B activation in macrophages.
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PMID:Role of protein kinase A in LPS-induced activation of NF-kappa B proteins of a mouse macrophage-like cell line, J774. 839 97

The human immunodeficiency virus type 1 (HIV-1)-encoded vpu product is a small class 1 integral membrane protein that is phosphorylated by the ubiquitous casein kinase II (CKII) in HIV-1-infected cells. The Vpu protein facilitates the release of budding virions from the surface of infected cells and delays the rate of syncytium formation. In this study, we investigated the role of phosphorylation in the biological activity of Vpu. Our results show that phosphorylation of Vpu occurs on serine residues at positions 52 and 56 located in a highly conserved dodecapeptide sequence. Mutation of either Ser 56, or both Ser 52 and Ser 56 impaired the ability of Vpu to delay the rate of syncytium formation while retaining virion release activity at levels comparable to vpu+ proviruses. Flow cytometry analysis indicates that the relative amounts of envelope glycoprotein gp120 expressed at the surface of cells transfected with these vpu mutant proviruses was two- to threefold greater than that observed on cells transfected with a vpu+ provirus. This increased expression of gp120 at the cell surface may explain the more rapid onset of syncytium formation observed in cell transfected with vpu mutant proviruses. These results suggest that Vpu-facilitated virion release and delayed cytopathic effect are the consequence of two distinct functional activities of the protein.
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PMID:Functional analysis of the phosphorylation sites on the human immunodeficiency virus type 1 Vpu protein. 854 40

HIV-1 associated central nervous system (CNS) disease involves neuronal damage and prominent reactive astrocytosis, the latter characterized by strong upregulation of the glial fibrillary acidic protein (GFAP) in astrocytes. Similar alterations are found in transgenic mice expressing the HIV-1 envelope protein gp120 in the CNS. Because alterations of astrocyte functions could contribute to neuronal impairment, we compared brains of gp120 transgenic mice and gp120-transfected C6 astrocytoma cells with controls and found that gp120 induced a prominent elevation of steady state GFAP mRNA levels, primarily due to transcript stabilization. Increased levels of GFAP mRNA were also found in nontransfected C6 cells exposed to recombinant gp120. Exposure of C6 cells or primary mouse astrocytes to soluble gp120 led to activation of PKC as indicated by redistribution and increase in PKC immunoreactivity at the single cell level. gp120 effects were diminished by inhibitors of protein kinase C (PKC) but not inhibitors of protein kinase A. PKC activity was upmodulated in gp120-transfected C6 cells and in the CNS of gp120 transgenic mice. Further, brain tissue from patients with HIV-1 encephalitis and from gp120 transgenic mice showed increased PKC immunoreactivity. Taken together, these results indicate that gp120-induced increases in PKC activity may contribute to the gliosis seen in gp120 transgenic mice as well as in HIV-1-infected humans and raise the question of whether dysregulation of signal transduction pathways represents a general mechanism of HIV-associated pathogenesis.
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PMID:Dysregulation of signal transduction pathways as a potential mechanism of nervous system alterations in HIV-1 gp120 transgenic mice and humans with HIV-1 encephalitis. 860 36

Vif is a 23-kDa protein encoded by human immunodeficiency virus, type 1 (HIV-1) which is important for virion infectivity. Here, we describe the phosphorylation of HIV-1 Vif and its role in HIV-1 replication. In vivo studies demonstrated that Vif is highly phosphorylated on serine and threonine residues. To identify phosphorylation sites and characterize the Vif kinase(s), Vif was expressed in Escherichia coli and purified for use as a substrate in in vitro kinase assays. The purified Vif protein was phosphorylated in vitro on serine and threonine residues by a kinase(s) present in both cytosol and membrane fractions. Phosphorylation of Vif was stimulated by phorbol 12-myristate 13-acetate and inhibited by staurosporine and hypericin, a drug with potent anti-HIV activity. The Vif kinase(s) was resistant to inhibitors of protein kinase C, cAMP-dependent kinase, and cGMP-dependent kinase, suggesting that it is distinct from these enzymes. To identify the phosphorylation sites, 32P-labeled Vif was digested by V8 protease and the peptides were resolved by reverse-phase high performance liquid chromatography. Radioactive peptide sequencing identified three phosphorylation sites within the C terminus, Ser144, Thr155, and Thr188. Two-dimensional tryptic phosphopeptide mapping indicated that these sites are also phosphorylated in vivo. Both Ser144 and Thr188 are contained in the recognition motifs (R/KXXS*/T* and R/KXXXS*/T*) used by serine/threonine protein kinases such as cGMP-dependent kinase and PKC. Ser144 is present in the motif SLQXLA, which is the most highly conserved sequence among all lentivirus Vif proteins. Mutation of Ser144 to alanine resulted in loss of Vif activity and >90% inhibition of HIV-1 replication. These studies suggest that phosphorylation of Vif by a serine/threonine protein kinase(s) plays an important role in regulating HIV-1 replication and infectivity.
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PMID:Phosphorylation of Vif and its role in HIV-1 replication. 862 71

