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Biomedical subjects

L Ratner

Publications and source records attributed to L Ratner.

At least 127 records · Page 7Linked to original sources
PubMed1991

Formation of noninfectious HIV-1 virus particles lacking a full-length envelope protein.

Deletions were constructed within a functional human immunodeficiency virus type 1 (HIV-1) proviral clone in order to assess the role of the envelope protein in virus particle formation. A graded exonuclease deletion technique was used to produce 12 clones with deletions of 175-308 nucleotides in the first conserved domain of envelope. This included 9 clones with frameshift deletions and 3 clones with in-frame deletions. Isogenic pairs of env deletion clones were produced with or without an additional deletion in the vif and vpr genes. Upon transfection, all clones produced virus particles, as determined by p24 antigen, reverse transcriptase, and sucrose gradient assays with conditioned media. Virus particles produced from clones with deletions in env or vif and vpr, or both regions, banded on sucrose gradients with a mobility similar to that of virus produced by the parental clone. The p24 gag capsid protein in the particles was resistant to trypsin, but the particles were disrupted by treatment with Triton X-100, suggesting the presence of a surrounding lipid bilayer. Furthermore, electron microscopic studies revealed both mature and immature virus particles derived from COS cells transfected with the env deletion clones. Cocultivation experiments with lymphoid cells and cells transfected with each of the env deletion clones demonstrated that the virus particles were noninfectious.

Animals↗
PubMed1991

Nucleotide sequence analysis of isolates of human T-lymphotropic virus type 1 of diverse geographical origins.

Nucleotide sequences for long terminal repeat (LTR), gag, the protease gene, and pol of a human T-lymphotropic virus type 1 (HTLV-1) isolate of probable Caribbean origin (HTLV-1CH) and a Zairian isolate (HTLV-1EL) were determined providing complete proviral sequences for these isolates. These sequences were compared with those available from previously analyzed isolates. Nucleotide sequence differences of 1.2-3.3% were identified among isolates for which complete genetic information was available. Nucleotide sequence diversity was distributed relatively evenly over the genome with 1.3-5.2% differences in the LTR, 1.1-2.9% differences in gag, 0.7-2.1% differences in the protease gene, 0.9-2.5% differences in pol, 0.9-2.4% differences in env, 0.0-1.4% differences in rex, and 0.1-2.6% differences in tax. There were 1.2-2.3% amino acid differences overall, with 0.8-1.6% nonconservative amino acid alterations. Nucleotide differences were not found in regions of the LTR which are important for transcriptional activity or Tax response. Within the Rex-response element, nucleotide differences were found predominantly in loop rather than stem structures, thus, maintaining the overall secondary structure necessary for Rex activity. Evolutionary tree analysis of the sequence differences suggests a predominant clustering of different HTLV1 strains according to geographical origin. An open reading frame was also identified on the minus DNA strand situated between the env and rex/tax genes which exhibits 0.1-6.9% nucleotide sequence variation among HTLV1 strains. The limited sequence variation among HTLV-1 strains is in striking contrast to the extensive heterogeneity seen among human immunodeficiency virus (HIV) strains.

Amino Acid Sequence↗
PubMed1991

Demonstration of two distinct cytopathic effects with syncytium formation-defective human immunodeficiency virus type 1 mutants.

