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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 1 (HIV-1) nucleocapsid protein p15 was produced as a fusion protein with glutathione S-transferase (GST) in Escherichia coli. Rapid purification of GST::p15 in an active form by one-step glutathione-agarose chromatography was accomplished in the presence of an antioxidant. Recombinant p15 fused to GST was shown to stimulate the dimerization of viral RNA. HIV-1 reverse transcriptase-catalyzed in vitro synthesis of minus-strand cDNA from synthetic human tRNA(Lys3UUU) and natural bovine tRNA(Lys3SUU) primer molecules was enhanced by GST::p15. GST produced in E.coli revealed no effect with respect to RNA dimerization and cDNA synthesis, demonstrating that both activities reside in the p15 portion of the fusion protein.
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PMID:Recombinant HIV-1 nucleocapsid protein p15 produced as a fusion protein with glutathione S-transferase in Escherichia coli mediates dimerization and enhances reverse transcription of retroviral RNA. 128 Feb 40

We present evidence that the HIV-1 Tat protein and the RNA-dependent cellular protein kinase, PKR, interact with each other both in vitro and in vivo. Using GST fusion chromatography, we demonstrate that PKR, interacts directly with the HIV-1 Tat protein. The region in Tat sufficient for binding PKR maps within amino acids 20 to 72. In in vitro assays, the two-exon form of Tat (Tat 86) was phosphorylated by PKR, while the one exon form of Tat (Tat 72) inhibited PKR autophosphorylation and substrate phosphorylation. The ability of Tat to interact with PKR was demonstrated in both yeast and mammalian cells. Expression of PKR in yeast results in a growth suppressor phenotype which was reversed by coexpression of a one exon form of Tat. Expression of Tat 72 in HeLa cells resulted in direct interaction with PKR as detected by coimmunprecipitation with a Tat antibody. Tat and PKR also form a coimmunoprecipitable complex in cell-free extracts prepared from productively infected T lymphocytes. The interaction of Tat with PKR provides a potential mechanism by which HIV could suppress the interferon system.
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PMID:HIV-1 Tat directly interacts with the interferon-induced, double-stranded RNA-dependent kinase, PKR. 749 66

Although the human immunodeficiency virus type 1 (HIV-1) nef gene still has no precisely defined function, in vivo studies have demonstrated that Nef is an important pathogenic determinant of HIV. In order to identify cellular proteins capable of binding to Nef, the HIV-1LAI nef gene product was expressed in the bacterial vector pGEX-2T as a glutathione S-transferase (GST)-Nef fusion protein. Deletion mutants corresponding to 86 and 35 N-terminal residues of the Nef protein were prepared. The GST-Nef constructs were used to identify cellular kinases capable of interacting with Nef. After incubation with a Jurkat cell lysate, the GST-Nef constructs immobilized on glutathione-agarose beads bound to cellular kinase(s) and were phosphorylated at three sites in vitro: one on threonine at position 15, one on serine between residues 1 and 35, and one on threonine between residues 36 and 86. The Nef-phosphorylating activity was inhibited by protein kinase C (PKC)-selective inhibitors. Cell fractionation showed that this Nef-binding kinase was mainly in the membrane-associated fraction. These results suggest that kinase(s) of the PKC family are specifically bound to and phosphorylate Nef in vitro. The interaction of Nef with cellular kinases and its phosphorylation may be important in mediating the effects of Nef in HIV-1 pathogenesis.
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PMID:In vitro binding and phosphorylation of human immunodeficiency virus type 1 Nef protein by serine/threonine protein kinase. 754 Jan 94

Earlier observations showed that the expression of recombinant protease of human immunodeficiency virus type-1 (HIV-1 PR) was usually in a low level, and its proteolytic activity and hydrophobicity were believed to be toxic for the host cells. Various constructs were investigated that contained an N-terminal extended HIV-1 PR gene (PR107) in order to find a system which can express this protease in high level. The constructs of PR107 gene expressed as fusion proteins either with glutathione S-transferase (GST) by pGEX-PR107 or with maltose-binding protein (MBP) by pMAL-PR107 showed that the full length of fusion protein exhibited self-cleavage in E. coli. The results from expression experiments indicated that the size of the fusion portion does not affect the self-processing of fused HIV-1 PR to release its mature form, despite the attachment of only one subunit of the dimeric protease to GST or MBP. The construct, pET-PR107, under the control of strong bacteriophage T7 promoter system, did not show clear advantages for expression of this HIV-1 PR. Comparing these three constructs, the pGEX-PR107 system showed the highest expression level. Quantitative immuno-blotting indicated that the amount of HIV-1 PR expressed by pGEX-PR107 was twice that expressed by pMAL-PR107, and thrice that expressed by pET-PR107. More than 1 mg of pure HIV-1 PR from per liter culture of E. coli. DH5 alpha containing pGEX-PR107 can be obtained via the purification procedures [Biochem. Mol. Biol. International, (1995) 35:899-912].(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Comparison of HIV-1 protease expression in different fusion forms. 766 45

