The human HIV-1 Rev binding-protein hRIP/Rab (HRB) maps to chromosome 2q36.
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Carrier Proteins↗
Biomedical subjects
J Hauber
Publications and source records attributed to J Hauber.
Probing the structure of the HIV-1 Rev trans-activator protein by functional analysis.
Human immunodeficiency virus type 1 (HIV-1) encodes a trans-acting regulatory protein, termed Rev, which is critically required for virus replication. Rev is a sequence-specific RNA binding protein which mediates the nuclear export of unspliced and incompletely spliced viral mRNAs encoding the viral structural proteins. While CD and fluorescence measurements have provided several possible structural models of Rev, all attempts employing X-ray crystallography and NMR techniques have so far failed to provide more accurate data. We present a new approach to validate alternative structural models of the N-terminal region of Rev which contains the nuclear localization/RNA binding domain. Points of contact between structural elements in a protein were determined by introduction of targeted amino acid substitutions and subsequent scoring of the biological activities. Our data resulted in the suggestion of a new and more refined model of HIV-1 Rev structure which to date has been impossible to obtain by other means.
Animals↗
Intracellular expression of cellular eIF-5A mutants inhibits HIV-1 replication in human T cells: a feasibility study.
Previously, we described two mutants of the cellular Rev co-factor, eukaryotic initiation factor 5A (eIF-5A M13 and M14), which suppress human immunodeficiency virus type 1 (HIV-1) SF2 replication in clonal T cell lines. This study introduced the notion that it is possible to develop gene therapies against infectious agents on the basis of mutant host factors required for viral replication. In this report, we provide further evidence to support this new paradigm and describe murine leukemia virus (MLV)-based retroviral vectors expressing three different eIF-5A mutants from the viral long terminal repeat (LTR). HIV-1 replication (SF2, HXB-3) was reduced up to 2 orders of magnitude in transduced, polyclonal T cell populations. All eIF-5A mutants also showed antiviral activity (approximately seven-fold reduction) in a chronic HIV-1 infection model. Expression of eIF-5A mutant M13 delta in peripheral blood lymphocytes (PBLs) showed no difference in proliferation and metabolic activity as determined in a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT)-assay, suggesting that expression of this type of mutant protein is not associated with cellular toxicity. In summary, these data suggest that gene therapy for HIV-1 infection can be developed on the basis of mutants of the Rev co-factor eIF-5A.
Blotting, Northern↗
The gene coding for human deoxyhypusine synthase (DHPS) maps to chromosome 19p13.11-p13.12.
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Animals↗
Inhibition of HIV-1 replication in lymphocytes by mutants of the Rev cofactor eIF-5A.
Eukaryotic initiation factor 5A(eIF-5A) is a cellular cofactor require d for the function of the human immunodeficiency virus type-1 (HIV-1) Rev trans-activator protein. The majority of a set of eIF-5A mutants did not support growth of yeast cells having an inactivated genomic copy of eIF-5A, indicating that the introduced mutation eliminated eIF-5A activity. Two nonfunctional mutants, eIF-5AM13 and eIF-5AM14, retained their binding capacity for the HIV-1 Rev response element:Rev complex. Both mutants were constitutively expressed in human T cells. When these T cells were infected with replication-competent HIV-1, virus replication was inhibited. The eIF-5AM13 and eIF5AM14 proteins blocked Rev trans-activation and Rev-mediated nuclear export.
Amino Acid Sequence↗
Molecular characterization of a cDNA encoding functional human deoxyhypusine synthase and chromosomal mapping of the corresponding gene locus.
Deoxyhypusine synthase is essentially required for the post-translational formation of hypusine, a modification of a specific lysine residue in eukaryotic initiation factor 5A, which appears to be pivotal for cell proliferation. From a human peripheral blood mononuclear cells cDNA library we isolated two independent sequences encoding biologically active deoxyhypusine synthase. DNA sequence analysis revealed a 369 amino acid protein with a molecular mass of 41.055 kDa. This recombinant deoxyhypusine synthase showed significant catalytic activity in synthesis of deoxyhypusine after in vitro transcription and translation as well as upon expression in Escherichia coli. Using a panel of somatic rodent-human cell hybrids we localized the deoxyhypusine synthase gene to human chromosome 19.
Amino Acid Sequence↗
Identification of a new member of the human eIF-5A gene family.
Using an oligodeoxynucleotide generated by rapid PCR amplification of 5'-cDNA ends (5'-RACE) as a detection probe, we have isolated a new genomic clone encoding the human eukaryotic initiation factor 5A (eIF-5A). Sequence analysis revealed that the eIF-5A coding region is identical to the corresponding cDNA but interrupted by three introns. In a plasmid shuffle experiment we show functional replacement of the essential homologous gene in Saccharomyces cerevisiae by this human eIF-5A.
