Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UMLS:C0019693 (HIV)
 170,526 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Human immunodeficiency virus type 1 (HIV-1) Tat protein is released from infected cells. Extracellular Tat enters the cell where it stimulates the transcriptional activity of HIV-long terminal repeat (LTR) and of endogenous genes. Heparin modulates the angiogenic (Albini, A., Benelli, R., Presta, M., Rusnati, M., Ziche, M., Rubartelli, A., Paglialunga, G., Bussolino, F., and Noonan, D. (1996) Oncogene 12, 289-297) and transcriptional (Mann, D. A., and Frankel, A. D. (1991) EMBO J. 10, 1733-1739) activity of extracellular Tat. Here we demonstrate that heparin binds specifically to recombinant HIV-1 Tat produced as glutathione S-transferase (GST) fusion protein and immobilized on glutathione-agarose beads. Heparin and heparan sulfate (HS), but not dermatan sulfate, chondroitin sulfates A and C, hyaluronic acid, and K5 polysaccharide, competed with 3H-labeled heparin for binding to immobilized GST-Tat and inhibited HIV-LTR transactivation induced by extracellular GST-Tat. Selective 2-O-, 6-O-, total-O-desulfation, or N-desulfation/N-acetylation dramatically reduced the capacity of heparin to bind GST-Tat. Totally-O-desulfated and 2-O-desulfated heparins also showed a reduced capacity to inhibit the transactivating activity of GST-Tat. Very low molecular weight heparins showed a significant decrease in their capacity to bind GST-Tat and to inhibit its LTR transactivating activity when compared with conventional 13.6-kDa heparin. However, when 3.0-kDa heparin was affinity chromatographed on immobilized GST-Tat to isolate binding and non-binding subfractions, the Tat-bound fraction was >/=1,000 times more potent than the unbound fraction in inhibiting the transactivating activity of GST-Tat. The results demonstrate that Tat interacts in a size-dependent manner with heparin/HS and that high affinity Tat-heparin interaction requires at least some 2-O-, 6-O-, and N-positions to be sulfated. The Tat binding activity of the glycosaminoglycans tested correlates with their capacity to affect the transactivating activity of extracellular Tat, indicating the possibility to design specific heparin/HS-like structures with Tat-antagonist activity.
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PMID:Interaction of HIV-1 Tat protein with heparin. Role of the backbone structure, sulfation, and size. 911 Oct 37

Human proliferation-associated protein p120 has previously been shown to localize to the nucleolus, and several functional domains of p120 have been elucidated. By using a nitrocellulose filter binding assay and a Northwestern blotting procedure this study shows that recombinant p120 binds to an rRNA fragment in vitro with a dissociation constant of 4 nM. The specific RNA-binding region of p120 (residues 1-57) was identified with glutathione S-transferase-fused p120 deletion constructs and Northwestern blotting procedures. This RNA-binding region of p120, which includes the nucleolar localization signal of p120, is similar to the arginine-rich RNA-binding regions found in other RNA-binding proteins such as HIV Rev and Tat. Experiments in vivo with HeLa cell nucleolar extracts showed that p120 was associated with the 60-80S pre-ribosomal particles. This association is disrupted by treatment with either RNase A or buffer of high ionic strength. These results suggest that p120 might be involved in rRNA/ribosome maturation, consistent with the role of the yeast homologue Nop2p in rRNA biogenesis.
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PMID:Nucleolar protein p120 contains an arginine-rich domain that binds to ribosomal RNA. 953 75

Nef is a membrane-associated cytoplasmic phosphoprotein that is well conserved among the different human (HIV-1 and HIV-2) and simian immunodeficiency viruses and has important roles in down-regulating the CD4 receptor and modulating T-cell signaling pathways. The ability to modulate T-cell signaling pathways suggests that Nef may physically interact with T-cell signaling proteins. In order to identify Nef binding proteins and map their site(s) of interaction, we targeted a highly conserved acidic sequence at the carboxyl-terminal region of Nef sharing striking similarity with an acidic sequence at the c-Raf1-binding site within the Ras effector region. Here, we used deletion and site-specific mutagenesis to generate mutant Nef proteins fused to bacterial glutathione S-transferase in in vitro precipitation assays and immunoblot analysis to map the specific interaction between the HIV-1LAI Nef and c-Raf1 to a conserved acidic sequence motif containing the core sequence Asp-Asp-X-X-X-Glu (position 174-179). Significantly, we demonstrate that substitution of the nonpolar glycine residue for either or both of the conserved negatively charged aspartic acid residues at positions 174 and 175 in the full-length recombinant Nef protein background completely abrogated binding of c-Raf1 in vitro. In addition, lysates from a permanent CEM T-cell line constitutively expressing the native HIV-1 Nef protein was used to coimmunoprecipitate a stable Nef-c-Raf1 complex, suggesting that molecular interactions between Nef and c-Raf1, an important downstream transducer of cell signaling through the c-Raf1-MAP kinase pathway, occur in vivo. This interaction may account for the Nef-induced perturbations of T-cell signaling and activation pathways in vitro and in vivo.
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PMID:Binding of c-Raf1 kinase to a conserved acidic sequence within the carboxyl-terminal region of the HIV-1 Nef protein. 962 70

