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Biomedical subjects

L Ratner

Publications and source records attributed to L Ratner.

At least 37 records · Page 2Linked to original sources
PubMed1994

Regulation of human immunodeficiency virus Nef protein by phosphorylation.

Human immunodeficiency virus isolates express a Nef protein with either an alanine or a threonine at amino acid residue 15. The threonine residue is a site for phosphorylation by protein kinase C. Jurkat T cells constitutively expressing the alanine variant of Nef exhibit the ability to downregulate the induction of transcription factors NF-kB and AP-1. In contrast, Jurkat cells with the threonine variant of Nef are at least partially restored in their ability to recruit NF-kB and AP-1.

Binding Sites↗
PubMed1994

Structure and expression of the human T-cell leukemia virus type 1 envelope protein.

The structure and expression of the HTLV-1 envelope protein was examined using T lymphoid cell lines infected with HTLV-1 and recombinant vaccinia viruses expressing the HTLV-1 envelope. Pulse-chase experiments demonstrated that the envelope precursor, gp62, had a half-life of 7-12 hr. N-glycosylation of the precursor protein was examined using tunicamycin and endoglycosidase H. These studies revealed that at least four and possibly five potential N-glycosylation sites were utilized. In addition, the envelope precursor was found to sediment on sucrose gradients as high-molecular-weight complexes, in positions consistent with the formation of dimers and smaller amounts of higher multimeric forms. Finally, the recombinant vaccinia system was used to express mutants designed to analyze the role in HTLV-1 envelope processing of the cytoplasmic tail and the membrane-spanning domain.

Base Sequence↗
PubMed1994

HIV-2 viral protein X association with the GAG p27 capsid protein.

VPX is a 16 kDa accessory protein expressed in cells infected with HIV-2 and most SIV strains and is packaged into virus particles. In order to define the requirements for incorporation of VPX into virions, VPX and HIV-2 GAG-POL were expressed independently from a vaccinia virus-based transient expression system. Under these conditions, VPX was exported from transfected cells only when coexpressed with the HIV-2 GAG-POL plasmid. A 27 kDa protein coprecipitating with VPX was found to have an identical electrophoretic mobility as the GAG capsid protein, and reacted with an anti-GAG antiserum. Coexpression of VPX and GAG-POL resulted in virus-like particles containing both proteins, as determined by sucrose gradient analyses. Expression of VPX and HIV-2 GAG without POL gave similar results. VPX association with HIV-2 GAG p27 capsid protein was specific, since no association was found with the HIV-1 GAG p25/p24 capsid protein.

Animals↗
PubMed1994

Particle assembly and Vpr expression in human immunodeficiency virus type 1-infected cells demonstrated by immunoelectron microscopy.

The 96 amino acid viral protein R (Vpr) of human immunodeficiency virus type 1 (HIV-1) was detected during virus assembly in intracellular vacuoles and at the plasma membrane on peripheral blood mononuclear cells. In both immature and mature virus particles, Vpr was located immediately beneath the viral envelope, colocalizing with the core structural protein, Gag p24. Vpr was present in intracellular HIV-1 wild-type virions at 50% of the level found in extracellular HIV-1 particles. Cells infected with HIV-1 strains with C-terminal truncations of Vpr manifested a different pattern of Vpr expression. A mutant with an alteration of amino acids 79 to 85 exhibited a 23% reduction in total levels of Vpr expression, but a marked accumulation of Vpr in intracellular rather than extracellular virions. A mutant with the last 17 amino acids of Vpr deleted expressed only 10% of wild-type levels of Vpr. These observations indicate that Vpr is incorporated into virions from the cytoplasmic aspect of either the vacuolar or plasma membrane. Furthermore, the proportion of Vpr on intracellular compared to extracellular virions is affected by a specific locus within the protein.

Amino Acid Sequence↗
PubMed1994

CD3-dependent lymphocyte activation by human T cell leukaemia virus type I-producing T cells.

Human T cell leukaemia virus type I-(HTLV-I) transformed cells are capable of stimulating the proliferation of normal T lymphocytes, or stimulating interleukin 2 expression in Jurkat T lymphoid cells. This effect is mediated by the CD2/lymphocyte function-associated antigen 3 (LFA-3) adhesion/signalling pathway. The current work demonstrates that CD3 is also required for this effect, suggesting that the T cell receptor (TCR)/CD3 complex is mediating this effect. However, this effect does not appear to be due to a superantigen since no change in TCR expression was found after HTLV-I-mediated proliferation, nor was proliferation inhibited by an antibody against the specific TCR expressed on Jurkat cells (TCR V beta 8).

