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J E Clements

Publications and source records attributed to J E Clements.

At least 109 records · Page 6Linked to original sources
PubMed1989

Sequences in the visna virus long terminal repeat that control transcriptional activity and respond to viral trans-activation: involvement of AP-1 sites in basal activity and trans-activation.

Visna virus is a pathogenic lentivirus of sheep whose gene expression is developmentally regulated in cells of the monocyte-macrophage lineage. Gene expression directed by the visna virus long terminal repeat (LTR) is increased in infected cells by a virus-encoded trans-acting protein. trans-Activation is mediated in part by increases in the steady-state level of mRNA. Deletion and linker-scanner mutants were constructed to locate sequences in the LTR that regulate transcription and are responsive to viral trans-activation. The activities of these mutants were tested by using them to drive transcription of the bacterial gene for chloramphenicol acetyltransferase in transient expression assays. Three regions located between-140 and the cap site were found to be important for basal transcriptional activity, and the importance of each region was found to be dependent on the cell type. Sequences responsive to viral trans-activation were found to be the same sequences required for basal transcriptional activity. The visna virus LTR contains six sequences that are homologous to the recognition site for cellular transcriptional factor AP-1 and a single sequence homologous to the recognition site for transcriptional factor AP-4. Both of these classes of binding sites appear to be important for regulating the basal level of transcription of visna virus. The AP-1-binding site most proximal to the TATA box was found to be one target for viral trans-activation. The visna virus promoter was found to be activated by serum; this serum response has also been mapped to the AP-1-related sequences in the LTR.

Base Sequence↗
PubMed1989

Regulation of the visna virus long terminal repeat in macrophages involves cellular factors that bind sequences containing AP-1 sites.

Visna virus gene expression is highly restricted in monocytes but is induced by monocyte-macrophage differentiation in vivo. Deletion and linker-scanning mutants, gel shift assays, and DNase I footprinting were used to identify sequences in the visna virus long terminal repeat involved in the developmental regulation of gene expression in the U937 monocytic cell line. We found that an AP-1 and an AP-4 binding site were critical for basal activity and that the AP-1 site was required for phorbol ester-inducible gene expression. These results suggest that cellular factors that interact with AP-1 sites are involved in the developmental regulation of visna virus gene expression in macrophages.

Base Sequence↗
PubMed1988

Hyaluronidase enhances cell fusion and synthesis of viral DNA during infection with caprine arthritis encephalitis virus.

Caprine arthritis encephalitis virus (CAEV) is a lentivirus which infects goats and causes chronic progressive arthritis after a prolonged incubation period. CAEV replicates productively in cultures of goat synovial membrane cells and causes cytopathic effects characterized by multinucleated giant cell formation. The enzyme hyaluronidase was found to accelerate this virus induced fusion of GSM cells. Hyaluronidase treatment also resulted in synthesis of increased levels of unintegrated viral DNA early after infection. However, there was no significant increase in viral RNA in the infected cells or in the amount of virus produced. These studies suggest that hyaluronidase facilitates the interaction of CAEV with the target cells. Further it suggests that only a few copies of viral DNA are required to achieve maximal levels of virus replication. Additional copies of viral DNA appear to be redundant not contributing to viral specific transcription or increased production of virus.

Animal Diseases↗
PubMed1988

Separate epitopes in the envelope of visna virus are responsible for fusion and neutralization: biological implications for anti-fusion antibodies in limiting virus replication.

Visna virus is a lentivirus which causes fusion of infected cells in vitro. Two types of fusion occur. Fusion from without requires no viral replication and a relatively high multiplicity of infection; fusion from within results from the replication of virus in cells. By using fusion from without as an assay, the mechanism of fusion by visna virus was investigated. Immune sera which contained both anti-fusion and neutralizing antibodies interacted with the virus with rapid kinetics in blocking fusion but relatively slow kinetics in the virus neutralization assay. By using visna virus and an antigenic variant, the epitopes responsible for fusion and virus neutralization were shown to be different. Antigenic variation of visna virus resulted in alteration of the neutralization epitope and conservation of the fusion epitope. This suggested that there were two populations of antibodies and that the viral epitopes for fusion and neutralization were separate. These data suggest that visna virus is capable of infecting cells via two pathways: one via the fusion site and the other via the viral epitope which mediates neutralization.

Antigens, Viral↗
PubMed1987

Pathogenesis of visna/maedi and caprine arthritis-encephalitis: new leads on the mechanism of restricted virus replication and persistent inflammation.