The mechanism of human immunodeficiency virus type 1 (HIV-1) cytopathicity is poorly understood and might involve formation of multinucleated giant cells (syncytia), single-cell lysis, or both. In order to determine the contributions of the fusion domain to syncytium formation, single-cell lysis, and viral infectivity and to clarify the molecular details of these events, insertion mutations were made in the portion of env encoding this sequence in the functional HIV-1 proviral clone HXB2. Viruses produced from these mutant clones were found to have a partial (F3) or complete (F6) loss of syncytium-forming ability in acutely infected CEM, Sup T1, and MT4 T-cell lines. During the early stage of acute infection by F6 virus, there was a loss of the syncytial cytopathic effect, which resulted in increased cell viability, and a 1.9- to 2.6-fold increase in virus yield in the cell lines tested. In the late stage of acute infection, the single-cell cytopathic effect of F6 virus was similar to that of the parental HXB2 virus. The F3 and F6 viruses were also found to have a 1.7- to 43-fold reduction in infectivity compared with the HXB2 virus. The mutant F3 and F6 and parental HXB2 envelope proteins were expressed in vaccinia virus, and the mutant envelope proteins were observed to be defective in their ability to form syncytia. BSC-40 cells infected with vaccinia virus recombinants revealed no differences in kinetics of cleavage, cell surface expression, or CD4 binding capacity of the mutant and parental envelope proteins. These results demonstrate that a loss of syncytium formation results in an attenuation of infectivity and a loss of the syncytial cytopathic effect without a loss of single-cell lysis. These mutants may reflect in tissue culture the changes observed in the HIV isolates in vivo during disease progression, which exhibit marked differences in syncytium production.

Amino Acid Sequence↗
PubMed1991

Simian immunodeficiency virus negative factor suppresses the level of viral mRNA in COS cells.

The nef gene is conserved among all human and simian lentiviruses. However, the amino acid similarity between simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 NEF is only 38%. To assess the role of SIV NEF on virus replication and compare its activity with that of its human immunodeficiency virus type 1 counterpart, we examined the activity of an intact nef gene from proviral clone pSIV 102, an isolate from SIV-MAC-251-infected cells. Proviral clone pSIV BA was constructed by introducing a premature termination codon at codon 40 of the nef gene without altering the predicted amino acid sequence of the overlapping env gene. These two clones were transfected into CD4- COS cells, and virus replication was monitored by p27 enzyme-linked immunosorbent assay kits. In seven independent experiments, clone pSIV BA afforded two- to sixfold greater levels of viral antigen compared with those in clone pSIV 102 and two- to sixfold-increased levels of viral mRNAs as indicated with Northern (RNA) blot and S1 nuclease protection analyses. Nuclear run-on assays demonstrated a two- to threefold increased rate of RNA synthesis with nuclei isolated from cells transfected with pSIV BA compared with that from cells transfected with pSIV 102. In contrast, there was no apparent destabilization of SIV mRNAs by NEF, as measured in dactinomycin-treated cells. This study demonstrates that SIV NEF is a negative regulator of virus replication and acts by suppressing the level of mRNA synthesis and accumulation in COS cells.

Amino Acid Sequence↗
PubMed1991

Temporal regulation of viral and cellular gene expression during human T-lymphotropic virus type I-mediated lymphocyte immortalization.

An autocrine mechanism of proliferation may play a significant role in the leukemogenesis of adult T-cell leukemia, a mature T-cell malignancy caused by human T-lymphotropic virus type I (HTLV-I). To further delineate the role of HTLV-I and the interleukin 2 (IL2) system in the transformation process, human primary lymphocytes were infected by cocultivation with lethally X-irradiated MT2 cells in the presence or absence of human rIL2; the polymerase chain amplification reaction was used to examine quantitatively the expression of HTLV-I, IL2, and IL2R alpha mRNAs during early and late proliferation phases that displayed polyclonal (days 7 to 49) and oligoclonal (days 100 to 150) proviral integration, respectively. IL2 mRNA and IL2 activity were transiently expressed during the polyclonal phase but were undetectable at later time points. IL2R alpha mRNA expression remained at a constitutively high level throughout the examined time course. Viral transcripts were detectable at each time point. Expression of the tax-rex mRNA was inversely related to IL2 mRNA levels; it was low early in the polyclonal phase but increased 30-fold with the development of oligoclonality. In addition, paraformaldehyde-fixed HTLV-I-producing cells activated peripheral blood lymphocytes. These data suggest that HTLV-I activates human T lymphocytes. However, IL2 expression is transient, indicating a limited involvement of an IL2 autocrine growth loop in the transformation process. Lastly, another viral determinant, in addition to the trans activator tax, may be important in HTLV-I-induced T-cell proliferation.

