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J E Clements

Publications and source records attributed to J E Clements.

At least 91 records · Page 5Linked to original sources
PubMed1991

Identification of the fusion domain in the visna virus transmembrane protein.

Visna virus, a lentivirus of sheep, causes fusion of susceptible cells. Fusion has previously been shown to be mediated by the viral envelope glycoprotein. The transmembrane protein of visna virus contains a hydrophobic region at its amino terminus. This region is similar to the fusion epitopes of the orthomyxoviruses and paramyxoviruses. This region is located in a position similar to that of the fusion epitopes in the transmembrane proteins of HIV-1 and SIV. To determine the role of this hydrophobic region in visna virus-induced cell fusion, a peptide of 24 amino acids corresponding to this region was synthesized. The peptide alone induces fusion of goat cells. Antibodies to this peptide inhibit both viral-induced cell fusion and peptide fusion in goat cells. Further, the direct fusion of cells by this peptide is a unique observation and may be useful for studying the fusion epitopes of other lentiviruses. Thus, this hydrophobic region appears to be one epitope responsible for visna virus-induced cell fusion.

Amino Acid Sequence↗
PubMed1991

Rhesus monkey macrophages infected with simian immunodeficiency virus cause rapid lysis of CD4-bearing lymphocytes.

Inoculation of simian immunodeficiency virus into cultures of primary rhesus monkey macrophages or CD4-bearing transformed T lymphocytes resulted in persistent infection, with minimal virus replication in the macrophages and extensive replication in the lymphocytes. However, uninfected T cells added to infected macrophages underwent rapid fusion and lysis and were almost completely eliminated without the production of virus particles. Lysis required direct contact between the T cells and the infected macrophages, which enabled binding between CD4 on the former and viral gp120 on the latter to occur. This process was blocked by soluble CD4 and dextran sulphate. Neutralizing antibodies in the serum of an infected macaque prevented cell fusion by preventing infection of the macrophages. However, these antibodies did not prevent fusion when added to previously infected macrophages. Infected macrophages were incorporated into the syncytia of lymphocytes and continued incorporation of new lymphocytes into the syncytia required infected macrophages to be metabolically active. One inference from these studies is that infected macrophages in vivo could help mediate the well known depletion of T4 cells in patients with AIDS.

Animals↗
PubMed1991

Analysis of Borna disease virus-specific RNAs in infected cells and tissues.

Borna disease virus (BDV) is an infectious agent that causes profound disturbances in motor function and behaviour in a wide range of animal species and possibly humans. The infectious nature of BDV has long been established, but the aetiological agent has not been isolated or classified. Recently, we have reported the isolation of BDV-specific cDNA clones using subtractive libraries constructed from mRNA from infected material. Here we describe studies on one of these cDNA clones, B8, and confirm its specificity by in situ hybridization on sections of BDV-infected brain. The complete nucleotide sequence of BDV-specific clone B8 was determined. Oligonucleotides of positive and negative polarity synthesized from sequences from the 5' and 3' ends, as well as the central part, of clone B8 identified both positive- and negative-strand BDV-specific RNAs in infected rat brain. All B8 sequences used as oligonucleotide probes were found to be contained in the larger positive- and negative-strand RNAs. Thus, the structure of the BDV-specific RNAs appears to be a nested set of multiple, overlapping subgenomic positive- and negative-strand RNA transcripts.

Amino Acid Sequence↗
PubMed1991

Comparison of the transcriptional activity of the long terminal repeats of simian immunodeficiency viruses SIVmac251 and SIVmac239 in T-cell lines and macrophage cell lines.