The Rev protein of HIV-1 is essential for the nuclear export of incompletely spliced viral mRNAs. This action depends on the mutationally defined Rev activation domain, which both binds the nucleoporin-like human cellular cofactor Rab/hRIP and also functions as a nuclear export signal. Protein kinase inhibitor alpha (PKI) also contains a potent nuclear export signal. However, PKI plays no role in nuclear RNA export and instead induces the nuclear export of a specific protein target, the catalytic subunit of cAMP-dependent protein kinase. Here, it is demonstrated that the nuclear export signal of PKI not only binds the Rab/hRIP cofactor specifically but also can effectively substitute for the Rev activation domain in mediating the nuclear export of HIV-1 mRNAs. We conclude that HIV-1 Rev and PKI act through an identical nuclear export pathway and that Rev, rather than using a dedicated RNA export pathway, is instead acting as an adaptor that allows viral mRNAs to access a cellular protein export pathway.
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PMID:Nuclear export of late HIV-1 mRNAs occurs via a cellular protein export pathway. 863 82

The serine/threonine protein kinase Raf-1 is a component of a conserved intracellular signaling cascade that controls responses to various extracellular stimuli. Transcription from several promoters, including the oncogene-responsive element in the polyomavirus enhancer, the c-fos promoter, as well as other AP-1- and Ets-dependent promoters, can be induced by Raf-1 kinase. Previously, we have shown that activated Raf-1 kinase transactivates the human immunodeficiency virus type 1 (HIV-1) long terminal repeat and have identified the NF-kappaB binding motif as a Raf-1-responsive element (RafRE). We now report that Raf-1 kinase-induced transactivation from the HIV RafRE involves the purine-rich-repeat-binding protein (GABP), which is composed of two distinct subunits (alpha and beta). GABP alpha is an Ets oncogene-related DNA-binding protein, and GABP beta contains four ankyrin-like repeats that have been shown to be essential in protein-protein interactions. In electrophoretic mobility shift assays using nuclear extracts from human Jurkat T cells, a protein-DNA complex which was supershifted with antiserum against GABP alpha and GABP beta was observed. Purified recombinant GABP alpha and beta interact with the HIV RafRE as judged from DNA binding assays. Cotransfection experiments with GABP alpha and beta and Raf-1 kinase demonstrate synergistic transactivation of the HIV-1 promoter. Point mutations in the HIV RafRE abolished the Raf-1 kinase as well as GABP alpha- and beta-induced transactivation. The observed Raf-1-GABP synergism presumably involves phosphorylation of GABP subunits, as treatment of cells with Raf-1 kinase activators serum and 12-O-tetradecanoylphorbol-13-acetate increases phosphorylation of GABP in vivo. However, GABP is not a target of Raf-1 kinase; instead, it is a substrate of mitogen-activated protein kinase (MAPK/ERK), since in vitro phosphorylation of GABP alpha and beta was achieved by the reconstituted protein kinase cascade but not with purified Raf-1 or MEK. These results suggest that Raf-1 kinase- induced activation of the HIV-1 promoter is mediated by the classical cytoplasmic cascade resulting in MAPK/ERK-mediated phosphorylation of GABP alpha and beta. Because the HIV RafRE corresponds to a region within the promoter which is essential for regulation of HIV-1 expression, the data indicate that in addition to NK-kappaB, GABP transcription factors are important for induced expression of HIV.
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PMID:Raf-1 kinase targets GA-binding protein in transcriptional regulation of the human immunodeficiency virus type 1 promoter. 864 52