Lentiviruses are unique retroviruses which cause diseases with long incubation periods and prolonged clinical courses. The prototype lentiviruses, visna/maedi of sheep and arthritis-encephalitis virus of goats (CAEV), infect cells of the monocyte-macrophage system and replicate at a restricted level in these cells. The virus life cycle is closely associated with maturational factors in the cells; monocytes support the early stages of the replication cycle which goes to completion only when the cells mature to macrophages. Virus replication in the monocyte-macrophage results in lesions characterized by mononuclear cell infiltration of the central nervous system (CNS), lungs, synovium and mammary gland and their draining lymph nodes. Co-cultivation of sheep or goat lymphocytes with macrophages infected with visna or CAE viruses results in production of a unique interferon (LV-IFN). LV-IFN is a non-glycosylated protein of 54,000 to 64,000 daltons and has biological properties which have several implications for pathogenesis. Firstly, it retards the rate of maturation of monocytes and thus indirectly slows the rate of virus replication. Second, it restricts the rate of virus replication in mature macrophages by preventing virus maturation. Third, it induces expression of class II (Ia) antigens of the major histocompatibility complex on cells of macrophage lineage. Thus, by curtailing virus replication and enhancing expression of MHC class II antigens, LV-IFN may contribute to the induction and augmentation of the host's lymphoproliferative response to the virus.

Animals↗
PubMed1987

Glutathione reductase activity and its relationship to pyridoxine phosphate activity in G6PD deficiency.

Per cent stimulation of GR activity by FAD in vitro and PNP oxidase activity were measured in G6PD deficiency, heterozygous beta-thalassaemia and controls. It is confirmed that, in contrast to the high stimulation of GR by FAD commonly found in in thalassaemia indicating red-cell deficiency of FAD, and shown here to be greater in the Italian subjects, GR is usually saturated with FAD in G6PD deficiency, leading to high in vitro activity. Unexpectedly, on the other hand, low FMN-dependent PNP oxidase activity due to red-cell deficiency of FMN, confirmed by response to oral riboflavin, was found in the majority of subjects with G6PD deficiency, similar to that found in heterozygous beta-thalassaemia. Whereas this is explained in thalassaemia by an inherited slow red-cell metabolism of riboflavin to FMN, it is suggested that in G6PD deficiency an increased rate of red-cell metabolism of FMN to FAD leads to the low FMN and high FAD. When G6PD deficiency occurs with heterozygous beta-thalassaemia, GR is usually saturated with FAD as in G6PD deficiency alone, unless there is an inherited, very slow red-cell metabolism of riboflavin to FMN. The part played by GR in haemolytic crises in G6PD deficiency is discussed.

Erythrocytes↗
PubMed1987

The visna virus genome: evidence for a hypervariable site in the env gene and sequence homology among lentivirus envelope proteins.

The complete nucleotide sequence of the visna virus 1514 genome was determined. Our sequence confirms the relationship of visna virus and other lentiviruses to human immunodeficiency virus (HIV) both at the level of sequence homology and of genomic organization. Sequence homology is shown to extend to the transmembrane proteins of lentivirus env genes; this homology is strongest in the extracellular domain, suggesting that close structural and functional similarities may also exist among these envelope proteins. Comparison of our data with the sequence of visna virus LV1-1, an antigenic variant derived from strain 1514, demonstrates that the rate of divergence has been about 1.7 x 10(-3) substitutions per nucleotide per year in vivo. This rate is orders of magnitude higher than that for most DNA genomes, but agrees well with estimates of the rate for HIV. A statistically significant cluster of mutations in the env gene appears to represent a hypervariable site and may correspond to the epitope responsible for the antigenic differences between 1514 and LV1-1. Analysis of the potential RNA folding pattern of the visna virus env gene shows that this hypervariable site falls within a region with little potential for intramolecular base pairing. This correlation of hypervariability with lack of RNA secondary structure is strengthened by the fact that it also holds for a hypervariable site in the env gene of HIV.

Animals↗
PubMed1987

Topographical rearrangements of visna virus envelope glycoprotein during antigenic drift.