Adult↗
PubMed1994

Identification of human immunodeficiency virus type 1 Gag protein domains essential to membrane binding and particle assembly.

Assembly of human immunodeficiency virus type 1 (HIV-1) particles occurs at the plasma membrane of infected cells. Myristylation of HIV-1 Gag precursor polyprotein Pr55Gag is required for stable membrane binding and for assembly of viral particles. We expressed a series of proteins representing major regions of the HIV-1 Gag protein both with and without an intact myristyl acceptor glycine and performed subcellular fractionation studies to identify additional regions critical for membrane binding. Myristylation-dependent binding of Pr55Gag was demonstrated by using the vaccinia virus/T7 hybrid system for protein expression. Domains within the matrix protein (MA) region downstream of the initial 15 amino acids were required for membrane binding which was resistant to a high salt concentration (1 M NaCl). A myristylated construct lacking most of the matrix protein did not associate with the plasma membrane but formed intracellular retrovirus-like particles. A nonmyristylated construct lacking most of the MA region also was demonstrated by electron microscopy to form intracellular particles. Retrovirus-like extracellular particles were produced with a Gag protein construct lacking all of p6 and most of the nucleocapsid region. These studies suggest that a domain within the MA region downstream from the myristylation site is required for transport of Gag polyprotein to the plasma membrane and that stable plasma membrane binding requires both myristic acid and a downstream MA domain. The carboxyl-terminal p6 region and most of the nucleocapsid region are not required for retrovirus-like particle formation.

Animals↗
PubMed1994

Human immunodeficiency virus type 1 Nef protein down-regulates transcription factors NF-kappa B and AP-1 in human T cells in vitro after T-cell receptor stimulation.

Human immunodeficiency virus type 1 (HIV-1) negative factor (Nef) has been shown to down-regulate the transcription factors NF-kappa B and AP-1 in vitro. To define the mechanism of action of the Nef protein, the signal transduction pathways which may be affected in T cells by constitutive expression of the nef gene were examined. Stimulation of T cells with tumor necrosis factor, interleukin-1, or lipopolysaccharide resulted in the recruitment of transcriptional factors to a similar level whether or not the cells expressed the nef gene. On the other hand, stimulation of T cells by mitogens or antibodies to the T-cell receptor (TCR)-CD3 complex resulted in the down-regulation of transcriptional factors NF-kappa B and AP-1 in cells expressing the nef gene compared with cells not expressing the nef gene. Because the Nef protein does not affect the surface expression of the CD3-TCR complex, we conclude that the Nef protein down-regulates the transcriptional factors NF-kappa B and AP-1 in T cells in vitro through an effect on the TCR-dependent signal transduction pathway.

Antigens, CD↗
PubMed1993

Highly localized tracks of human immunodeficiency virus type 1 Nef in the nucleus of cells of a human CD4+ T-cell line.

A human T-cell line constitutively expressing the nef gene from the human immunodeficiency virus type 1 SF2 isolate was used to examine the distribution of the Nef protein in the nucleus. High-resolution immunogold labeling/electron microscopic studies with polyclonal anti-Nef antibodies on nef+ and nef- cells revealed that a small fraction of Nef is in the nucleus and it is localized in specific curvilinear tracks that extend between the nuclear envelope and the nucleoplasm. An examination of the sequence of the SF2 nef gene revealed a putative nuclear targeting sequence that was previously found in several other eukaryotic nucleoplasmic proteins. The nuclear localization of Nef suggests a potential nuclear function for this protein. The presence of Nef in distinct nuclear tracks suggests that Nef is transported along a specific pathway that extends from the nuclear envelope into the nucleoplasm. A previous study [Meier, U. T. & Blobel, G. (1992) Cell 70, 127-138] has shown that the nucleolar protein of rat liver cells (Nopp140) shuttles from the nucleolus to the nuclear envelope on distinct tracks. The present study has suggested that the transport of a nucleoplasmic protein may also occur on distinct nuclear pathways.

Amino Acid Sequence↗
PubMed1993

HIV-1 Nef protein inhibits the recruitment of AP-1 DNA-binding activity in human T-cells.

