Biomedical subjects

J Mous

Publications and source records attributed to J Mous.

At least 19 recordsLinked to original sources

PubMed1992

CD4 epitope masking by gp120/anti-gp120 antibody complexes. A potential mechanism for CD4+ cell function down-regulation in AIDS patients.

The in vitro suppressive effect of gp120 and gp120/anti-gp120 antibody is well known but not yet proven to operate in vivo. We report findings consistent with the presence of gp120/anti-gp120 antibody complexes on CD4+ lymphocytes from HIV-infected patients with advanced disease. PBMC from most AIDS patients showed selective masking of the CD4 epitope associated with the gp120 binding site; immunoprecipitation of PBMC with anti-CD4 mAb disclosed high amounts of IgG bound to CD4 receptors. Antibodies against HIV env proteins, but not other HIV products or CD4 Ag, were detected in purified CD4+ cell culture supernatants; in vitro culture was associated with normalization of both CD4 expression in PBMC and the lymphocyte proliferative response to anti-CD3. gp120 presence could not be directly demonstrated, but findings strongly suggested that CD4+ lymphocytes from most HIV-infected patients with advanced disease were covered with gp120/anti-gp120 antibody complexes, which are responsible for down-regulation of surface CD4 expression as well as functional lymphocyte impairment; this event may represent an important mechanism in the pathogenesis of HIV-associated immunodeficiency.

Acquired Immunodeficiency Syndrome↗

PubMed1992

Identification of amino acid residues critical for endonuclease and integration activities of HIV-1 IN protein in vitro.

HIV-IN protein, tagged with a hexahistidine tail was expressed in Escherichia coli and purified by a one-step nickel chelate affinity chromatography procedure. The purified IN protein was characterized in terms of its endonuclease and integrase properties in vitro. Specific cleavage and integration of HIV U5 LTR ends were observed in the presence of 2-5 mM Mg2+ or Ca2+. In the presence of 2 mM Mn2+, cleavage and integration occurred at additional sites indicating a decreased specificity. The properties of mutant IN proteins were examined in vitro. Deletion of 39 amino acids from the N-terminus and a minimum of 25 residues from the C-terminus impaired IN-mediated cleavage and integration activities. The results of site-directed mutagenesis experiments showed that residues critical for IN function are highly conserved. Mutations of conserved residues Asp64, Pro109, Asp116, and Glu152 adversely affected IN function in vitro. Mutations of nonconserved residues Gly189 and Thr112 had no effect. Mutation of a conserved Thr115 to Ala caused a near complete loss of Mg(2+)-dependent integration activity, but only partially effected endonucleolytic cleavage activity of IN. These results suggest that not all conserved residues are involved in both endonucleolytic cleavage and integration activities of HIV-IN.

Amino Acid Sequence↗

PubMed1992

Progression of early steps of human immunodeficiency virus type 1 replication in the presence of an inhibitor of viral protease.

We have evaluated a possible role for human immunodeficiency virus type 1 protease during early steps of replication. For these studies, a specific inhibitor of human immunodeficiency virus protease, Ro31-8959, was used. Synthesis of viral cDNA, its integration into cellular DNA, and its transcription were determined during a one-step, acute infection of MT-4 cells. No consistent difference in any of these parameters was noted between control-infected cultures and those treated with protease inhibitor. However, no infectious progeny virus was produced in treated cultures, and thus spread of infection was severely restricted. Our results do not support an essential activity of viral protease in early steps of replication but are in line with its established role in gag and gag-pol processing and in maturation to infectious progeny virus.

Antiviral Agents↗

PubMed1990

CD4-affinity purification of recombinant and native HIV gp120 and comparison of the affinity constants for the receptor.

Soluble CD4-immunoglobulin chimeric proteins were covalently attached to CNBr-activated Sepharose. This affinity matrix was used to establish a powerful new method to isolate different species of the HIV external glycoprotein gp120 from cell-free culture supernatants. Recombinant gp120 was expressed in Baculovirus-infected insect cells and isolated from cell-free culture supernatants. The recombinant protein has an apparent molecular mass of 130 kDa. These two gp120 species were shown to be of identical molecular size after complete deglycosylation achieved by endoglycosidase treatment, and they bound to CD4-H gamma l with the same binding constant, that was reported for native forms of gp120 and CD4. Thus the different glycosylation of gp120 does not influence its affinity to CD4 and the gp120-CD4 complex can be reversibly dissociated.

Blotting, Western↗

PubMed1990

Expression of biologically active human platelet-derived growth factor (PDGF) B-receptor in Xenopus laevis oocytes.