Visna virus undergoes antigenic drift during persistent infection in sheep and thus eludes neutralizing antibodies directed against its major envelope glycoprotein, gp135. Antigenic variants contain point mutations in the 3' end of the genome, presumably within the envelope glycoprotein gene. To localize the changes in the viral proteins of antigenic mutants, we isolated 35 monoclonal antibodies (MAbs) against the envelope glycoprotein gp135 or the major core protein p27 of visna virus. The MAbs defined five partially overlapping epitopes on gp135. We used the MAbs and polyclonal immune sera directed against visna virus, gp135, or p27 in enzyme-linked immunosorbent assays to compare visna virus (strain 1514) with antigenic mutants (LV1-1 to LV1-6) previously isolated from a single sheep persistently infected with plaque-purified strain 1514. Polyclonal immune sera and anti-core p27 MAbs failed to distinguish antigenic differences among the viruses. By contrast, the anti-gp135 MAbs detected changes in all five epitopes of the envelope glycoprotein. Three gp135 epitopes, prominently exposed on strain 1514, were lost or obscured on the mutants; two covert gp135 epitopes, poorly exposed on strain 1514, were reciprocally revealed on the mutants. Even virus LV1-2, which is indistinguishable from parental strain 1514 by serum neutralization tests and which differs from it by only two unique oligonucleotides on RNase-T1 fingerprinting, displayed global changes in gp135. Our data suggest that visna virus variants may emerge more frequently during persistent infection than can be detected by serological tests involving the use of polyclonal immune sera, and the extent of phenotypic changes in their envelope glycoproteins may be greater than predicted by the small number of genetic changes previously observed. We suggest that topographical rearrangements in the three-dimensional structure of gp135 may magnify the primary amino acid sequence changes caused by point mutations in the env gene. This may complicate strategies to construct lentiviral vaccines by using the envelope glycoprotein.

Animals↗
PubMed1987

Visna virus exhibits a complex transcriptional pattern: one aspect of gene expression shared with the acquired immunodeficiency syndrome retrovirus.

A complex pattern of gene expression was found for visna virus in a highly permissive cell culture system in vitro. In addition to the genomic RNA (9.4 kilobases [kb]), five other mRNAs were detected. The three large RNA transcripts (5.0 kb and a doublet at 4.3 kb) arise by a single splicing event joining 5' sequences to sequences located at positions 3' to the pol gene. The two smallest transcripts (1.8 and 1.5 kb) are at least doubly spliced mRNAs which contain sequences derived from the 5' end of the genome, the region between the pol and env genes, and 3' terminal sequences. In addition to this complex pattern of transcription, the mRNAs appear to be regulated temporally. The 1.5-kb mRNA appears 6 h later than the other transcripts. The significance of this complex pattern of gene expression in the unique aspects of the lentivirus life cycle and pathogenesis is considered.

Endonucleases↗
PubMed1986

Human T-cell lymphotropic virus type III shares sequence homology with a family of pathogenic lentiviruses.

The etiologic agent of the acquired immune deficiency syndrome, human T-cell lymphotropic virus type III (HTLV-III), has recently been shown to morphologically resemble and share sequence homology with visna virus, a pathogenic lentivirus. Molecular hybridization, heteroduplex mapping, and DNA sequence analyses were used to compare HTLV-III to other lentiviruses of domestic animals, including visna, caprine arthritis encephalitis, and equine infectious anemia viruses. Hybridization results showed that a substantial amount of sequence homology exists between each of these viruses and HTLV-III. In addition, a closer relationship was found between visna and caprine arthritis encephalitis viruses than for any of the other lentiviruses studied. These results, along with nucleotide and amino acid sequence comparisons, have been used in a comprehensive effort to derive a systematic relationship for lentiviruses and to provide further evidence for classifying HTLV-III with the Lentivirinae subfamily of retroviruses. This relationship predicts that similarities in biology and disease process can be expected between HTLV-III and other Lentivirinae members.

Cloning, Molecular↗
PubMed1986

Tropism of sheep lentiviruses for monocytes: susceptibility to infection and virus gene expression increase during maturation of monocytes to macrophages.

Visna lentiviruses have a natural tropism for cells of the macrophage lineage of sheep and goats, but virus replication in these cells in vivo is restricted so that only small quantities of virus are produced. One restricting factor suggested in previous studies is that virus replication is dependent on the maturity of the cells: the more mature the cell, the less restrictive the replication of the virus. Since monocytes in peripheral blood are precursors of macrophages, we investigated the effect of cell maturation on virus replication under limited control conditions in vitro by inoculating blood leukocytes with virus and retarding the maturation of monocytes to macrophages during cultivation in serum-free medium. Using enzyme markers that identified the cells in their resting monocytic stage (peroxidase) and mature macrophage stage (acid phosphatase) along with quantitative in situ hybridization and immunocytochemistry with viral reagents to trace the efficiency of virus replication, we correlated virus replication with cell maturation. Only a few monocytes were susceptible to infection, and virus replication did not extend beyond a low level of transcription of viral RNA. In the acid phosphatase-positive, maturing macrophage, susceptibility of the cells to infection was increased and virus replication was greatly amplified to the level of translation of viral polypeptides. However, virus maturation was delayed by 3 days until further cell maturation had occurred. Thus, the entire life cycle of the virus, from its attachment to the target cell to its maturation in the cell, was dependent on the level of maturation/differentiation of the monocytic cell.