Amino Acid Sequence↗
[High-grade coronary stenosis due to a bronchogenic cyst].
A 32-year-old woman without preceding illness or significant cardiovascular risk factors was admitted to hospital because of a syncope after physical exertion. Transoesophageal echocardiography, performed to exclude aortic dissection, showed as an incidental finding a cystic space-occupying mass, 5 x 6 cm in diameter, dorsal to the aortic root and main pulmonary artery. Exercise ECG had S-T segment depressions of about 0.3 mV, associated with anginal symptoms, so that inadequate coronary artery flow on exertion was suspected. Subsequent coronary angiography demonstrated a funnel-shaped 80% stenosis at the origin of the main left coronary artery, while all other coronary arteries were normal and smooth-walled. A causal connection between the two findings was assumed and quickly undertaken surgical intervention revealed an intrapericardial bronchogenic cyst which, presumably as a result of its size and location, had pressed on the main stem of the left coronary artery and caused the stenosis of its ostium. The cyst was completely resected and angioplasty of the coronary artery ostium was performed to ensure its patency. 3 months postoperatively the patient underwent an exercise test of up to 125 Watt without signs of ischaemia. This case presents a previously unreported complication of a bronchogenic cyst.
Adult↗
The eukaryotic cofactor for the human immunodeficiency virus type 1 (HIV-1) rev protein, eIF-5A, maps to chromosome 17p12-p13: three eIF-5A pseudogenes map to 10q23.3, 17q25, and 19q13.2.
The eukaryotic initiation factor 5A (eIF-5A) has been identified as an essential cofactor for the HIV-1 transactivator protein Rev. Rev plays a key role in the complex regulation of HIV-1 gene expression and thereby in the generation of infectious virus particles. Expression of eIF-5A is vital for Rev function, and inhibition of this interaction leads to a block of the viral replication cycle. In humans, four different eIF-5A genes have been identified. One codes for the eIF-5A protein and the other three are pseudogenes. Using a panel of somatic rodent-human cell hybrids in combination with fluorescence in situ hybridization analysis, we show that the four genes map to three different chromosomes. The coding eIF-5A gene (EIF5A) maps to 17p12-p13, and the three pseudogenes EIF5AP1, EIF5AP2, and EIF5AP3 map to 10q23.3, 17q25, and 19q13.2, respectively. This is the first localization report for a eukaryotic cofactor for a regulatory HIV-1 protein.
Base Sequence↗
Nucleocytoplasmic transport of the Rev protein of human immunodeficiency virus type 1 is dependent on the activation domain of the protein.
The human immunodeficiency virus type 1 (HIV-1) regulatory protein Rev, which is required for the cytoplasmic expression of unspliced and incompletely spliced viral mRNAs, is located predominantly in the nucleolus. In this study, we show that Rev translocates from the nucleolus to the cytoplasm in HeLa and COS cells transfected with Rev under conditions where rRNA synthesis is inhibited (e.g., with actinomycin D). Dominant-negative mutants with mutations in the activation domain of Rev, which are known to inhibit wild-type Rev function in trans, are unable to leave the nucleus upon actinomycin D treatment. More importantly, when present in excess, these mutants inhibit the translocation of wild-type Rev. This correlation of inhibitory activities suggests that Rev function depends on its transport to and presence (at least transient) in the cytoplasm. In this context, we discuss the possibility that Rev is actively involved in the transport of HIV-1-specific mRNAs containing the Rev response element (a highly structured RNA sequence, which is specifically recognized by the Rev trans-activator). We also discuss the potential of nucleocytoplasmic export of Rev as a target for anti-HIV chemotherapy.
Amino Acid Sequence↗
[Pacemaker therapy for the sick sinus node syndrome. Does the atrially involved pacemaker system lower the frequency of atrial fibrillation and thromboembolic complications as well as mortality?].
