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Biomedical subjects

S Oroszlan

Publications and source records attributed to S Oroszlan.

At least 55 records · Page 3Linked to original sources
PubMed1992

Identification of a major growth factor for AIDS-Kaposi's sarcoma cells as oncostatin M.

Conditioned medium from human T cell leukemia virus type 2 (HTLV-II)-infected T cells supports the growth and long-term culture of cells derived from acquired immunodeficiency syndrome (AIDS)-associated Kaposi's sarcoma lesions (AIDS-KS cells). A protein of 30 kilodaltons was purified from conditioned medium that supports the growth of AIDS-KS cells. The amino-terminal sequence of this protein was identical to the amino-terminal sequence of Oncostatin M, a glycoprotein that inhibits the growth of a variety of cancer cells. Oncostatin M from conditioned medium stimulated a twofold increase in the growth of AIDS-KS cells at a concentration of less than 1 nanogram of the protein per milliliter of medium.

Acquired Immunodeficiency Syndrome↗
PubMed1992

Purification of immature cores of mouse mammary tumor virus and immunolocalization of protein domains.

The immature capsids of the mouse mammary tumor virus (MMTV), known as intracytoplasmic A particles, have been isolated from murine L1210 leukemia cells. The diameter of the isolated particles was 80 nm as determined by negative staining. Two polypeptides of 77 and 110 kDa were found to be their major polypeptide components, in agreement with the expected sizes of the Gag and Gag-Pro precursor polypeptides of the mature MMTV proteins. Both polypeptides were recognized by antibodies directed toward the matrix (p10) and capsid (p27) proteins of MMTV. Immunogold labeling of p10 on isolated A particles, visualized by negative staining, showed that this protein is located at the surface of the immature capsids, whereas p27 can be detected only in broken or disrupted particles, suggesting that it has an internal location. These observations were confirmed by immunolabeling of both proteins on thin sections of A particle-producing cells. In addition, the viral protease had a more internal position than p27. Since the sequential order of the viral proteins in the Gag precursor is p10-pp21-p27-p14 and that in Gag-Pro is p10-pp21-p27-p30-protease, our results demonstrate the radial organization of the polypeptide precursors forming the intracytoplasmic A particles.

Amino Acid Sequence↗
PubMed1991

HIV-1 proteinase is required for synthesis of pro-viral DNA.

HIV-1 proteinase activity is thought to occur primarily post-integration by cleaving the viral Gag and Gag-Pol polyproteins. Its role in the pre-integration stages of viral replication, however, has not been studied in detail. Here we report that a synthetic peptide analogue, UK-88,947, which is a specific inhibitor of purified HIV-1 proteinase, inhibits the processing of the viral polyproteins in cultures of HIV-1 infected cells and prevents the formation of mature, infectious virions. Analysis of DNA from HIV-1 infected cells treated with UK-88,947 showed that viral DNA synthesis was inhibited when the compound was added to cultures one hour before infection. Similar results were obtained when AZT was used. Neither HIV-1 reverse transcriptase or the replication of FIV are inhibited by UK-88,947.

Amino Acid Sequence↗
PubMed1991

Autoprocessing of the HIV-1 protease using purified wild-type and mutated fusion proteins expressed at high levels in Escherichia coli.

Various constructs of the human immunodeficiency virus, type 1 (HIV-1) protease containing flanking Pol region sequences were expressed as fusion proteins with the maltose-binding protein of the malE gene of Escherichia coli. The full-length fusion proteins did not exhibit self-processing in E. coli, thereby allowing rapid purification by affinity chromatography on cross-linked amylose columns. Denaturation of the fusion protein in 5 M urea, followed by renaturation, resulted in efficient site-specific autoprocessing to release the 11-kDa protease. Rapid purification involving two column steps gave an HIV-1 protease preparations of greater than 95% purity (specific activity approximately 8500 pmol.min-1.micrograms protease-1) with an overall yield of about 1 mg/l culture. Incubation of an inactive mutant protease fusion protein with the purified wild-type protease resulted in specific trans cleavage and release of the mutant protease. Analysis of products of the HIV-1 fusion proteins containing mutations at either the N- or the C-terminal protease cleavage sites indicated that blocking one of the cleavage sites influences the cleavage at the non-mutated site. Such mutated full-length and truncated protease fusion proteins possess very low levels of proteolytic activity (approximately 5 pmol.min-1.micrograms protein-1).

ATP-Binding Cassette Transporters↗
PubMed1991

Interaction of C-terminal sequences of human immunodeficiency virus reverse transcriptase with template primer.