Human immunodeficiency virus type 1 (HIV-1) Nef protein causes the loss of cell surface CD4 and interleukin-2 (IL-2) receptor (Tac) from peripheral blood mononuclear cells (PBMC) and CD4+ T-cell lines. As both CD4 and the IL-2 receptor play crucial roles in antigen-driven helper T-cell signalling and T-cell proliferation, respectively, the role of Nef in the viral life cycle may be to perturb signalling pathways emanating from these receptors. However, the intracellular targets for Nef that result in receptor down-regulation are unknown. Using a recombinant glutathione S-transferase-full-length 27 kDa Nef (Nef27) fusion protein, produced in Escherichia coli by translation from the first start codon of HIV-1 nef clone pNL4-3, as an affinity reagent to probe cytoplasmic extracts of MT-2 cells and PBMC, we have shown interaction with at least seven host cell protein species ranging from 24 to 75 kDa. Immunoblotting identified four of these proteins as p56lck, CD4, p53, and p44mapk/erk1, all of which are intimately involved in intracellular signalling. To assess the relevance of these interactions and further define the biochemical activity of Nef in signal transduction pathways, highly purified Nef27 protein was introduced directly into PBMC by electroporation. Nef27-treated PBMC showed reduced proliferative responsiveness to exogenous recombinant IL-2. Normally, stimulation of T-cells by IL-2 or phorbol 12-myristate 13-acetate provokes both augmentation of p56lck activity and corresponding posttranslational modification of p56lck. These changes were also inhibited by treatment of PBMC with Nef, suggesting that Nef interferes with activation of p56lck and as a consequence of signalling via the IL-2 receptor. Further evidence for Nef interfering with cell proliferation was the decreased production of the proto-oncogene c-myb, which is required for cell cycle progression, in Nef-treated MT-2 cells. In contrast to the binding characteristics and biological effects of Nef27, the alternate 25-kDa isoform of Nef (Nef25) produced by translation from the second start codon of HIV nef pNL4-3 (57 nucleotide residues downstream) was shown to interact with only three cellular proteins of approximately 26, 28, and 56 kDa from PBMC and MT-2 cells, one of which was identified as p56lck. Also, proliferation and posttranslational modification of p56lck in response to IL-2 stimulation were not profoundly affected by treatment of PBMC with Nef25 compared with Nef27.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Human immunodeficiency virus type 1 Nef protein inhibits activation pathways in peripheral blood mononuclear cells and T-cell lines. 785 25

The selective encapsidation of retroviral RNA requires sequences in the Gag protein, as well as a cis-acting RNA packaging signal (psi site) near the 5' end of the genomic transcript. Gag protein of human immunodeficiency virus type 1 (HIV-1) has recently been found to bind specifically to the HIV-1 psi element in vitro. Here we report studies aimed at mapping features within the genetically defined psi locus that are required for binding of HIV-1 Gag or of its processed nucleocapsid derivative. The full-length HIV-1 Gag (p55) and nucleocapsid (p15) sequences were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. In a gel shift assay containing excess competitor tRNA, affinity-purified GST-p15 and GST-p55 proteins bound to a 206-nucleotide psi RNA element spanning the major splice donor and gag start codons but did not bind to antisense psi transcripts. Quantitative filter-binding assays revealed that both GST-p55 and GST-p15 bound to this RNA sequence with identical affinities (apparent Kd congruent to 5 x 10(-8) M), indicating that all major determinants of psi binding affinity reside within the nucleocapsid portion of Gag. Chemical and RNase accessibility mapping, coupled with computerized sequence analysis, suggested a model for psi RNA structure comprising four independent stem-loops. Filter-binding studies revealed that RNAs corresponding to three of these hypothetical stem-loops can each function as a independent Gag binding site and that each is bound with approximately fourfold-lower apparent affinity than the full-length psi locus. Interaction of Gag with these regions is likely to play a major role in directing HIV-1 RNA encapsidation in vivo.
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PMID:RNA secondary structure and binding sites for gag gene products in the 5' packaging signal of human immunodeficiency virus type 1. 788 56

Human immunodeficiency virus type 1 (HIV-1) and HIV-2 encode related transcriptional activators known as Tat-1 and Tat-2, respectively, that are required for efficient viral replication. The Tat proteins have been studied extensively, and it appears that their mechanism of action is unique to the primate immunodeficiency viruses or a few distantly related lentiviruses. Here we describe a collection of 24 wild-type and mutant Tat-1 and Tat-2 proteins that are expressed in Escherichia coli as fusions with glutathione S-transferase (GST). The GST-Tat fusions can be used for biochemical studies after simple purification from E. coli lysates in a single step under nondenaturing conditions. The availability of these GST-Tat fusions should be useful to investigators examining biochemical properties of Tat-1 and Tat-2 proteins. E. coli cultures harboring GST-Tat fusions described here are available through the National Institute of Health AIDS Research and Reference Reagent Program.
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PMID:Wild-type and mutant HIV-1 and HIV-2 Tat proteins expressed in Escherichia coli as fusions with glutathione S-transferase. 793 78

