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B Krust

Publications and source records attributed to B Krust.

At least 37 records · Page 2Linked to original sources
PubMed1996

Specific binding of adenosine deaminase but not HIV-1 transactivator protein Tat to human CD26.

Adenosine deaminase (ADA) and the HIV-1 transactivator protein Tat have been reported to bind to human CD26, also known as dipeptidyl peptidase IV (DPP IV). In order to demonstrate the specificity of such binding under native conditions of CD26, i.e., when expressed on the cell surface, we established murine cell lines expressing transfected human CD26, either wild-type or mutated at its serine-630, which inactivates the DPP IV activity. This experimental system is advantageous since murine ADA does not bind human CD26, whereas human and bovine ADA bind. Consequently, murine cell clones expressing either the wild-type or mutated form of human CD26 were found to bind specifically bovine 125I-labeled ADA with a high affinity (KD = 12 +/- 2 nM and 11 +/- 4 nM, respectively). No specific binding of 125I-labeled ADA was observed to murine clones not expressing human CD26. The binding of 125I-labeled ADA to CD26 was further characterized by the use of monoclonal antibodies specific to human CD26. The results obtained were in accord with those reported previously using other experimental models. These observations indicated that the murine cells expressing human CD26 provide a highly suitable model to investigate the potential binding of HIV-1 Tat to CD26. In contrast to previously published results, however, we could not demonstrate a specific interaction between Tat and human CD26. The 125I-labeled ADA-specific binding to human CD26 was not affected by Tat, even at concentrations which induced cell death. Similarly, the binding of several monoclonal antibodies to human CD26 was not modified by the addition of Tat. More significantly, Tat binding to different murine cell clones (human CD26 negative or positive) was found not to be correlated with the expression of human CD26. Finally, the toxic effect of Tat on the growth of different murine cell clones was independent of human CD26 expression. Taken together, these observations further confirm the specific binding of ADA to human CD26 and point out that CD26 is not the target of HIV-1 Tat protein.

Adenosine Deaminase↗
PubMed1996

Inhibition of HIV infection by pseudopeptides blocking viral envelope glycoprotein-mediated membrane fusion and cell death.

The RP dipeptide motif is highly conserved in the third hypervariable region (V3 loop) of the extracellular envelope glycoprotein of different types of HIV isolates. In view of this, we have designed and synthesized a construction referred to as "template assembled synthetic peptide" (TASP), in which a lysine-rich short polypeptide was used as a template to covalently anchor arrays of tripeptides, such as RPR, RPK, or KPR. The pentavalent presentation, 5(RPR)-, 5(RPK)-, or 5(KPR)-TASP, molecules manifested maximum inhibitory activity on HIV infection with a 50% inhibitory concentration value of 1-5 microM, respectively. Structure and inhibitory-activity relationship studies using analogs of 5(KPR)-TASP indicated that the positively charged side chains of the K and R residues in the tripeptide molecules are critical for the optimal inhibitory activity of the pentavalent construct. Interestingly, replacement of L-amino acid residues by D-amino acids or reduction of the peptide bond between the first two amino acids of the tripeptide generated peptide-TASP analogs active at sub-microM, concentrations. The anti-HIV action of the peptide-TASP constructs is specific, since they inhibit infection of several types of CD4-expressing cells by HIV-1 Lai and HIV-2 EHO but not by the simian SIV-mac isolate. Our results suggest that these inhibitors block three post-CD4 binding functions of the HIV envelope glycoproteins, mediation of viral entry, syncytium formation, and triggering cell death by apoptosis. As the peptide-TASP derivatives with unnatural amino acid sequences in the tripeptide moiety retain full inhibitory activity, they should provide potent protease-resistant peptide inhibitors as potential therapeutic agents for treatment of AIDS patients.

Amino Acid Sequence↗
PubMed1994

Specific inhibition of viral protein synthesis in HIV-infected cells in response to interferon treatment.