The U3 regions of the long terminal repeats (LTRs) of simian immunodeficiency viruses SIVmac251 and SIVmac239 were analyzed for basal transcriptional activity and for interaction with cellular factors in the T-cell line HUT-78 and the monocyte/macrophage cell line U937. A number of 5' deletions and mutations were made in the U3 regions of the two LTRs, and these constructs were placed upstream of a plasmid containing the bacterial chloramphenicol acetyltransferase reporter gene. The nucleotide sequences between -225 and +18 were sufficient to maintain full transcriptional activity of both LTRs in HUT-78 and U937 cells. Nucleotide sequence analysis revealed several differences between SIVmac251 and SIVmac239 within this region. Analysis of deletion mutants revealed that an additional removal of bases, from -124 to -225, had little effect on the transcriptional activity of the clone 239 LTR, whereas this deletion resulted in a significant reduction of activity in the clone 251 LTR. DNase protection assays using nuclear extracts from HUT-78 and U937 cells showed that bases within this region bound cellular factors. In addition, the NF-kappa B site was protected in DNase assays with HUT-78 cells and 12-O-tetradecanoylphorbol-13-acetate-treated U937 cells. An additional DNase footprint was detected in SIVmac239, at -52 to -38, just upstream of the TATA box. This site overlaps the 3' half of the 3'-most Sp-1 site and is downstream of 11 bases that are found in SIVmac239 but not SIVmac251. Thus, differences in the sequences in the U3 region of the LTRs of SIVmac251 and SIVmac239 have been identified which appear to alter the transcriptional activity of these promoters as well as changing the interaction of cellular proteins with sequences in the LTRs.

Base Sequence↗
PubMed1991

Identification of cell membrane proteins that bind visna virus.

Visna virus infects cells of ovine origin by attaching to a cell surface receptor via its envelope glycoprotein. The identity of the visna virus receptor is not known. To identify the molecule responsible for binding the virus to target cells, virus overlay protein blot assays were used to examine the molecular weights of cell surface molecules which bind purified virus. Molecules on the surface of goat synovial membrane (GSM) cells and sheep choroid plexus (SCP) cells of approximately 15, 30, and 50 kDa bound to visna virus. The binding of visna virus to these proteins was reduced by preincubating virus with neutralizing antibodies. 125I-labeled cell membrane preparations of GSM and SCP cells were used to affinity purify these virus-binding proteins. These proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had molecular masses of 15, 30, and 50 kDa. Antibodies to the 50-kDa protein bound to the surface of both live SCP and GSM cells in immunofluorescence assays. In addition, antibodies to the 50-kDa protein blocked the binding of [35S]methionine-labeled visna virus to SCP cells in culture. Antibodies raised against the 15- and 30-kDa proteins did not block virus binding to cells. The blocking activity of antibody of the 50-kDa protein provided data that this protein is the molecule which visna virus recognizes and binds to on the surface of target cells.

Animals↗
PubMed1991

Molecular and immunopathological studies of borna disease virus infection in rats.

Borna disease virus is an agent distinct from all known viruses. Pathogenesis of its infection is also unique. This review highlights several aspects of the biology of this viral infection and the preliminary characterization of the agent. BDV can be used to answer important questions in neurobiology. These include neuroinvasiveness and neurotropism of viral agents, CD4+ T cell-mediated immunopathology and tolerance in newborn animals to a persistent viral infection in the CNS and behavioral diseases and eating disorders induced by neurotropic viruses. This review is dedicated to Prof. Dr. Rott on occasion of his 65th birthday in recognition of his immense contributions to studies on Borna disease and also for his success focusing the attention of the scientific community to this still evolving unique viral disease.

Animals↗
PubMed1990

A borna virus cDNA encoding a protein recognized by antibodies in humans with behavioral diseases.

Borna disease virus (BDV) causes a rare neurological disease in horses and sheep. The virus has not been classified because neither an infectious particle nor a specific nucleic acid had been identified. To identify the genome of BDV, a subtractive complementary DNA expression library was constructed with polyadenylate-selected RNA from a BDV-infected MDCK cell line. A clone (B8) was isolated that specifically hybridized to RNA isolated from BDV-infected brain tissue and BDV-infected cell lines. This clone hybridized to four BDV-specific positive strand RNAs (10.5, 3.6, 2.1, and 0.85 kilobases) and one negative strand RNA (10.5 kilobases) in BDV-infected rat brain. Nucleotide sequence analysis of the clone suggested that it represented a full-length messenger RNA which contained several open reading frames. In vitro transcription and translation of the clone resulted in the synthesis of the 14- and 24-kilodalton BDV-specific proteins. The 24-kilodalton protein, when translated in vitro from the clone, was recognized by antibodies in the sera of patients (three of seven) with behavioral disorders. This BDV-specific clone will provide the means to isolate the other BDV-specific nucleic acids and to identify the virus responsible for Borna disease. In addition, the significance of BDV or a BDV-related virus as a human pathogen can now be more directly examined.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence and transcriptional analysis of molecular clones of CAEV which generate infectious virus.