The nef gene is conserved throughout the primate lentivirus family. Although dispensable in vitro, an important role for nef in vivo is suggested by the failure of SIV nef mutants to establish persistent viraemia. Although the biochemical function of the Nef protein remains equivocal, a consistent theme has emerged with the reproducible observation that Nef expression results in the down-modulation of the cell surface marker CD4. Down-modulation requires amino acid sequences within the cytoplasmic domain of CD4 but occurs by a mechanism distinct from the normal serine phosphorylation-dependent pathway. As CD4 is a transmembrane glycoprotein and Nef a myristoylated protein targeted to the cytoplasmic face of the plasma membrane we considered that a direct interaction between Nef and CD4 might play a role in down-modulation. Here we demonstrate that a baculovirus-expressed Nef-GST fusion protein interacts specifically with CD4. This interaction requires co-expression in the same cell and is dependent on Nef myristoylation. The site of Nef interaction maps to the cytoplasmic domain of CD4, as a deletion mutant lacking this domain fails to interact with Nef. This observation sheds new light on the biochemical function of Nef and offers new opportunities for the future development of HIV chemotherapy.
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PMID:Myristoylation-dependent binding of HIV-1 Nef to CD4. 805 54

The present study was designed to determine the antibody specificity for the human immunodeficiency virus type 1 (HIV-1) V3 domains of infectious and noninfectious virions present in the serum of AIDS patients. To accomplish this, HIV-1 was isolated in the presence of autologous antibodies from the serum samples of six AIDS patients in HIV-1-negative donor peripheral blood mononuclear cells by short-term cultivation. The isolated virus, defined as the infectious cell-free virus (iCFV), was characterized by sequence analysis of the proviral DNA coding for the third hypervariable (V3) region of the external glycoprotein gp120. This was carried out by amplifying and cloning the V3 region. In all six cases studied, 20 randomly selected V3 clones derived from the proviral DNA of the iCFV, 20 clones from patient cell-free virus, and 20 clones from cell-integrated virus were sequenced to study the distribution and frequency of the intrapatient virus population. The number of major virus variants in the six patients ranged from three to nine. The various V3 sequences found in the AIDS patients showed the typical amino acid pattern of the syncytium-inducing and non-syncytium-inducing viral phenotypes characteristic for the late stage of infection. However, only one patient-specific iCFV variant was detected within the 20 V3 clones analyzed per virus isolation. For the six patients a total of 34 V3-loop variants, either iCFV or non-iCFV, was observed. All 34 V3-loop sequences were expressed as glutathione-S-transferase fusion proteins (V3-GST). The autologous antibody response to the V3-GST fusion proteins was studied by Western immunoblot analysis. A strong antibody response to almost all non-iCFV V3-GST proteins was found in the sera of the six patients. In contrast, the autologous antibody response to the six iCFV V3 loops was undetectable (in four patients) or very faint (in two patients) compared with that to the non-iCFV V3 loops. Five of the six iCFV loops showed positively charged amino acids at positions strongly associated with the syncytium-inducing phenotype. These findings suggest that our in vitro isolation system selects for virions which are not recognized by V3-specific antibodies and are infectious both in vitro and in vivo.
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PMID:Antibodies of symptomatic human immunodeficiency virus type 1-infected individuals are directed to the V3 domain of noninfectious and not of infectious virions present in autologous serum. 818 27

Packaging of retroviral genomic RNA during virion assembly is thought to be mediated by specific interactions between the gag polyprotein and RNA sequences (often termed the psi or E region) near the 5' end of the genome. For many retroviruses, including human immunodeficiency virus type 1 (HIV-1), the portions of the gag protein and the RNA that are required for this interaction remain poorly defined. We have used an RNA gel mobility shift assay to measure the in vitro binding of purified glutathione S-transferase-HIV-1 gag fusion proteins to RNA riboprobes. Both the complete gag polyprotein and the nucleocapsid (NC) protein alone were found to bind specifically to an HIV-1 riboprobe. Either Cys-His box of NC could be removed without eliminating specific binding to the psi riboprobe, but portions of gag containing only the MA and CA proteins without NC did not bind to RNA. There were at least two binding sites in HIV-1 genomic RNA that bound to the gag polyprotein: one entirely 5' to gag and one entirely within gag. The HIV-1 NC protein bound to riboprobes containing other retroviral psi sequences almost as well as to the HIV-1 psi riboprobe.
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PMID:Specific binding of human immunodeficiency virus type 1 gag polyprotein and nucleocapsid protein to viral RNAs detected by RNA mobility shift assays. 823 Apr 41


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