Vpr is a 96-amino-acid protein encoded by human immunodeficiency virus type 1 (HIV-1) that prevents proliferation of infected cells. We have established a system for infection of 100% of a T-cell population with HIV and use this system to show that within the context of HIV-1 infection, Vpr is primarily cytostatic rather than cytotoxic. Vpr acts upstream of dephosphorylation of the mitotic cyclin-dependent kinase, and causes infected cells to accumulate in the G2 stage of the cell cycle. However, some HIV-1 infected cells increase in ploidy and size, accumulating DNA to an 8N level. Furthermore, the mechanism of the Vpr mitotic block is qualitatively different from that of G2 DNA damage checkpoint control.
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PMID:Human immunodeficiency virus type 1 cell cycle control: Vpr is cytostatic and mediates G2 accumulation by a mechanism which differs from DNA damage checkpoint control. 864 59

Persistent human immunodeficiency virus (HIV) infection of human monocytes and macrophages increases I kappa B alpha degradation, resulting in the activation of NF-kappa B, a key transcription factor in the regulation of the HIV long terminal repeat. The signal transduction pathways leading to NF-kappa B activation in cells of the monocytic lineage, especially those regulated by HIV infection, and their relevance in regulating viral persistence remain unknown. Both p21ras and its downstream Raf-1 kinase participate in the transduction of signals initiated from a variety of cell surface receptors and in the regulation of transcription factors. We have studied whether the Ras-Raf pathway is functional and participates in HIV-mediated NF-kappa B activation in monocytic cells. Constitutively active p21ras (v-H-Ras) activated NF- kappa B-dependent transcription and induces the nuclear translocation of a bona fide p65/p50 heterodimer by targeting I kappa B alpha. In addition, the constitutively active form of Raf (RafBXB) also increases the NF-kappa B-dependent transcriptional activity. Because of the similarity between HIV and Ras-Raf-induced NF-kappa B activation in monocytic cells, we next tested whether HIV-induced NF-kappa B activation was mediated by the Ras-Raf signal transduction pathway. Negative dominant forms of both Ras (Ras N17) and Raf (Raf 301) decreased the HIV- but not lipopolysaccharide-dependent NF-kappa B activation in U937 cells. Moreover, Raf-1 kinase activity was greater in HIV-infected than uninfected monocytic cells in in vitro kinase assays. Altogether, these results indicate that the Ras-Raf pathway is unregulated in HIV monocytic cells and participates in the virus-induced activation of NF-kappa B.
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PMID:The Ras-Raf pathway is activated in human immunodeficiency virus-infected monocytes and particpates in the activation of NF-kappa B. 864 60

The HIV-1 Vpu protein induces the proteolysis of CD4 in the endoplasmic reticulum (ER) and enhances the release of virus particles from the plasma membrane. The two biological activities of HIV-1 Vpu appear to be reconstituted in distinct membrane compartments of the mammalian cell. We carried out experiments to understand the role of Vpu sequences in membrane trafficking of the Vpu protein and to gain insights into Vpu-mediated proteolytic reactions. To this end, we generated CD4/Vpu hybrid proteins and analyzed their biochemical and biological properties in HeLa cells. We show here that all hybrid proteins are delivered to the plasma membrane undergoing endo-H-resistant modifications in the Golgi complex. Importantly, a hybrid protein bearing the CD4 extracellular domain and full-length Vpu induced the degradation of HIV envelope glycoproteins bearing the transmembrane and cytoplasmic domains of CD4 (Vpu-responsive elements, VRE). Glycoproteins lacking the VRE are stable under these conditions. In addition, a hybrid protein having the extracellular-transmembrane domains of CD4 and the Vpu cytoplasmic domain was only partially active in inducing the degradation of Vpu-sensitive proteins. These results suggest that the Vpu transmembrane domain is capable of regulating Vpu activity in the cell. Mutational studies have further demonstrated that casein kinase-2 phosphorylation is critically important in the degradation reaction, but does not regulate membrane trafficking of the CD4/Vpu hybrid proteins. We also show that the CD4 extracellular domain appended to the Vpu protein is protected from degradation while existing in a complex with Vpu-sensitive ectodomains. Taken together, these studies have revealed that the Vpu protein does not possess sequences that have the ability to sequester CD4 in the intracellular compartments of mammalian cells and that the Vpu protein tethered to the CD4 extracellular domain was biologically active in inducing the degradation of VRE-bearing glycoproteins in the ER.
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PMID:The human immunodeficiency virus type 1 Vpu protein tethered to the CD4 extracellular domain is localized to the plasma membrane and is biologically active in the secretory pathway of mammalian cells: implications for the mechanisms of Vpu function. 865 6

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