The human immunodeficiency virus type 1 long terminal repeat, HIV-1-LTR, contains binding sites for several cellular transcription factors which contribute to HIV-1 gene expression. Our previous studies on the function of the HIV-1-encoded Nef protein suggested that Nef may be an inhibitor HIV-1 transcription. To determine whether Nef affects the binding of cellular factors implicated in HIV-1 regulation, 32P-labeled oligonucleotides corresponding to the binding sites were incubated with nuclear extracts prepared from Nef-expressing T-cell lines that were not stimulated or were stimulated with T-cell mitogens. We found that Nef inhibited the recruitment of AP-1 DNA-binding activity in mitogen-stimulated human T-cells. Additionally, Nef expressing cells were transiently transfected with a plasmid in which HIV-1 AP-1 DNA recognition sequences were cloned downstream of the chloramphenicol acetyltransferase (CAT) gene. Mitogen-mediated transcriptional activation of the CAT gene in this construct was inhibited in Nef-expressing cells but not in control cells. These studies suggest that, by inhibiting AP-1 activation, Nef may play a role in regulating HIV-1 gene expression in infected T-cells.

Base Sequence↗
PubMed1993

Myristoylation-enhanced binding of the HIV-1 Nef protein to T cell skeletal matrix.

The negative factor, Nef, of HIV-1 was found to associate to an extent of 16-42% with the detergent insoluble cytoskeletal fraction of T lymphocytes. Furthermore, Escherichia coli expressed Nef protein was found to bind during in vitro reactions with the cytoskeletal matrix to an extent of 30-50%. Cytoskeletal association of Nef was significantly enhanced by myristoylation. The specificity of the myristoylation-enhanced binding was demonstrated by the lack of an effect of myristoylation on binding of the HIV-1 Gag protein to the cytoskeleton. Cytoskeletal binding was saturable, and inhibited by high concentrations of sodium chloride, or with SDS or urea. Binding of Nef to the cytoskeletal matrix may be important in mediating its effects on HIV-1 replication.

Cloning, Molecular↗
PubMed1993

Identification of HIV1 determinants for T lymphoid cell line infection.

Determinants responsible for HIV-1 infection of T lymphoid cell lines were identified by functional analysis of chimeric proviral clones derived from T-cell line tropic-(HXB2) and non-T-cell line-tropic isolates (YU2, ADA). Replacement of the HXB2 V3 envelope loop sequence with that derived from YU2 resulted in a virus that is no longer T cell line-tropic. However, the reciprocal replacement using HXB2 V3 loop sequences did not confer upon either ADA or YU2 envelope proteins the ability to infect T cell lines. Furthermore, the resultant viruses were incapable of infection of primary lymphocytes. Single, double, and multiple point mutations made within the V3 loop sequence did not result in change in tropism, although mutations involving residue 275 resulted in a virus that was incapable of infecting primary lymphocytes but retained the ability to infect Jurkat T lymphoid cells. These results suggest that the V3 envelope determinant is necessary for T cell line infection, but other determinant(s) in envelope are also necessary to obtain infectious virus expression.

Amino Acid Sequence↗
PubMed1993

Role of HIV-1 envelope V3 loop cleavage in cell tropism.

The envelope protein is an important determinant of HIV-1 cell-specific tropism. The gp160 envelope precursor proteins from macrophage-tropic or T lymphoid cell line-tropic strains of HIV-1 were expressed in recombinant vaccinia virus-infected cell lines or primary lymphocytes or macrophages. No significant differences in the kinetics of synthesis of gp160, processing into gp120 and gp41 proteins, N-linked glycosylation, or release of gp120 into the medium were noted with the different envelope proteins. However, gp120 envelope protein shed into the medium was found to be at least partially cleaved at a site within the V3 loop. The gp120 envelope proteins from macrophage-tropic isolates exhibited lower rates of cleavage than those from lymphoid cell line-tropic strains in all cell types examined. Cell-free protease digestion studies also demonstrated relative resistance of the envelopes from macrophage-tropic compared to lymphoid cell line-tropic strains. All recombinant envelope proteins were recognized by monoclonal antibodies directed at gp41 or the C-terminal gp120 epitopes, and no differences in binding to CD4 were noted.

Amino Acid Sequence↗
PubMed1993

Alterations in spliced and unspliced HIV-1-specific RNA detection in peripheral blood mononuclear cells of individuals with varying CD4-positive lymphocyte counts.