Heparin binds extracellular HIV-1 Tat protein and modulates its HIV long terminal repeat (LTR)-transactivating activity (M. Rusnati, D. Coltrini, P. Oreste, G. Zoppetti, A. Albini, D. Noonan, F. d'Adda di Fagagna, M. Giacca, and M. Presta (1997) J. Biol. Chem. 272, 11313-11320). On this basis, the glutathione S-transferase (GST)-TatR49/52/53/55/56/57A mutant, in which six arginine residues within the basic domain of Tat were mutagenized to alanine residues, was compared with GST-Tat for its capacity to bind immobilized heparin. Dissociation of the GST-TatR49/52/53/55/56/57A.heparin complex occurred at ionic strength significantly lower than that required to dissociate the GST-Tat.heparin complex. Accordingly, heparin binds immobilized GST-Tat and GST-TatR49/52/53/55/56/57A with a dissociation constant equal to 0.3 and 1.0 microM, respectively. Also, the synthetic basic domain Tat-(41-60) competes with GST-Tat for heparin binding. Suramin inhibits [3H]heparin/Tat interaction, 125I-GST-Tat internalization, and the LTR-transactivating activity of extracellular Tat in HL3T1 cells and prevents 125I-GST-Tat binding and cell proliferation in Tat-overexpressing T53 cells. The suramin derivative 14C-PNU 145156E binds immobilized GST-Tat with a dissociation constant 5 times higher than heparin and is unable to bind GST-TatR49/52/53/55/56/57A. Although heparin was an antagonist more potent than suramin, modifications of the backbone structure in selected suramin derivatives originated Tat antagonists whose potency was close to that shown by heparin. In conclusion, suramin derivatives bind the basic domain of Tat, prevent Tat/heparin and Tat/cell surface interactions, and inhibit the biological activity of extracellular Tat. Our data demonstrate that tailored polysulfonated compounds represent potent extracellular Tat inhibitors of possible therapeutic value.
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PMID:The basic domain in HIV-1 Tat protein as a target for polysulfonated heparin-mimicking extracellular Tat antagonists. 963 53

GST-Gag(p55) binds specifically to HIV-1 RNA sequences 1-406, in vitro, with a Kd of about 50 nM. This RNA transcript contains a number of stem loop (SL) structures. The binding is due to the Gag moiety of the fusion protein, not GST. There is a high affinity binding site for Gag in an RNA containing nucleotides 325-362. SL4 is predicted by both biochemical studies and computer folding to be located between nucleotides 335 and 358. An RNA transcript ending at nucleotide 335 does not bind Gag. The deletion of nucleotides 334-358 from HIV-1 RNAs does not affect Gag binding. Digestions with RNase V1 and T1 show that nucleotides 297-300 in SL2, 310, 312, 313, 315, 317, 318, 325 in SL3, and 342 and 343 in SL4 are protected in the presence of Gag. The cleavage of nucleotides 348-351 in SL4 by RNAse V1 is enhanced by Gag binding. At least two Gag binding sites are therefore located in the leader RNA. Those located 5' of nucleotide 335 require the presence of additional 3' sequences.
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PMID:HIV-1 Gag binds specifically to RNA stem-loops in the 5' leader sequence. 965 23

The HIV-1 promoter was used as a model to identify transcription factors involved in LPS-dependent transcription in RAW 264 murine macrophages. Expression plasmids for Ets-2 and PU.1 trans-activated the HIV-1 LTR and recombinant PU.1 and an Ets-2 DNA binding domain/GST fusion protein bound to the 5' kappa B site of the HIV-1 enhancer. Ets-2 mRNA was LPS-inducible in RAW 264 cells and LPS stimulated phosphorylation of threonine 72 residue within the Ets-2 pointed domain. Induction of Ets-2 and other LPS-responsive transcription factors was also observed upon addition of plasmid DNA, which complicates interpretation of transient transfections. The proximal promoter region, containing two Sp1 sites, was also LPS-responsive. We propose that the kappa B elements and the tandem Sp1 sites act as LPS response elements and that kappa B-mediated LPS action involves Ets and rel factors.
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PMID:Involvement of Ets, rel and Sp1-like proteins in lipopolysaccharide-mediated activation of the HIV-1 LTR in macrophages. 965 43

The transmembrane protein of HIV-1, gp41, mediates fusion between membranes of the virus and target cell. Strong interaction between the helical regions in the ectodomain of gp41 has been exploited to develop a method that can detect a potential inhibitor against gp41. The N-terminus coiled-coil or the C-terminus helical sequences within the ectodomain of gp41 were inserted into the C-terminus of thioredoxin (Trx) or glutathione S-transferase (GST) to generate the fusion proteins, Trx-N and GST-C, respectively. The inserted sequences of GST-C and Trx-N cause the two proteins to interact with each other and to form a complex. Furthermore, GST-C binds specifically to the surface-coated Trx-N, and the amount of attached GST-C is detected by an ELISA assay using anti-GST antibodies. Peptides derived from the helical regions of gp41 compete with GST-C for binding to Trx-N as well as prevent the gp41-mediated cell fusion. This in vitro assay system can be applied to screening compounds that have an inhibitory activity against gp41.
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PMID:Development of an in vitro assay system for screening of gp41 inhibitory compounds. 989 25