We have raised a rabbit monospecific antibody (designated C2003) against a synthetic peptide (CTP66) derived from a conserved sequence in the C-terminal portion of the p66 component of human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) (DeVico, A.L., Copeland, T.D., Veronese, F.D., Oroszlan, S., Gallo, R. C., and Sarngadharan, M. G. (1989) AIDS Res. Hum. Retroviruses 5, 51-60). This antibody directly inhibits the polymerase activity of HIV-1 RT and of RTs from a variety of retroviruses. HIV-1 RT is protected from this inhibition by preincubation of the enzyme with template primer prior to treatment with the antibody. Such protection is abrogated when the pretreatment is conducted under conditions of high ionic strength. Kinetic studies showed that the antibody-mediated inhibition is competitive with respect to template primer concentration. These results indicate that C2003 antibody acts to interfere with the template binding function of the enzyme and further indicates that conserved residues recognized by the antibody may be directly involved in this function.

Amino Acid Sequence↗
PubMed1991

Comparison of the HIV-1 and HIV-2 proteinases using oligopeptide substrates representing cleavage sites in Gag and Gag-Pol polyproteins.

The substrate specificity of the human immunodeficiency virus type 1 (HIV-1) and type 2 (HIV-2) proteinases was compared using oligopeptides corresponding to cleavage sites in the Gag and Gag-Pol polyproteins of both viruses. All peptides mimicking cleavage sites at the junction of major functional protein domains were correctly cleaved by both enzymes. However, some other peptides thought to represent secondary cleavage sites remained intact. The kinetic parameters (Km and kcat) obtained for the different substrates showed several hundred-fold variation but were similar for the same substrate.

Amino Acid Sequence↗
PubMed1991

The effect of salt on the Michaelis Menten constant of the HIV-1 protease correlates with the Hofmeister series.

The effect of different types of salt on the proteolytic activity of HIV-1 protease was studied. At a similar ionic strength, the enzyme activity changed according to the salting out effect of the ions used (Hofmeister series). Kinetic studies showed that a stronger salting out effect of the ions rather than the higher ionic strength per se increased the affinity to the substrate (Km) but in general did not alter the Kcat value.

Amino Acid Sequence↗
PubMed1991

Purification of HIV-1 wild-type protease and characterization of proteolytically inactive HIV-1 protease mutants by pepstatin A affinity chromatography.

Recombinant wild-type protease of human immunodeficiency virus, type 1 (HIV-1) expressed in E. coli was purified by pepstatin A affinity chromatography. An 88-fold purification was achieved giving a protease preparation with a specific enzymatic activity of approximately 3700 pmol/min/micrograms. Two proteolytically inactive HIV-1 mutant proteases (Arg-87----Lys; Asn-88----Glu) were found to bind to pepstatin A agarose, and and they were purified as the wild-type protease. A third mutant protease Arg-87----Glu) was apparently unable to bind to pepstatin A under similar conditions. Binding to pepstatin A indicates the binding ability of the substrate binding site and the ability to form dimers. These features may be used to purify and to characterize other mutated HIV-1 proteases.

Amino Acid Sequence↗
PubMed1991

Studies on the role of the S4 substrate binding site of HIV proteinases.

Kinetic analysis of the hydrolysis of the peptide H-Val-Ser-Gln-Asn-Tyr*Pro-Ile-Val-Gln-NH2 and its analogs obtained by varying the length and introducing substitutions at the P4 site was carried out with both HIV-1 and HIV-2 proteinases. Deletion of the terminal Val and Gln had only moderate effect on the substrate hydrolysis, while the deletion of the P4. Ser as well as P'3 Val greatly reduced the substrate hydrolysis. This is predicted to be due to the loss of interactions between main chains of the enzyme and the substrate. Substitution of the P4 Ser by amino acids having high frequency of occurrence in beta turns resulted in good substrates, while large amino acids were unfavorable in this position. The two proteinases acted similarly, except for substrates having Thr, Val and Leu substitutions, which were better accommodated in the HIV-2 substrate binding pocket.

Amino Acid Sequence↗
PubMed1991

In situ processing of a retroviral nucleocapsid protein by the viral proteinase.

The proteolytic processing pathway of the nucleocapsid protein (NC) by the viral proteinase within intact capsids of equine infectious anemia virus (EIAV) is presented. The cleavage sites are located at the carboxyl side of the first cysteine residue within the zinc-finger domains. EIAV is used as a model to predict similar NC cleavages in other retroviruses, including human immunodeficiency virus (HIV). The observed cleavages suggest a previously unrecognized function of the retroviral proteinase that may be crucial for replication during the early stages of the virus life-cycle (i.e. reverse transcription/integration).