Platelet-derived growth factor (PDGF) consists of three different isoforms, PDGF-AA, PDGF-AB and PDGF-BB, which bind to at least two types of receptors: the B-receptor, to which only PDGF-BB binds, and the A/B receptor, to which all three isoforms bind. Microinjection of synthetic mRNA in Xenopus laevis oocytes was used to obtain cell-surface expression of the human PDGF B-receptor. The production of receptor molecules of correct size (190 kd) was demonstrated by specific immunoprecipitation; the binding properties of the membrane- associated PDGF B-receptor were investigated with highly purified recombinant [125I] labeled human PDGF-BB and -AA. Unlike Swiss mouse 3T3 cells, which possess both B- and A/B-receptors and, therefore, bind both isoforms with high affinity, the mRNA-injected oocytes bound only the BB isoform. Mock-injected oocytes showed no specific binding.

Animals↗

PubMed1990

HIV-1 RT-associated ribonuclease H displays both endonuclease and 3'----5' exonuclease activity.

We have analysed the mechanism of ribonuclease H (RNaseH) induced cleavage of a defined RNA-DNA hybrid by human immuno-deficiency virus (HIV-1) reverse transcriptase (RT). An in vitro transcribed RNA labelled at the 3' end was hybridized to a pentadecameric DNA oligonucleotide complementary to an internal region of the RNA. Upon incubation of this RNA-DNA hybrid with recombinant p66 or p66/p51 HIV-1 reverse transcriptase, RT-RNaseH mediated cleavage is observed at most nucleotides within the short hybridized stretch, resulting in a spectrum of RNA fragments extending from the 3' label to this region and differing in length by one nucleotide. The same RNA, this time labelled at the 5' end, yields only one or two major cleavage products corresponding to RNA species extending from the 5' label to the middle of the hybridized region. Such a result can be explained by the action of both endonuclease and 3'----5' exonuclease activities inherent to the C-terminal domain of p66 RT. To investigate how RNaseH cleavage is coupled to reverse transcription, a combination of deoxynucleoside triphosphates was used which allowed controlled extension of the primer DNA. Concomitantly with the elongation of the oligonucleotide primer, RNaseH cleavage proceeds towards the 5' end of the RNA with identical increments, suggesting a simultaneous action of both activities.

Base Sequence↗

PubMed1990

Mutation of conserved N-glycosylation sites around the CD4-binding site of human immunodeficiency virus type 1 GP120 affects viral infectivity.

Infection by the human immunodeficiency virus type 1 (HIV-1) is initiated through interaction of its exterior envelope glycoprotein gp120 with the CD4 receptor on target cells. To address the possible role of N-glycosylation of HIV-1 gp120 in binding CD4, we mutated different conserved N-glycosylation site Asn-residues in the vicinity of the putative CD4 binding site, as single mutations or in combinations. Authentic and mutant gp120 proteins were produced using the baculovirus expression system. All mutant proteins were produced and secreted at similar levels and could be immunoprecipitated with an HIV(+)-serum. Furthermore, all glycosylation mutants retained the full capacity to bind CD4 except for a triple mutant which showed reduced binding. Different gp120 mutant genes were then introduced in an infectious proviral DNA clone. Upon transfection of MT-2 cells, the authentic HIV-1 clone induced maximal virus production after 6 days. In the case of the triple glycosylation mutant, comparable virus production was first reached after a delay of about 12 days. Moreover, in contrast to native HIV, the mutant virus induced no typical multinucleated giant cells. These results suggest that the attached carbohydrates around the CD4-binding site of gp120, may contribute to the generation of this protein domain required for high affinity receptor interaction.

Acquired Immunodeficiency Syndrome↗

PubMed1990

Molecular cloning and characterization of a German HIV-1 isolate.

The genetic diversity of HIV-1 is well documented. Except for the HIV-1 isolate LAV-1BRU, no nucleic acid sequence of a European isolate of HIV-1 has been published to date. To further investigate the extent of the genetic variability and the evolution of HIV-1, we have isolated, cloned, and subsequently sequenced HIV-1 from a German patient with AIDS-related complex. Comparative studies of the nucleic acid sequence revealed that this isolate, designated HAN2, is highly divergent from the North American and African subtypes of HIV-1 and may represent a European subtype of HIV-1. Furthermore, a full-length molecular clone was derived from this isolate which was infectious in human T-cell lines. Therefore this new isolate will be particularly useful for studies on the genetic evolution and biology of HIV-1 as well as for testing antiviral substances and for developing vaccines.

Amino Acid Sequence↗

PubMed1990

Genomic divergence of an HIV-2 from a German AIDS patient probably infected in Mali.