The mechanism of action of different types of interferons (IFN-alpha, -beta, and -gamma) against human immunodeficiency virus (HIV)-1 infection was investigated in chronically infected monocytoid U937 cells and during an acute infection of the T lymphoblastoid CEM cells. Two chronically infected U937 cell populations, obtained independently (referred to as type A and B cells), were analyzed for their response to IFNs. In type A cells, IFNs mainly inhibited virus particle release, whereas in type B cells, the anti-HIV effect of IFNs cells was found to be largely due to a specific inhibition of viral protein synthesis without any apparent effect on total cellular protein synthesis. Interestingly, such a differential inhibition of HIV protein synthesis could also be demonstrated in acutely infected CEM cells in response to treatment with IFN-alpha. Both in chronically infected U937 type B and acutely infected CEM cells, equivalent amounts of nuclear and cytoplasmic HIV-1 mRNA were detected in control and IFN-treated cells in spite of at least 80% inhibition of HIV protein synthesis. Analysis of the distribution of cellular and viral mRNAs on polysomes in HIV-1-infected cells demonstrated that IFN treatment induces a specific block on viral mRNA translation. These results indicate that the antiviral mechanism of IFN on later stages of HIV replication cycle may be partly due to the inhibition of HIV mRNA translation, besides an effect on virus budding or release.

HIV-1↗
PubMed1994

HIV-2 EHO isolate has a divergent envelope gene and induces single cell killing by apoptosis.

The human immunodeficiency virus type 2 (HIV-2)-related isolate, referred to as HIV-2 EHO, has been isolated from an Ivory Coast patient with acquired immunodeficiency syndrome (AIDS). Infection of CD4 expressing cells with this highly infectious virus mediates a cytopathic effect characterized by single-cell killing as a consequence of apoptosis. Nucleotide sequence analysis of the HIV-2 EHO genome revealed a significant degree of divergence of its envelope gene from that of other known HIV-2 strains. This divergence for the deduced amino acid sequence corresponding to the extracellular envelope glycoprotein was 26 to 30%. These unique genetic and biological properties suggest that the HIV-2 EHO isolate is a distinct prototype in the HIV-2 family.

Acquired Immunodeficiency Syndrome↗
PubMed1993

T cell activation antigen, CD26, as a cofactor for entry of HIV in CD4+ cells.

The CD4 molecule is essential for binding HIV particles, but is not sufficient for efficient viral entry and infection. The cofactor was shown to be dipeptidyl peptidase IV (DPP IV), also known as CD26. This serine protease cleaves its substrates at specific motifs; such motifs area also highly conserved in the V3 loops of HIV-1, HIV-2, and related simian isolates. Entry of HIV-1 or HIV-2 into T lymphoblastoid and monocytoid cell lines was inhibited by a specific monoclonal antibody against DPP IV or specific peptide inhibitors of this protease. Coexpression of human CD4 and CD26 in murine NIH 3T3 cells rendered them permissive to infection by HIV-1 and HIV-2. These observations could provide the basis for developing simple and specific inhibitors of HIV and open a possibility for vaccine development.

3T3 Cells↗
PubMed1993

Inhibition of entry of HIV into cells by poly(A).poly(U).

Polyadenylic-polyuridylic acid referred to as poly(A).poly(U), is a synthetic double-stranded RNA that has been shown to manifest both antitumoral and immunodulatory activities. Previously, we have reported that poly(A).poly(U) inhibits HIV infection in cell cultures. Here we provide direct evidence to demonstrate that the inhibitory action of poly(A).poly(U) is through its capacity to prevent entry of HIV particles into CD4-positive T lymphocytes. Such inhibition of HIV entry is also observed in the case of other polyanions such as heparin, dextran sulfate, and poly(I).poly(C). The mechanism of inhibition appears to occur postbinding of HIV particles to the CD4 receptor molecules, because the binding of the external envelope glycoprotein of HIV-1 (gp120) is not affected significantly in the presence of poly(A).poly(U) or other polyanions. These results confirm the potential of poly(A).poly(U) as an antiviral drug against HIV infection.