Amino Acid Sequence↗
PubMed1991

Synthesis of homologous peptides using fragment condensation: analogs of an HIV proteinase substrate.

Two protected peptides Boc-Val-Ser(Bzl)-Gln-Asn-Tyr(BrZ)OH and Boc-Val-Ser(Bzl)-Gln-Asn-Tyr(BrZ)-ProOH were synthesized on a resin substituted by 9-(hydroxymethyl)-2-fluoreneacetic acid. After cleavage with piperidine/DMF, desalting, and activation, these peptides were used for the synthesis of 11 analogs of an HIV proteinase nonapeptide substrate Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-NH2 using fragment condensation in solid phase. The fragment condensation was made in an ultrasonic bath. Using only 2 equivalents of the activated peptide in a DMF solution, this reaction was complete in 2 h. All nonapeptides were assayed as substrates for HIV-1 and HIV-2 proteinases.

Amino Acid Sequence↗
PubMed1991

The topoisomerase I inhibitor, camptothecin, inhibits equine infectious anemia virus replication in chronically infected CF2Th cells.

Camptothecin (CPT), a topoisomerase I-specific inhibitor, was found in this study to inhibit the replication of equine infectious anemia virus (EIAV) in chronically infected CF2Th cells (designated CF2Th/EIAV). By measuring viral reverse transcriptase activity in the culture medium, we demonstrated that treatment for 1 h with noncytotoxic doses of this drug inhibited production by 32 to 52%, whereas continuous exposure to this drug resulted in an 85 to 92% inhibition. No effect on the viability or growth rate of the cells was detected in any of these treatments. Indirect immunofluorescence analysis of the CPT-treated CF2Th/EIAV cells with anti-p26 capsid protein antibodies showed 60 to 85% reduction in the immunofluorescence-positive cells following drug treatment, and radioimmunoprecipitation analysis of these cells showed a comparable decrease of the pr55gag precursor protein. These data suggest that CPT acts as an anti-EIAV agent to block virus replication in the chronically infected cells.

Animals↗
PubMed1990

Selective phosphinate transition-state analogue inhibitors of the protease of human immunodeficiency virus.

The phosphinic acid isosteres of di-, tetra- and hexapeptides containing a hydrophobic amino acid side chains at the P1-P'1 positions are powerful inhibitors of Human Immunodeficiency Virus protease. Ki's ranged from 0.4 nM to 26 microM at pH 6.5 and were lower at pH 4.5. The compounds showed no activity against trypsin, weak activity against renin at pH 6.5, moderate activity against pepsin at pH 2.0 (Ki values in the microM range) and substantial activity against cathepsin D at pH 3.5 (Ki values from 9 to 300 nM).

Cathepsin D↗
PubMed1990

Substitution of proline with pipecolic acid at the scissile bond converts a peptide substrate of HIV proteinase into a selective inhibitor.

The nonapeptide H-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-NH2 containing the retroviral Tyr-Pro cleavage site is a good substrate for the proteinase of human immunodeficiency viruses but it is not readily hydrolyzed by other nonviral proteinases including the structurally related pepsin-like aspartic proteinases. Replacing the Pro by L-pipecolic acid (2-piperidinecarboxylic acid) converted the substrate into an effective inhibitor of HIV-1 and HIV-2 proteinases with IC50 of approximately 1 microM. This compound showed a high degree of selectivity in that it did not inhibit cathepsin D and renin.

Amino Acid Sequence↗
PubMed1990

Topoisomerase I activity associated with human immunodeficiency virus (HIV) particles and equine infectious anemia virus core.

In the present study, we found a topoisomerase I (topo I) activity in two strains of human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) particles. The topo I activity was located in the EIAV cores and differed from the cellular topo I in its ionic requirements and response to ATP, indicating that these were two distinct forms of this enzyme. Topo I activity was removed from the viral lysates and viral cores by anti-topo I antiserum. The only protein recognized by this antiserum was an 11.5 kd protein in HIV lysate and 11 kd in EIAV lysate. We showed that the 11 kd protein recognized by the anti-topo I antiserum is the EIAV p11 nucleocapsid protein. Furthermore, purified topo I protein blocked the binding of the antibodies to the p11 protein and vice versa, purified p11 protein blocked the binding of these antibodies to the cellular topo I. These results suggest that the EIAV p11 nucleocapsid protein and the cellular topo I share similar epitopes.

Antibodies↗