Base Sequence↗
PubMed1990

Familial adult T-cell leukemia/lymphoma.

Clinical and laboratory data are described for two siblings who both developed adult T-cell leukemia/lymphoma resulting from infection by human T lymphotropic virus type I (HTLV-I). These findings suggest that genetic factors or virus-specific factors may determine which HTLV-I-infected individuals will develop leukemia.

Adult↗
PubMed1990

Alpha 1-antitrypsin phenotypes in patients with abdominal aortic aneurysms.

The alpha 1-antitrypsin phenotypes were determined in 47 patients with abdominal aortic aneurysms. The MM phenotype was present in 85%, the MS phenotype in 4%, the MZ phenotype in 11%, and the ZZ phenotype not present in any patients. The MZ phenotype occurred significantly more often in patients with aneurysms than would be expected in the general population. The current data would support a genetic mechanism of either autosomal-dominant or multifactorial influences but not an X-linked mechanism for families with a history of abdominal aortic aneurysms.

Aorta, Abdominal↗
PubMed1990

Myristoylation-dependent replication and assembly of human immunodeficiency virus 1.

Covalent linkage of myristic acid to the N-terminal glycine residue of Pr55gag, the precursor of the major structural proteins of human immunodeficiency virus 1 (HIV-1), facilitates an essential step in virus assembly and propagation. Substitution of the myristoyl-acceptor glycine with alanine, in a functional clone of HIV-1, eliminates virus replication. Complementation of this defect, in trans, restores infectious particle production. The nonmyristoylated (myr-) gag precursor accumulates in infected cells and is not processed into the mature capsid components of the intact virion. However, myr- Pr55gag can be processed by purified HIV protease in vitro, demonstrating that the myristoyl moiety is not required for cleavage by the protease. Myristoylation of Pr55gag is not necessary for localization but is required for stable membrane association and assembly of HIV-1.

Alanine↗
PubMed1990

Limited sequence heterogeneity among biologically distinct human immunodeficiency virus type 1 isolates from individuals involved in a clustered infectious outbreak.

Human immunodeficiency virus type 1 isolates were obtained over a 3-year period from blood, brain, and lung of three patients in a clustered infectious outbreak. This included a blood donor who was initially asymptomatic but subsequently developed AIDS-related complex and two neonatal transfusion recipients who developed AIDS. Isolates from brain and lung replicated to greater than 30-fold higher levels in primary monocyte cultures than did those from blood; no growth differences on primary lymphocytes were observed. Thirteen clones were obtained from seven isolates, and env sequences were determined. The predicted amino acid sequences among these clones differed by only 0.01% but differed by 15-27% when compared to previously sequenced isolates from other patients. The level of envelope amino acid sequence divergence noted among these isolates is considerably lower than that previously reported for other human immunodeficiency virus isolates. No differences in the envelope unique to lung or brain isolates compared to blood isolates were noted. This study provides evidence that mutations in the envelope may not be necessary for disease progression and that other portions of the viral genome may contribute to cell-specific tropism.

AIDS-Related Complex↗
PubMed1990

Attenuation of HIV-1 infectivity by an inhibitor of oligosaccharide processing.