Antiviral Agents↗
PubMed1993

Membrane expression of HIV envelope glycoproteins triggers apoptosis in CD4 cells.

The cytopathic effect of HIV-1 and HIV-2 in CD4+ lymphocytes has been shown to be associated with apoptosis or programmed cell death. Using different experimental conditions, we demonstrate here that apoptosis is triggered by cell membrane expression of the mature HIV envelope glycoproteins, gp120-gp41 complex, and their interaction with CD4 receptor molecules. Viral entry alone did not induce apoptosis but virus replication was required in order to produce the gp120-gp41 complex. Indeed, expression of the HIV env gene alone in the CD4+ T cell line (CEM) was sufficient for the induction of apoptosis. In general, syncytium formation and apoptosis induction were closely associated as both events require functional envelope glycoproteins and CD4 molecules. Nevertheless, apoptosis but not syncytium formation was suppressed by a monoclonal antibody against CD4 that does not affect gp120 binding. Furthermore, single-cell killing by apoptosis was observed in infected cell cultures treated with a monoclonal antibody against gp41, which completely abolishes the formation of syncytia. These results indicate that apoptosis is not the consequence of toxic effects induced by the formation of syncytia but is triggered by the HIV envelope glycoproteins. Therefore, cell death during HIV infection in CD4+ lymphocyte cultures is due to a specific event triggered by the gp120-gp41 heterodimer complex programming death in metabolically active cells.

Amino Acid Sequence↗
PubMed1992

Antiviral action of polyadenylic-polyuridylic acid against HIV in cell cultures.

Polyadenylic-polyuridylic acid referred to as poly(A).poly(U) is a synthetic double-stranded RNA which has been shown to manifest both antitumoral and immunomodulatory activities. Here we used this agent to demonstrate its antiviral activity against the human immunodeficiency virus (HIV-1 and HIV-2). Treatment of cells with poly(A).poly(U) resulted in a significant delay in the development of the HIV-specific cytopathic effect characterized by the formation of syncytia and cell lysis. Furthermore, the production of virus measured by the concentration of the HIV major core protein was reduced by 90-95%. Under these experimental conditions, the synthesis of HIV proteins was reduced at least tenfold whereas the metabolism and proliferation of cells apparently were not affected. The inhibitory action of poly(A).poly(U) seems to be at the level of viral entry into cells. Combined treatment of infected cells with poly(A).poly(U) and azidothymidine (AZT) resulted in a 4-5-fold synergistic inhibitory effect. Previously, no toxicity has been observed in cancer patients with long-term treatment with poly(A).poly(U). In view of this and the significant anti-HIV effect, poly(A).poly(U) provides a potential candidate as a therapeutic drug in AIDS disease.

Antiviral Agents↗
PubMed1991

The cytopathic effect of HIV is associated with apoptosis.

Large amounts of histones, H1, H2A, H2B, H3, and H4, were observed in total extracts of T4 lymphocytes and derived cell lines infected with the human immunodeficiency virus (HIV) type 1 or type 2. These histones were simply detectable by analysis of crude cellular extracts by polyacrylamide gel electrophoresis in SDS and staining the proteins with Coomassie blue or by immunoblot assays using specific polyclonal antibodies. The histones were found to be localized in the nucleoplasm, bound to low molecular weight (LMW) DNA in the form of nucleosomes. The mechanism responsible for the accumulation of nucleosomes during HIV infection was found to be due to fragmentation of cellular DNA, a mechanism referred to as apoptosis or programmed cell death in which a nuclear endonuclease becomes activated and cleaves DNA at internucleosomal regions. Accordingly, the LMW DNA accumulated in the course of infection was found to have a characteristic pattern of nucleosomal ladder and its accumulation was reduced in the presence of zinc, a known inhibitor of the endonuclease. Routinely in acute HIV infections, the accumulation of nucleosomes was observed at least 24 hr before lysis of infected cells. In a particular HIV-1 infection, in which the first signals of the cytopathic effect (vacuolization of cells and appearance of syncytia) was observed at Days 6-7 whereas maximal virus production occurred at Days 10-17, the accumulation of nucleosomes was at its maximal level already on Day 6 postinfection. In the nucleoplasm of chronically infected cells producing virus but not manifesting a cytopathic effect, no LMW DNA or histones were detectable. These observations indicate that the cytopathic effect of HIV infection is associated with apoptosis. The detection of histones and oligonucleosomal DNA fragments in the nucleoplasm can be used as a convenient marker for chromatin fragmentation during this process.