The lentivirus caprine arthritis-encephalitis virus (CAEV) is closely related by nucleotide sequence homology to visna virus and other sheep lentiviruses and shows less similarity to the other animal and human lentiviruses. The genomic organization of CAEV is very similar to that of visna virus and the South African ovine maedi visna virus (SA-OMVV) as well as to those of other primate lentiviruses. The CAEV genome includes the small open reading frames (ORF) between pol and env which are the hallmarks of the lentivirus genomes. The most striking difference in the organization of CAEV is in the env gene. The Env polyproteins of visna virus and the related SA-OMVV contain 20 amino acids between the translational start and the signal peptide not present in CAEV. In addition to nucleotide sequence analysis, the transcriptional products of CAEV were determined by Northern analysis. The viral mRNA present in cells transfected with the infectious clone reveal a pattern characteristic of the mRNAs observed in other lentivirus infections. The putative tat ORF of CAEV could be identified by genomic location and amino acid homology to the visna virus tat gene. However, the CAEV rev gene could not be identified in a similar fashion. Thus, to determine the location of the rev ORF cDNA clones were obtained by PCR amplification of the mRNA from infected cells. To determine if a Rev response element was contained in the CAEV genome, secondary structural analysis of the viral RNA was performed. A stable stem loop structure which is similar in location, stability, and configuration to that determined for the Rev response element of HIV was found.

Amino Acid Sequence↗
PubMed1990

Visna virus encodes a post-transcriptional regulator of viral structural gene expression.

Visna virus is an ungulate lentivirus that is distantly related to the primate lentiviruses, including human immunodeficiency virus type 1 (HIV-1). Replication of HIV-1 and of other complex primate retroviruses, including human T-cell leukemia virus type I (HTLV-I), requires the expression in trans of a virally encoded post-transcriptional activator of viral structural gene expression termed Rev (HIV-1) or Rex (HTLV-I). We demonstrate that the previously defined L open reading frame of visna virus encodes a protein, here termed Rev-V, that is required for the cytoplasmic expression of the incompletely spliced RNA that encodes the viral envelope protein. Transactivation by Rev-V was shown to require a cis-acting target sequence that coincides with a predicted RNA secondary structure located within the visna virus env gene. However, Rev-V was unable to function by using the structurally similar RNA target sequences previously defined for Rev or Rex and, therefore, displays a distinct sequence specificity. Remarkably, substitution of this visna virus target sequence in place of the HIV-1 Rev response element permitted the Rev-V protein to efficiently rescue the expression of HIV-1 structural proteins, including Gag, from a Rev- proviral clone. These results suggest that the post-transcriptional regulation of viral structural gene expression may be a characteristic feature of complex retroviruses.

Animals↗
PubMed1990

Inhibition of lymphoproliferation and protein kinase C by synthetic peptides with sequence identity to the transmembrane and Q proteins of visna virus.

A peptide sequence in the transmembrane protein of visna virus has been identified that bears a high degree of similarity to a sequence within the transmembrane protein gp41 of human immunodeficiency virus that we have previously shown to be immunosuppressive. Also within the Q (vif/sor) open reading frame of the visna virus genome is a sequence that is highly similar to the immunosuppressive sequence from the retroviral transmembrane protein p15E. We synthesized peptides containing these visna virus sequences and tested them for immunosuppressive activity, comparing them with their human immunodeficiency virus and leukemia retrovirus counterparts. Both the Q- and transmembrane-derived visna virus peptides inhibited lymphoproliferation stimulated by either interleukin-2 or the T-cell antigen receptor in a dose-dependent and sequence-specific manner. The two visna virus peptides also inhibited the enzymatic activity of protein kinase C, thus providing a possible molecular mechanism by which they inhibit immune function.