Epithelin/granulin growth factor is synthesized as a 593 amino acid precursor protein that contains 7.5 imperfectly conserved repeats of approximately 57 amino acids. Processed epithelin/granulin peptides have been isolated from vertebrate/invertebrate species and are growth factors implicated in epithelial and haemic cell function. Here they are identified as Human Immunodeficiency Virus (HIV) Tat binding proteins using the yeast two-hybrid assay. Intracellularly in yeast, mutation of selected cysteines in an epithelin/granulin dimeric repeat caused loss of binding to Tat exon 1. In vitro binding of HIV-1 and HIV-2 Tat to epithelin/granulin dimeric and monomeric repeats was also observed by GST-glutathione bead "pulldown" assays. Because Tat is actively secreted from HIV-infected cells and has been shown to serve as a mitogenic factor for angiogenesis and for Kaposi-like cells, our observations suggest that epithelin/granulin growth factors may function as biologically important extracellular Tat co-factors.
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PMID:Epithelin/granulin growth factors: extracellular cofactors for HIV-1 and HIV-2 Tat proteins. 1007 80

The active form of HIV-1 reverse transcriptase (RT) is a p66/p51 heterodimer, in which the p51 subunit is generated by C-terminal proteolytic cleavage of p66. A well-known problem of p66 recombinant expression is partial cleavage of a 15-kDa peptide from the C-terminus by host proteases that can not be completely suppressed. In order to analyse the contribution of specific residues to a particular function in one distinct subunit, an expression and purification system is required that selects for the combination of the two individual subunits with the desired substitutions. We reconstituted the p66/p51 heterodimer from subunits coexpressed in Escherichia coli as an N-terminal fusion protein of glutathione S-transferase (GST) with p51 and a C-terminally His-tagged p66, respectively. The two-plasmid coexpression system ensures convenience for gene manipulation while degradation is reduced to a minimum, as dimerization protects the protein from further proteolysis. The combination of glutathione-agarose, phenyl-superose and Ni/nitrilotriacetate affinity chromatography allows rapid and selective purification of the desired subunit combination. Truncated forms of p51 are efficiently removed. Mobility-shift assay revealed that the preparations are free of p66 homodimer. In a successful test of the novel expression system, mixed reconstituted RTs with p51 selectively mutated in a putative nucleic acid binding motif (the so called helix clamp) show reduced binding of dsDNA in mobility-shift assays. This indicates the p51 subunit has an active role in DNA binding
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PMID:Mixed reconstitution of mutated subunits of HIV-1 reverse transcriptase coexpressed in Escherichia coli - two tags tie it up. 1010 27

Monocytic cells exhibit constitutive NF-kappaB activation upon infection with human immunodeficiency virus-1 (HIV-1). Because IkappaBbeta has been implicated in maintaining NF-kappaB.DNA binding, we sought to investigate whether IkappaBbeta was involved in maintaining persistent NF-kappaB activation in HIV-1-infected monocytic cell lines. IkappaBbeta was present in the nucleus of HIV-1-infected cells and participated in the ternary complex formation with NF-kappaB and DNA. In contrast to uninfected cells, the addition of recombinant glutathione S-transferase-IkappaBalpha protein to preformed NF-kappaB.DNA complexes from HIV-1-infected cell extracts did not completely dissociate the complexes, suggesting that IkappaBbeta may protect NF-kappaB complexes from IkappaBalpha-mediated dissociation. Immunodepletion of IkappaBbeta resulted in an NF-kappaB.DNA binding complex that was sensitive to IkappaBalpha-mediated dissociation, thus demonstrating the protective role of IkappaBbeta. In addition, co-transfection studies with an NF-kappaB-dependent reporter construct demonstrated that IkappaBbeta co-expression partially alleviated inhibition of NF-kappaB-mediated gene expression by IkappaBalpha, implying that IkappaBbeta can maintain transcriptionally active NF-kappaB.DNA complexes. Furthermore, constitutive phosphorylation of IkappaBalpha was observed. Immunoprecipitation of the IkappaB kinase (IKK) complex followed by in vitro analysis of kinase activity demonstrated that IKK was constitutively activated in HIV-1-infected myeloid cells. Thus, virus-induced constitutive IKK activation, coupled with the maintenance of a ternary NF-kappaB.DNA complex by IkappaBbeta, maintains persistent NF-kappaB activity in HIV-1-infected myeloid cells.
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PMID:Nuclear IkappaBbeta maintains persistent NF-kappaB activation in HIV-1-infected myeloid cells. 1022 51


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