CD4-Positive T-Lymphocytes↗
PubMed1990

Production of a non-functional nef protein in human immunodeficiency virus type 1-infected CEM cells.

The nef gene product of the human immunodeficiency virus (HIV) is suggested to be a negative factor involved in down-regulating viral expression by a mechanism in which the correct conformation of the nef protein is essential. The nef protein expressed by vaccinia virus recombinants is phosphorylated by protein kinase C. We investigated the synthesis of the nef protein and its state of phosphorylation during HIV-1 infection of a T4 cell line (CEM cells). Maximum synthesis of viral proteins occurred 3 days after infection, when more than 90% of cells were producing viral proteins. The synthesis of the nef protein was detected in parallel with the env and gag proteins. As expected, the nef protein was myristylated but not phosphorylated, and its half-life was less than 1 h. By the use of the polymerase chain reaction technique, we isolated and sequenced the nef gene of this HIV-1 stock. Two significant mutations were observed. Firstly, threonine, at amino acid number 15, the site of phosphorylation by protein kinase C, was mutated into an alanine, and secondly aspartic acid of the tetrapeptide WRFD, which is probably involved in GTP binding, was mutated into an asparagine. The mutated nef gene was expressed in a vaccinia virus system, in which it was not phosphorylated and its half-life was dramatically reduced compared to the wild-type nef gene product. Furthermore, down-regulation of CD4 cell surface expression was no longer affected by the mutated nef gene. These results emphasize that phosphorylation of the nef protein provides an efficient test to monitor its biological activity.

Amino Acid Sequence↗
PubMed1990

Transmembrane envelope glycoproteins of human immunodeficiency virus type 2 and simian immunodeficiency virus SIV-mac exist as homodimers.

An 80-kilodalton glycoprotein (gp80) was produced in human immunodeficiency virus type 2 (HIV-2)-infected cells along with three envelope glycoproteins that we have recently reported: the extracellular glycoprotein (gp125), the envelope glycoprotein precursor (gp140), and the transient dimeric form of the precursor (gp300). gp125 and gp80 were detectable after the synthesis of gp140 and the formation of gp300. Using a specific monoclonal antibody, we showed here that gp80 is a dimeric form of the transmembrane glycoprotein gp36 of HIV-2. Dimerization of the envelope glycoprotein precursor and dimeric forms of the transmembrane glycoproteins were also observed in cells infected with simian immunodeficiency virus (SIV-mac), a virus closely related to HIV-2. Under routine conditions of our experiments (i.e., extraction by 1% Triton X-100 before polyacrylamide gel electrophoresis in sodium dodecyl sulfate [SDS]), monomeric forms of the transmembrane glycoprotein of HIV-2 and SIV-mac were only seldomly observed. Dimeric forms of the envelope precursors and the transmembrane glycoproteins are probably stabilized by extraction in the nonionic detergent Triton X-100 since such dimeric forms resist dissociation during subsequent electrophoresis in the presence of the ionic detergent SDS. However, the dissociation of these dimeric forms might occur when samples are prepared by extraction directly in 1% SDS or by incubation of the purified dimers at acidic pH. Dimerization of the envelope precursor might be required for its processing to give the mature envelope proteins, whereas the transmembrane dimer might be essential for optimal structure of the virion and thus its infectivity.

Amino Acid Sequence↗
PubMed1989

Characterization of an HIV-2-related virus with a smaller sized extracellular envelope glycoprotein.