The complete nucleotide sequence of an HIV-2 isolate derived from a German AIDS patient with predominantly neurological symptoms is reported. The HIV-2BEN sequence is highly divergent from those of previously described HIV-2 and SIV strains. Evolutionary tree analysis of eight HIV-2 sequences reveals the existence of three HIV-2 groups. HIV-2BEN belongs to a group with two isolates from Ghana and The Gambia. Based on a comparison of HIV-2BEN with six HIV-2 isolates, SIVsmm and SIVmac, the variability of the structural env and gag proteins is similar within the HIV-2/SIVsmm/mac and HIV-1 groups. In contrast, the regulatory HIV-1 proteins are more highly conserved than those from HIV-2 strains. Multiple sequence alignments reveal that some domains of the envelope and regulatory proteins are well conserved among HIV-1, HIV-2/SIVsmm/mac, SIVagm and SIVmnd. The identification of conserved domains within the external glycoprotein could help to develop broadly active vaccines.

Acquired Immunodeficiency Syndrome↗

PubMed1989

Comparison of the human immunodeficiency virus type 1 and 2 proteases by hybrid gene construction and trans-complementation.

To determine the cleavage specificity of the proteases of the type 1 and 2 human immunodeficiency viruses (HIV-1, HIV-2), we interchanged this domain of the polymerase (pol) genes and analyzed the maturation programs of the chimeric polyproteins in an Escherichia coli expression system. In both cases, release of reverse transcriptase and integrase was observed, together with the respective 10-kDa protease form resulting from autocatalysis, although the maturation proceeded less efficiently compared to the homologous system. In further experiments, the ability of both HIV-1 and HIV-2 proteases to release in vivo gag p24 from an in-frame fusion of the full length gag and protease precursors was analyzed. In either case, p24 was released, albeit with greater efficiency in the heterologous gene construction. In vitro mixed lysate experiments with the HIV-1 gag precursor furthermore demonstrate that both enzymes respond to the aspartyl proteinase inhibitor pepstatin A. Taken together, these results illustrate that although different cleavage recognition sequences exist for HIV-1 and -2, they are amenable to the proteases of both viruses, but additionally that subtle differences in the mode of action of both enzymes are observable.

Catalysis↗

PubMed1988

Active site mutagenesis of the AIDS virus protease and its alleviation by trans complementation.

Replacement of the putative active site Asp residue of cloned HIV-1 protease with Ala yields a molecule incapable of autocatalytic processing. Similarly, protease/reverse transcriptase and protease/reverse transcriptase/endonuclease polyproteins containing the same mutation accumulate as enzymatically inert polyproteins. Introduction of a second, wild-type, copy of protease in trans alleviates this defect, leading in the case of individually cloned protease to cleavage of the mutant protein, and with the polyprotein mutants to release of the reverse transcriptase and endonuclease polypeptides, the former of which recover enzymatic activity. In related experiments, a similar inhibition and trans-complementation of a genetically engineered gag--protease fusion protein was observed.

Amino Acid Sequence↗

PubMed1988

Processing protease and reverse transcriptase from human immunodeficiency virus type I polyprotein in Escherichia coli.

Expression of the human immunodeficiency virus type I pol open reading frame in Escherichia coli led to several protease-mediated processing steps of the pol precursor polyprotein. Accumulation of two polypeptides with molecular sizes of 64 and 52 kilodaltons, with which reverse transcriptase activity is associated, was observed. The protease moiety of the precursor polyprotein accumulated as a 10-kilodalton species as a result of two specific cleavages. Furthermore, a single-amino-acid substitution in the putative active site of protease totally abolished processing of the precursor polyprotein.

Amino Acid Sequence↗

PubMed1988

A single 66-kilodalton polypeptide processed from the human immunodeficiency virus type 2 pol polyprotein in Escherichia coli displays reverse transcriptase activity.

We have cloned the entire pol gene of human immunodeficiency virus type 2 into a high-level Escherichia coli expression system. Induction of cultures containing the recombinant plasmid, p2RTL1, leads to rapid accumulation of polypeptides of 66, 54, and 34 kilodaltons. We have designated the larger polypeptides reverse transcriptase, and we have designated the smaller polypeptide endonuclease. Purification of reverse transcriptase via ion-exchange and affinity chromatography yields the 66-kilodalton polypeptide, with which reverse transcriptase activity is associated. Purified enzyme furthermore displays a higher apparent molecular weight than its counterpart from human immunodeficiency virus type 1.

Escherichia coli↗

PubMed1987

A new ELISA test for HIV antibodies using a bacterially produced viral env gene product.

Screening tests for antibodies to the human immunodeficiency virus (HIV), based on the indirect ELISA principle using viral preparations as antigen, yield a substantial number of false-positive and false-negative results. These failures are due to the lack of certain viral polypeptides or contaminating cellular polypeptides in viral preparations. Therefore, the accuracy of the screening tests should be improved by using highly purified, synthetic viral antigens. With establishment of such an ELISA antigen in mind, we examined a bacterially synthesized polypeptide [ENV(80)] that corresponds to 80 conserved amino acids of the HIV gp41 transmembrane glycoprotein. ENV(80) was expressed as a DHFR fusion protein in Escherichia coli. Results obtained by HIV ELISA and immunoprecipitation with 497 serum samples from various groups at risk of AIDS were compared with those obtained with the ENV(80) ELISA. The ENV(80) ELISA was found to be superior to the H9/HTLV-III ELISA with respect to sensitivity and specificity and is almost equivalent in accuracy to immunoprecipitation.