Amino Acid Sequence↗
PubMed1989

Characterization of a cDNA clone encoding the visna virus transactivating protein.

The unique pathogenesis of lentiviral infections in humans and ruminant animals may be explained, in part, by the complex mechanisms regulating transcription and translation of their viral genes. This report demonstrates that a visna virus-encoded protein transactivates viral gene expression. A 1.4-kilobase cDNA clone encodes two distinct proteins with apparent molecular masses of 21.5 and 10 kDa. We demonstrate that the 10-kDa species is the visna virus transactivating (Tat) protein; the other species may be analogous to the rev (formerly art or trs) gene product of human immunodeficiency virus.

Amino Acid Sequence↗
PubMed1989

Rapid uptake and clearance of pyridoxine by red blood cells in vivo.

There is rapid pyridoxine (PN) uptake in vitro into red cells where it is converted to pyridoxal (PL) forms. To assess uptake in vivo, the equivalent of 48.6 and 118 mumol PN were given intravenously to a healthy female subject. Vitamin B-6 compounds were measured by a Lactobacillus casei microbiological assay in blood taken 1-60 min after injection. After either injection there was a considerable amount of PN in the red cells at 1 min but by 3 min a large amount of that PN had disappeared, mostly unaccounted for by conversion to PL forms. Although there was considerably less PN at 1 min in both red cells and plasma after the smaller injection, in the next 2 min similar amounts had left the red cells (4.59 and 4.30 mumol) and plasma (9.37 and 10.09 mumol), respectively, after the injections. Red cells, as well as plasma, may be transporting PN to other sites of metabolism in tissues.

Adult↗
PubMed1989

Genetic and other influences on red-cell flavin enzymes, pyridoxine phosphate oxidase and glutathione reductase in families with beta-thalassaemia.

In 18 beta-thalassaemia families from the Ferrara area the incidence of an inherited low flavin mononucleotide (FMN)-dependent pyridoxine phosphate (PNP) oxidase activity, a sensitive indicator of red-cell FMN deficiency, is higher in related members in these families than in the unrelated spouses and controls subjects without family history of thalassaemia. This suggests slower red-cell riboflavin metabolism in thalassaemia families, which may have resulted from selection in combination with thalassaemia by malaria. However, there was a markedly higher incidence of red-cell flavin adenine dinucleotide (FAD) deficiency in thalassaemia heterozygotes than in their normal relatives. This was indicated by higher stimulation of FAD-dependent glutathione reductase (GR) activity by FAD and lower GR activity per red cell, and suggests a marked additive effect by thalassaemia on the red cell FAD deficiency that results from the inherited slow riboflavin metabolism. There is evidence that diversion of FAD to other FAD-dependent enzymes might be an important factor.

Adult↗
PubMed1989

The visna virus long terminal repeat directs expression of a reporter gene in activated macrophages, lymphocytes, and the central nervous systems of transgenic mice.

Visna virus is a lentivirus which causes a slow progressive disease involving the immune system and the central nervous system. To determine the role of the viral long terminal repeat (LTR) in targeting the virus to specific host cells and tissues, transgenic mice were constructed which contained the visna virus LTR directing expression of the bacterial gene encoding chloramphenicol acetyltransferase (CAT). Analysis of the transgenic mouse tissues for CAT activity revealed that the viral LTR was responsible, in part, for the tropism of visna virus for macrophages and the central nervous system. Expression of the LTR required the macrophage to be in an activated state both in vivo and in vitro. Thioglycolate activation of peritoneal macrophages in vivo and 12-O-tetradecanoylphorbol 13-acetate treatment in vitro induced expression of the visna virus LTR. Lymphocytes from the spleens of the transgenic mice expressed CAT activity, suggesting that visna virus was able to replicate in lymphocytes, as did human immunodeficiency virus and simian immunodeficiency virus. These studies demonstrated that the lentivirus LTR was responsible, in part, for cell and tissue tropism in vivo.

Animals↗