A new isolate of the human immunodeficiency virus (HIV) related to the HIV-2 strain was isolated from peripheral blood lymphocytes of an Ivory Coast patient with AIDS. This isolate referred to as HIV-2 EHO could be differentiated by its envelope precursor and external glycoprotein which are 20-kDa smaller than those of HIV-2 ROD isolate. Furthermore, the apparent molecular weight of the major core protein of HIV-2 EHO is 27 kDa instead of 26 kDa as in HIV-2 ROD isolate. In addition, the product of the vpx gene which is a characteristic feature of the HIV-2 strain, is 14 kDa in HIV-2 EHO compared with 16 kDa in HIV-2 ROD. In contrast to these, the envelope precursor of HIV-2 EHO forms a transient dimer its maturation as is the case for HIV-2 ROD. In both cases the transmembrane proteins are 36 kDa and exists as homodimers of 80 kDa. Endoglycosidase H digestion experiments indicated that the 20-kDa difference between the two HIV-2 isolates is not due to a difference in the number of N-linked oligosaccharide chains per polypeptide, since deglycosylated envelope precursors of HIV-2 ROD and EHO have an apparent molecular weight of 80 and 60 kDa, respectively. Partial proteolysis of the envelope precursors from the two isolates with Staphylococcus aureus V8 protease gave a distinct pattern of polypeptides. These results suggest that the differences between the external envelope proteins of the two HIV-2 isolates are due to their amino acid composition. Accordingly, polyclonal antibodies raised against HIV-2 ROD envelope do not recognize the corresponding envelope proteins of HIV-2 EHO by immunoblotting and immunoprecipitation assays. These data illustrate that analysis of viral proteins could be useful for a rapid characterization of new viral isolates and show the heterogeneity of HIV-2 isolates in West Africa.

Acquired Immunodeficiency Syndrome↗
PubMed1989

Characterization of human immunodeficiency virus type 2 envelope glycoproteins: dimerization of the glycoprotein precursor during processing.

Four glycoproteins with apparent molecular weights of 300,000, 140,000, 125,000, and 36,000 (gp300, gp140, gp125, and gp36) were detectable in human immunodeficiency virus type 2 (HIV-2)-infected cells. gp125 and gp36 are the external and transmembrane components, respectively, of the envelope glycoproteins of HIV-2 mature virions. gp300 and gp140 are only detectable in virus-infected cells. They have identical isoelectric points, suggesting that gp300 might be a dimeric form of the immature precursor, gp140. The purified gp300 can be dissociated in a slightly acidic buffer to give rise to monomers of 140,000 molecular weight. Such dissociated monomers and the purified gp140 showed identical patterns of polypeptides after partial proteolysis with Staphylococcus aureus V8 protease. Pulse-chase experiments indicated that gp300 is formed after synthesis of gp140 and before the detection of the mature external envelope glycoprotein, gp125. These results were confirmed by using various inhibitors of glycosylation and inhibitors of trimming enzymes. Dimer formation of the envelope glycoprotein precursor was also observed in cells infected with simian immunodeficiency virus (SIV), a virus closely related to HIV-2. On the other hand, the envelope glycoprotein precursor of HIV-1 did not form a dimer during its processing. Therefore, dimer formation seems to be a specific property of HIV-2 and SIV envelope gene expression. Such transient dimerization of the glycoprotein precursor might be required for its efficient transport to the Golgi apparatus and for its processing.

Glycoproteins↗
PubMed1989

Cell surface expression of several species of human immunodeficiency virus type 1 major core protein.

The major core protein (p25) of the human immunodeficiency virus type 1 (HIV-1) was characterized by two-dimensional-gel isoelectric focusing. The p25 detectable in HIV-1-infected cells is composed of four species with related isoelectric points. This is due in part to the phosphorylated state of p25. The four species of p25 are expressed on the cell surfaces of infected cells, but only the two most basic species are incorporated into the HIV-1 virion. These findings emphasize the importance of p25 in understanding infection with HIV and might have implications for the development of vaccines.