A series of inhibitors of trimming glucosidases and mannosidases were examined for antiviral activity toward HIV-1. N-butyl deoxynojirimycin (N-buDNJ) was found to be the most potent agent studied. Treatment of acutely infected lymphoid cells with 2.0 mM N-buDNJ reduced virus yield more than 90%, without affecting cell growth. Though lower concentrations of N-buDNJ (0.002-0.2 mM) did not affect HIV-1 production, there was complete inhibition of syncytia formation. Treatment of chronically infected lymphoid cells with 0.1-1.0 mM N-buDNJ resulted in no significant change in virus production, but 80% reduction of infectivity. The attenuation in HIV-1 infectivity was due at least partially to diminished binding to CD4+ lymphoid cells. Chronically infected lymphoid cells treated with 0.02-1.0 mM N-buDNJ for at least 3 days were markedly impaired in their ability to form syncytia with uninfected lymphoid cells. N-buDNJ treatment of HIV-1 infected cells resulted in both a reduction in the cell surface envelope proteins, and an increase in their apparent molecular weight. These results show that N-buDNJ can be used to impair the infectivity of HIV-1 without significant toxicity.

1-Deoxynojirimycin↗
PubMed1990

Immunoblastic lymphoma of T-cell type in a chronically immunosuppressed renal transplant recipient.

A case of non-Hodgkin's lymphoma of T-cell type occurring in a renal transplant recipient is described. This lymphoma was classified as large cell, immunoblastic type and presented as a mediastinal mass, although it was demonstrated to be disseminated at autopsy two weeks later. Lymphoma cells expressed the immunologic profile of a mature, activated cytotoxic/suppressor T-lymphocyte. T-cell receptor gene rearrangement and karyotypic studies confirmed a clonal T-cell genotype. Molecular studies failed to demonstrate any association with Epstein-Barr virus or human T-cell lymphotropic virus type I or type II. Pathogenesis of lymphomas in this clinical setting is discussed.

Adult↗
PubMed1990

Deletion of Alu sequences in the fifth c-sis intron in individuals with meningiomas.

An abnormality in the c-sis protooncogene was identified in leukocyte DNA from members of a family predisposed to the development of meningioma, and was found to be associated with the development of the tumor in those individuals. Molecular analysis of this abnormality demonstrated a deletion within the fifth intron of the c-sis gene. The normal c-sis gene has an Alu sequence in this region which includes two perfect 130 nucleotide repeated sequences separated by 5 bp. The deleted c-sis allele is missing precisely one copy of the 130 bp repeat and the intervening 5 bp. An identical deletion was also found in DNA from 1 of 13 sporadic meningiomas.

Alleles↗
PubMed1989

Regulation of expression of the c-sis proto-oncogene.

Regulation of expression of platelet derived growth factor polypeptide B encoded by the c-sis proto-oncogene is important in a number of physiological and pathological conditions. Sequences in the 1028 nucleotide long 5' untranslated region of the c-sis mRNA were found to inhibit protein synthesis. The inhibition is relieved by deletion of nucleotides 154-378 or 398-475. Sequences within 375 nucleotides upstream of the RNA initiation site are important for transcriptional activity. Sequences in two portions of this region, between -375 and -235 nucleotides and between -235 and -99 nucleotides relative to the RNA CAP site are important for full activity. A transcriptional enhancer activity is demonstrated by its ability to increase the activity of the human T lymphotropic virus type (HTLV) I promoter at a distance and in an orientation-independent manner. Furthermore, sequences upstream of the c-sis RNA CAP site respond to the HTLV I transactivator protein to increase RNA synthesis from either the c-sis or HTLV I promoter.

Base Composition↗
PubMed1989

Analysis of the function of viral protein X (VPX) of HIV-2.

To investigate the function of vpx, a gene in HIV-2 and SIV, but not in HIV-1, three site-directed mutants (pMX) were constructed from a functional proviral HIV-2 plasmid clone (pSE). Transfection of COS-1 cells with all three mutants as well as pSE gave rise to equivalent amounts of virus. Each virus could be passaged in H9 and CEM lymphoid cell lines, peripheral blood lymphocytes, and monocytes with equal efficiency and demonstrated similar cytopathic effects. Hybridization data with DAN from the infected cells demonstrated the presence of similar levels of viral sequences and the mutations in each of the MX-infected cell lines. Immunoprecipitation analysis demonstrated a 16-kDa VPX protein in cells infected with SE virus, as well as in the virus particles, but not in cells infected with MX viruses or the particles themselves. However, equivalent levels of gag and env proteins were demonstrated in all infected cells and virion preparations. These data suggest that VPX is dispensable for virus replication and cytopathicity.