Acquired Immunodeficiency Syndrome↗

PubMed1987

Expression of biologically active human T-cell lymphotropic virus type III reverse transcriptase in Bacillus subtilis.

A 2.4-kb DNA fragment from the pol region of the human T-cell lymphotropic virus, encoding the protease, reverse transcriptase and a portion of the endonuclease N-terminus was stably introduced into a high-level Bacillus subtilis expression system under inducible control of the Escherichia coli lac regulatory elements. The major expression plasmid, pRTL11, contains a bacteriophage T5 promoter/lac operator element, which is controlled by lac repressor, supplied by the secondary plasmid, pBL1. Upon IPTG induction, a 90-kDa polyprotein is synthesised and subsequently proteolytically cleaved to reveal 64-kDa and 52-kDa polypeptides. Partial purification reveals that reverse transcriptase activity co-migrates with these two polypeptides.

Bacillus subtilis↗

PubMed1986

Glyceraldehyde-3-phosphate dehydrogenase is a nonhistone protein and a possible activator of transcription in neurons.

A single-stranded DNA-binding protein of Mr 35,000 (35K protein) was isolated from calf cerebral cortex by affinity chromatography on immobilized double-stranded and single-stranded DNA. Its localization in the nuclear compartment was demonstrated by immunohistochemistry. Previous studies had uncovered a homologous nonhistone chromosomal protein in the nuclei of rat cerebral cortex neurons, cerebellar neurons, oligodendrocytes, and liver cells. The rat protein accumulated in the nuclear compartment of neurons in exact temporal coincidence with the arrest of cell division and the initiation of terminal differentiation. Therefore, in the present work, the 35K protein was tested for an activating role in RNA transcription. During the course of this study we became aware that the 35K protein was identical to a glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12). When authentic GAPDH from rabbit skeletal muscle was injected into Xenopus laevis oocytes, it greatly stimulated RNA polymerase II transcription, whereas the 35K protein from calf brain did not. This apparent discrepancy was partially resolved by the finding that rabbit muscle GAPDH could be fractionated into two components by affinity chromatography on single-stranded DNA cellulose. Only 5% of the applied protein was retained on the column and could be eluted with a shallow salt gradient identical to the one used for the isolation of the 35K protein. This single-stranded DNA-binding component of rabbit muscle GAPDH did not stimulate transcription. Apparently, the 35K protein from calf brain corresponded to this single-stranded DNA-binding subfraction, which explained its failure to activate transcription.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1985

Stimulation of sea urchin H2B histone gene transcription by a chromatin-associated protein fraction depends on gene sequences downstream of the transcription start site.

We isolated a chromosomal protein fraction derived from chromatin of sea urchin embryos which specifically stimulated the expression of the histone H2B gene by a factor of 5- to 10-fold when the complete sea urchin histone gene repeat h22 was injected in Xenopus laevis oocyte nuclei. Gene manipulation experiments revealed the existence of two different target sites in the H2B gene which appear to mediate the response to injection of the stimulatory sea urchin chromatin-associated proteins; both are located downstream of the transcription initiation site. The first sequence element which is shown to be implicated is within, or at least includes, the H2B 5' untranslated leader sequence between nucleotides 11 and 76. The second element resides within an H2B DNA segment located near the 3' end of the gene, extending from 90 base pairs upstream of the mRNA 3' terminus to 140 base pairs in the spacer sequences downstream.

Animals↗

PubMed1984

Processing and nucleo-cytoplasmic transport of histone gene transcripts.

Precursors of Xenopus and sea urchin histone mRNAs were synthesized in vitro with the SP6 transcription system, and their maturation and nucleo-cytoplasmic transport was studied by frog oocyte injection. 3' processing is most rapid for homologous histone messenger sequences and does not require either genuine 5' or specific 3' ends of the precursor, but capping of the 5' terminus strongly influences the efficiency of 3' processing. No generation of 5' histone mRNA ends can be detected when precursors containing 5' spacer sequence extensions are injected into the oocyte nucleus. This finding may have some implications for the question whether histone gene transcription could be polycistronic. Using a novel oocyte technique, we have separated nuclei from cytoplasm and have studied the time course of exit of the processed RNA from the oocyte nucleus into the cytoplasm. The results suggest that RNA maturation and nucleo-cytoplasmic transport are not temporally coupled processes.

Animals↗