Antigens, Surface↗
PubMed1988

Characterization of a monoclonal antibody specific for the HIV-1 precursor glycoprotein.

HIV-1 infected Molt4-T4 cells provide an efficient system for the production of cellular precursor gp160 of HIV envelope glycoproteins, gp120 and gp41. The precursor gp160 was purified on an immuno-affinity column containing antibodies from sera of HIV-1-seropositive patients. The precursor gp160 was then isolated by preparative polyacrylamide gel electrophoresis. Two out of four Balb/c mice, immunized with these purified preparations of gp160, developed specific circulating antibodies. A hybridoma cell line was subsequently isolated producing monoclonal antibody KL49/19 (IgG1, K) specific for gp160. This monoclonal antibody can specifically immunoprecipitate gp160, existing in HIV-1-infected cells. In an immunoblotting assay, it identifies mainly gp160 and shows a slight affinity for the mature glycoprotein, gp120. The monoclonal antibody is probably directed against an epitope in the polypeptide residue of gp160 since it can recognize a deglycosylated polypeptide of molecular weight 90,000, a product of gp160 digestion by endoglycosidase H (Endo H). It does not cross-react with any protein of HIV-2 by immunoblot or immunoprecipitation assays. By virtue of its specificity, the monoclonal antibody KL49/19 might provide a powerful probe with which to detect gp160 in cells which might partially express the HIV-1 genes.

Animals↗
PubMed1985

Identification and antigenicity of the major envelope glycoprotein of lymphadenopathy-associated virus.

The major envelope glycoprotein of the causative agent of Acquired Immune Deficiency Syndrome (AIDS) lymphadenopathy-associated virus (LAV) has been identified and characterized. The glycoprotein has an apparent molecular weight of 110,000-120,000 under denaturing conditions in polyacrylamide gel electrophoresis. Upon deglycosylation by a specific endoglycosydase, its size is reduced to 80,000. Cellular precursors of this glycoprotein have been detected with apparent molecular weight of 150,000 and 135,000. Nearly all AIDS and pre-AIDS patients have detectable antibodies against this viral glycoprotein.

Carbon Radioisotopes↗
PubMed1985

Monoclonal antibodies to an interferon-induced Mr 68,000 protein and their use for the detection of double-stranded RNA-dependent protein kinase in human cells.

Extracts from interferon-treated human cells show an enhanced level of a double-stranded RNA-dependent protein kinase activity that is manifested by the phosphorylation of an endogenous Mr 69,000-72,000 protein in its phosphate-saturated state. By using a highly purified protein kinase fraction from interferon-treated human Daudi cells, we can now describe the preparation of murine monoclonal antibodies directed against this phosphoprotein, the Mr of which in its native state is found to be 68,000. These monoclonal antibodies (class IgG1) can identify the electrophoresed protein (p68) in polyacrylamide gels by the electrophoretic transfer blotting technique. Immunoprecipitates formed after incubation of extracts from interferon-treated human cells with the monoclonal antibodies can be conveniently recovered by protein A-Sepharose. Such immune complex preparations have associated protein kinase activity--i.e., addition of [gamma-32P]ATP results in the phosphorylation of p68 and added substrates, calf thymus histone, and eukaryotic initiation factor 2. Immune complex preparations from [35S]methionine-labeled extracts show the specific immunoprecipitation of p68. In addition, several other [35S]methionine-labeled proteins are bound unspecifically in these immune complexes prepared under similar experimental conditions as for the assay of protein kinase activity. These unspecifically bound proteins can be washed out by using a buffer containing detergents or high concentrations of KCl and magnesium acetate. Immune complex preparations washed similarly with these buffers still retain p68 but lose their capacity to phosphorylate p68 or exogenous substrates. These results indicate that p68 by itself has no protein kinase activity. The induction of [35S]methionine-labeled p68 in Daudi cells occurs with as little as 1 unit of human alpha interferon, with maximal synthesis between 6 to 9 hr after the addition of interferon. Actinomycin D blocks this induction.

Adenosine Triphosphate↗