Amino Acid Sequence↗
PubMed1989

Replication of human immunodeficiency virus 1 and Moloney murine leukemia virus is inhibited by different heteroatom-containing analogs of myristic acid.

Myristoyl-CoA:protein N-myristoyltransferase (NMT; EC 2.3.1.97) catalyzes the cotranslational linkage of myristate to the N-terminal glycine residues of several cellular, viral, and oncoproteins. We have recently synthesized a series of sulfur- and oxygen-substituted analogs of myristic acid that are similar in length to the 14:0 fatty acid yet have hydrophobicities equivalent to dodecanoate or decanoate. Previous in vitro enzyme assays and metabolic labeling studies indicate that some of these analogs are excellent substrates for NMT and are incorporated into subsets of cellular N-myristoyl proteins. Their sequence-specific incorporation probably arises from cooperative interactions between the acyl CoA and peptide binding sites of NMT. The human immunodeficiency virus 1 (HIV-1) and Moloney murine leukemia virus (MoMLV) depend on myristoylation of gag polyprotein precursors for assembly. We have tested four analogs--12-methoxydodecanoic acid, 10-propoxydecanoic acid, 5-octyloxypentanoic acid, and 11-ethylthioundecanoic acid--for their ability to block replication of these retroviruses. All reduce HIV-1 replication when incubated with CD4+ H9 cells for 10 days at 10-100 microM. 12-Methoxydodecanoic acid is most effective, producing a concentration-dependent decrease in (i) reverse transcriptase activity (to levels that were 5-10% of control at 20-40 microM), (ii) p24 levels, and (iii) syncytia formation. This degree of inhibition of HIV-1 replication is equivalent to that seen with 5 microM 3'-azido-3'-deoxythymidine and is accomplished without apparent toxicity, as measured by cell viability, protein, and nucleic acid synthesis. 5-Octyloxypentanoic acid inhibits MoMLV assembly in a dose-dependent fashion without accompanying cellular toxicity, while 12-methoxydodecanoic acid has no effect. These data suggest that the use of cellular NMT activity to deliver analogs of myristate with altered physical-chemical properties to proteins that undergo this cotranslational modification may represent an effective anti-viral therapeutic strategy as well as a way to investigate the role of covalently bound fatty acid in viral assembly.

Animals↗
PubMed1989

Human immunodeficiency virus type 1 negative factor is a transcriptional silencer.

The negative factor (nef) of human immunodeficiency virus (HIV) type 1 acts to down-regulate virus replication. To decipher the step in the virus life cycle affected by nef, functional proviral clones with (pHIV F-) or without (pHIV F+) a deletion mutation in the nef gene were constructed. In CD4+ cells, 30- to 50-fold more virus was produced over the course of 18-20 days with cultures infected with F- compared to F+ virus. In CD4- cell lines, 2- to 10-fold greater virus production was found from cultures transfected with pHIV F- than those transfected with pHIV F+. The negative regulatory effects of nef on pHIV F- could be supplied in trans with a plasmid expressing only the nef gene product. Virus produced by COS-1 cells transfected with pHIV F- or pHIV F+ showed similar binding, uptake, uncoating, and reverse transcription. Analysis of HIV-1 RNA and structural protein levels and rates of viral RNA synthesis in CD4- cells also showed 2- to 10-fold higher levels in cells transfected with pHIV F- compared to pHIV F+. The activity of a HIV-1-chloramphenicol acetyltransferase (CAT) plasmid was also suppressed by nef, whereas other CAT plasmids were unaffected. These findings demonstrate that nef acts as a specific silencer of HIV-1 transcription. This activity may be critical for maintenance of HIV-1 latency in vivo.

Animals↗