Between 1986 and 1992, pacemakers were implanted in 307 patients with symptoms caused by the sick sinus syndrome (SSS). 301 patients were regularly followed up (161 men, 146 women, mean age 72.9 [27-91] years) of whom 180 had a VVI, 65 and AAI and 58 a DDD/DDI pacemaker. Mean follow-up period was 58.3 months for VVI-stimulated patients and 35.6 months for atrial paced patients. The data were analysed retrospectively to ascertain whether a change in pacemaker treatment to a more physiological system produced any lowering in the mortality rate, incidence of permanent atrial fibrillation (AF), and thromboembolic phenomena. The annual mortality rate of the VVI-stimulated patients was 6.9%, that of atrial paced patients 2.8%. Age, abnormal ventricular function, survived resuscitation and diabetes mellitus each correlated with a shortened life expectancy already at the time of implantation, regardless of the pacemaker mode. Permanent AF was more frequent during VVI stimulation (16% vs 7%), especially if it had been preceded by intermittent AF (26% vs 13%). But there was no significant difference with regard to transitory cerebral ischaemic episodes and peripheral arterial emboli (15% vs 10%). Fewer patients with atrial pacing went into heart failure (20% vs 30%). Four patients developed a high-grade atrioventricular (a-v) block on AAI stimulation (annual incidence 2.4%). - These observations suggest that patients with SSS should always have atrial paced pacemaker systems. If a-v conduction is disturbed, a bifocal pacemaker is the system of choice.
Adult↗
Induced gene expression of the hypusine-containing protein eukaryotic initiation factor 5A in activated human T lymphocytes.
The hypusine-containing protein eukaryotic initiation factor 5A (eIF-5A) is a cellular cofactor critically required for the function of the Rev transactivator protein of human immunodeficiency virus type 1 (HIV-1). eIF-5A localizes in the nuclear and cytoplasmic compartments of mammalian cells, suggesting possible activities on the level of regulated mRNA transport and/or protein translation. In this report we show that eIF-5A gene expression is constitutively low but inducible with T-lymphocyte-specific stimuli in human peripheral blood mononuclear cells (PBMCs) of healthy individuals. In contrast, eIF-5A is constitutively expressed at high levels in human cell lines as well as in various human organs. Comparison of eIF-5A levels in the PBMCs of uninfected and HIV-1-infected donors shows a significant upregulation of eIF-5A gene expression in the PBMCs of HIV-1 patients, compatible with a possible role of eIF-5A in HIV-1 replication during T-cell activation.
Base Sequence↗
The function of the hypusine-containing proteins of yeast and other eukaryotes is well conserved.
The hypusine-containing protein (Hypp) is highly conserved in evolution, from man to archaebacteria, but is not found in eubacteria. Hypp is essential for the viability for yeast cells, where two forms are encoded by the genes HYP1 and HYP2. The hypusine-containing protein Hyp2p, encoded by the HYP2 gene in yeast, is present under both aerobic and anaerobic conditions, whereas Hyp1p synthesis is restricted to anaerobiosis. hyp1 disruption mutants grown under anaerobic conditions reveal no detectable alteration in phenotype relative to wild-type strains. We demonstrate that either Hyp1p or Hyp2p alone is sufficient for normal growth under both metabolic conditions. Moreover, Hypp from various eukaryotic species (slime mold, alfalfa and man) carries the lysine to hypusine modification when expressed in yeast and can substitute functionally for Hyp2p in strains disrupted for HYP2, indicating a highly conserved function of this protein. In contrast, the archaebacterial Hypp expressed in yeast is neither modified by hypusine, nor does it allow growth of cells deficient for yeast Hypp.
Anaerobiosis↗
The genomic structure encoding human initiation factor eIF-5A.
A genomic clone encoding the human eukaryotic initiation factor 5A (eIF-5A) was isolated and its entire nucleotide sequence was determined. The whole eIF-5A coding region is not interrupted by introns. The functional eIF-5A gene is highly homologous to the corresponding complementary DNA. One single 1.4-kb transcript thereof is expressed in human cell lines. Furthermore, we also isolated and sequenced two additional eIF-5A-related sequences which are, by expression and sequence analyses, identified as pseudogenes of the functional eIF-5A. The sequence homology between these pseudogenes and the functional eIF-5A is 71 and 87%.
Base Sequence↗
Constitutive expression of chimeric neo-Rev response element transcripts suppresses HIV-1 replication in human CD4+ T lymphocytes.
We have previously reported that chimeric neomycin phosphotransferase (neo)-Rev response element (RRE) transcripts suppress the function of the human immunodeficiency virus type 1 (HIV-1) Rev trans-activator protein in HeLa cells. In an extension of these experiments, human CD4+ CEM cells (G418-resistant cell populations and clonal isolates) stably expressing chimeric neo-RRE genes (2, 3, or 6 RRE copies) were generated using retroviral-mediated gene transfer. The transduced CEM clones were infected with the HIV-1 HTLVIIIB isolate and the following three phenotypes were observed: (i) the transduced CEM cells were readily infected with HIV-1 indistinguishable from the control CEM cells; (ii) the appearance of HIV-1 replication markers was significantly delayed; (iii) no signs of HIV-1 replication were detectable although proviral HIV-1 DNA sequences could be detected in these cells. Furthermore, HIV antigen expression was limited in neo-resistant CEM cell populations inoculated with the HIV-1 HTLVIIIB isolate. Only 10% of the CEM-pX17-3xRRE cells and 20% of the CEM-pX17-2xRRE cells displayed HIV-1 antigens 43 days after challenge and had retained CD4 surface expression on 47% and 64% of the cells, respectively. In sharp contrast, 80% of the CEM-pX17 or the CEM-pX17-6xRRE cells expressed HIV-1 antigens but no CD4 antigens were detectable in these cultures. These results clearly indicate that RRE decoys could be developed into an effective somatic gene therapy approach against HIV-1 induced acquired immunodeficiency syndrome (AIDS).