Reverse transcriptase-polymerase chain amplification reactions (RT-PCR) were used to identify transcripts for HIV-1 structural and regulatory proteins in peripheral blood mononuclear cells of a cohort of 48 patients. At least one set of PCR primers was capable of detecting HIV-1 transcripts in 94% of patients. Unspliced gag-pol transcripts were detected with gag or pol primer sets in 60 and 63% of samples, respectively. A significant inverse correlation was noted between transcript identification with the gag primer set and the number of CD4-positive lymphocytes in the blood sample and the clinical stage of infection. Single-spliced env transcripts were identified in 44% of individuals. Multiple-spliced tat or nef transcripts were detected in 6.2 and 53% of individuals, respectively. These findings indicate that viral transcripts are expressed throughout the course of HIV-1 infection.

Base Sequence↗
PubMed1993

Mechanism of action of N-butyl deoxynojirimycin in inhibiting HIV-1 infection and activity in combination with nucleoside analogs.

The effects on HIV-1 infection of a glucosidase inhibitor, N-butyl deoxynojirimycin (N-buDNJ), were examined. The combinations of N-buDNJ and nucleoside analogs dideoxyinosine (DDI), dideoxycytidine (DDC), or azidothymidine (AZT) were examined in an acute infection assay. The combination of N-buDNJ and nucleoside analog reduced the yield of reverse transcriptase activity more than did either agent alone, and the effects on the number of infectious virus particles were additive or synergistic. In studies of the mechanism whereby N-buDNJ alters HIV-1 envelope fusion activity, no effects on CD4 binding were detected. However, cleavage within the V3 loop of gp120 was reduced by N-buDNJ treatment, possibly reflecting an altered conformation of this region of the envelope protein.

1-Deoxynojirimycin↗
PubMed1993

Cell surface proteins binding to recombinant soluble HIV-1 and HIV-2 transmembrane proteins.

OBJECTIVE: To further characterize cell surface proteins binding to recombinant soluble (rs) forms of the transmembrane glycoproteins gp41 of HIV-1 (rsgp41) and gp36 of HIV-2 (rsgp36). METHODS: Various human and murine cell lines of different lineages were surface-labelled with 125I. rsgp41 and rsgp36 were bound to CnBr-Sepharose and used as an affinity matrix for the surface-labelled cell lysates. The bound cell surface proteins were separated on sodium dodecyl sulphate polyacrylamide gel electrophoresis under reducing conditions. A rabbit serum was produced against one of the cell surface proteins and flow cytometry used to compare the results with those obtained from affinity chromatography. RESULTS: We have confirmed and extended the results obtained by Qureshi et al. [1]. In addition to the 44kD protein, we identified cell surface proteins with molecular weights of 98 and 106 kD binding with high affinity to both rsgp41 and rsgp36. We have demonstrated differences between human and murine cell lines in the expression of the cell surface proteins that interact with rsgp41 and rsgp36. Furthermore, a correlation between the level of rsgp41 and rsgp36 binding proteins, detected either by affinity chromatography or by reactivity with an antiserum directed against one of the cell surface binding components was shown. CONCLUSIONS: Three cell surface proteins, with molecular weights of 44, 98 and 106 kD, bind with high affinity to rs forms of gp41 and gp36. Their expression decreases from a T-lymphoid cell line, to a monoblastoid cell line, to a cell line representing mature monocytes. Human T-cell lymphotropic virus-infected cell lines show a predominance of the 44 kD protein. There are species-specific differences, in that murine cell lines lack the 44 kD protein.

Animals↗
PubMed1993

Human immunodeficiency virus type 1 viral protein R localization in infected cells and virions.

The subcellular localization of human immunodeficiency virus type 1 (HIV-1) viral protein R (Vpr) was examined by subcellular fractionation. In HIV-1-infected peripheral blood mononuclear cells, Vpr was found in the nuclear and membrane fractions as well as the conditioned medium. Expression of Vpr without other HIV-1 proteins, in two different eukaryotic expression systems, demonstrated a predominant localization of Vpr in the nuclear matrix and chromatin extract fractions. Deletion of the carboxyl-terminal 19-amino-acid arginine-rich sequence impaired Vpr nuclear localization. Indirect immunofluorescence confirmed the nuclear localization of Vpr and also indicated a perinuclear location. Expression of Vpr alone did not result in export of the protein from the cell, but when coexpressed with the Gag protein, Vpr was exported and found in virus-like particles. A truncated Gag protein, missing the p6 sequence and a portion of the p9 sequence, was incapable of exporting Vpr from the cell. Regulation of Vpr localization may be important in the influence of this protein on virus replication.

Amino Acid Sequence↗