Base Sequence↗
Scanning mutagenesis of the arginine-rich region of the human immunodeficiency virus type 1 Rev trans activator.
The structural proteins of human immunodeficiency virus type 1, for example, Gag and Env, are encoded by unspliced and incompletely spliced viral transcripts. The expression of these mRNAs in the cytoplasm, along with their commensurate translation, is absolutely dependent on the virally encoded Rev trans activator. Previous studies have demonstrated that Rev binds directly to its substrate mRNAs via an arginine-rich element that also serves as its nuclear localization sequence. In an attempt to define the specific amino acid residues that are important for in vivo activity, we have constructed a series of missense mutations that scan across this region. Our data demonstrate that all eight arginine residues within this element can, individually, be substituted for either leucine or lysine with no apparent loss of function. Importantly, these findings suggest that no single amino acid within the arginine-rich domain of Rev is, by itself, essential for activity and that considerable functional redundancy is therefore likely to exist within this region. Interestingly, one mutant in which a tryptophan had been substituted for a serine failed to accumulate exclusively in the nucleus but still bound RNA in a manner that was indistinguishable from that of the wild-type protein. This observation indicates that features of the arginine-rich region that are additional to those required for RNA binding are important for Rev's correct accumulation in the nucleus.
Amino Acid Sequence↗
Inhibition of human immunodeficiency virus type 1 replication in human T cells by retroviral-mediated gene transfer of a dominant-negative Rev trans-activator.
Human immunodeficiency virus type 1 (HIV-1) is the causative agent of the acquired immunodeficiency syndrome (AIDS). Currently, no satisfactory treatment for this viral disease is available. Somatic gene therapy has been proposed as an alternative to conventional therapies. Several antiviral gene therapy approaches including ribozymes, antisense inhibition, and RNA-decoy strategies, as well as dominant-negative mutants of HIV-1 proteins (Gag, Tat, and Rev) have been suggested. To prove the concept of trans-dominant inhibition of HIV-1 replication, we transduced CEM cells with a retroviral vector encoding a dominant-negative rev gene. Amplification of integrase-specific proviral sequences from high molecular weight DNA indicated successful HIV-1 human T-lymphotropic virus type IIIB (HTLV-IIIB) infection of all cells. In contrast to CEM cells and CEM cells expressing the rev wild-type (wt) gene, infection of two CEM-RevM10 clones with HIV-1 did not result in the release of significant levels of p24 Gag antigen as measured by antigen capture assay, indicating a block in HIV-1 replication due to the presence of the trans-dominant Rev protein. Furthermore, the parental CEM cells as well as CEM cells expressing the Rev wt protein were effectively killed in the course of the HIV-1 infection, whereas all CEM cells expressing the RevM10 protein were unaffected in their growth rate.
Base Sequence↗
Expression of chimeric neo-Rev response element sequences interferes with Rev-dependent HIV-1 Gag expression.
Recombinant plasmids containing reiterated human immunodeficiency virus type 1 (HIV-1) Rev response element (RRE) sequences were constructed to suppress Rev-dependent HIV-1 Gag expression. The mammalian expression vectors pMAMneo containing one, three, or six repeats of the RRE sequence were cotransfected with a HIV-1 HTLV-IIIB proviral DNA into HeLa cells. All three RRE expression plasmids reduced replication of HIV-1 with similar efficacy. Furthermore, the chimeric expression vector pCMV neoRRE6 x ----(containing six copies of the RRE sequence) was used to establish HeLa cell lines constitutively expressing RRE. A plasmid encoding a Rev-dependent HIV-1 p24 Gag protein was cotransfected with the wild-type Rev expression plasmid into three different RRE-expressing HeLa cell lines. p24 Gag protein production in the culture supernatants of the HeLaneoRRE cells was compared with two neo-expressing cell lines. Although all cell lines (HeLaneoRRE, HeLaneo) displayed similar transfection efficiencies, p24 Gag protein synthesis was markedly reduced in the RRE-expressing cell lines in comparison to the control cells.
Base Sequence↗