Animals↗
PubMed1986

Sequence homology between cloned caprine arthritis encephalitis virus and visna virus, two neurotropic lentiviruses.

Caprine arthritis encephalitis virus (CAEV) is an exogenous, nononcogenic retrovirus which causes neurological disease and crippling arthritis in goats. A complete CAEV genome was cloned from unintegrated viral DNA in two fragments of 9.4 and 0.4 kilobases in length, respectively. The biological activity of these clones was tested by ligation of the fragments followed by transfection onto goat synovial membrane cells; infectious virus was recovered. Cloned CAEV and visna virus, a related neurotropic virus of sheep, were compared by heteroduplex and molecular hybridization analyses. These data demonstrated that the greatest overall conservation of nucleotide sequences occurred in the gag and pol gene regions and two smaller regions, sor and the putative tat gene. The region of greatest divergence occurred in the env gene and, in particular, was localized primarily in the region coding for the glycosylated outer membrane protein. These findings and the recently demonstrated genetic relationship of visna virus, CAEV, and human T-cell lymphotropic virus type III, the etiologic agent of the acquired immune deficiency syndrome, may have important implications concerning the biological properties of these related viruses for human and veterinary medicine.

Animals↗
PubMed1986

Nucleotide sequence and transcriptional activity of the caprine arthritis-encephalitis virus long terminal repeat.

Caprine arthritis-encephalitis virus (CAEV) and visna virus are pathogenic lentiviruses of goats and sheep which share morphologic features and sequence homology with human T-cell lymphotropic virus type III (HTLV-III), the etiologic agent of the acquired immune deficiency syndrome. The nucleotide sequence of the CAEV long terminal repeat (LTR) was determined, and it was found to be 450 base pairs long, with U3, R, and U5 regions of 287, 85, and 78 base pairs, respectively. Portions of the CAEV LTR are closely homologous to analogous regions of visna virus. The CAEV LTR is not significantly homologous with the HTLV-III LTR; however, like HTLV-III, visna virus, and equine infectious anemia virus, CAEV uses tRNA lysine as a primer for reverse transcription. The transcriptional activity of the CAEV and visna virus LTRs was measured by a chloramphenicol acetyltransferase assay, and the activity of the visna virus LTR was generally higher in a variety of uninfected cell types. Infection of cells with visna virus markedly increased gene expression directed by either the CAEV or visna virus LTR, but in contrast, infection of cells with CAEV had little effect on the activity of either LTR. The lack of trans-activation by CAEV, a virus which causes debilitating arthritis and encephalitis in goats, suggests that trans-activation may not be a general property of pathogenic lentiviruses.

Acetyltransferases↗
PubMed1985

Restricted replication of lentiviruses. Visna viruses induce a unique interferon during interaction between lymphocytes and infected macrophages.

Lentivirus infections are characterized by a persistent, restricted type of virus replication in tissues. Using sheep and goat lentiviruses, whose target cells in vivo are macrophages, we explored virus-host cell interactions to determine whether an interferon (IFN) is produced during virus replication in vivo which causes restricted replication. We show that the lentiviruses were incapable of inducing IFN directly in any infected cell, including macrophages and lymphocytes. However, after infection with these viruses, sheep and goat macrophages acquired a factor that triggered IFN production by T lymphocytes. Only sheep/goat lentiviruses were capable of inducing the factor and, although these viruses replicated productively in various cell cultures of the natural host animal, only infected macrophages developed the IFN-inducing factor. The factor was produced continuously and was strictly cell associated, requiring direct contact with lymphocytes. The lymphocytes responded with a single, sudden release of IFN beginning 7 h after cocultivation and reaching peak values at 48 h, after which they ceased production and became refractory. IFN production was not immunologically specific and did not require histocompatibility between donors of the two cell types. The IFN is a nonglycosylated protein of molecular weight 54,000-64,000, and is stable to heat and acid treatments. These findings identify a unique IFN and a new method for virus induction of IFN. The novel two-stage process of induction provides a mechanism for local amplification and continuity of production of IFN in vivo. This is compatible with infection in the animal whose lentivirus-induced pathologic lesions consist of accumulations of lymphocytes and infected macrophages in target tissues.

Animals↗
PubMed1985

Persistent expression of Ia antigen and viral genome in visna-maedi virus-induced inflammatory cells. Possible role of lentivirus-induced interferon.

In this study we investigated the pathogenesis of the lymphoproliferative response in the chronic-active visna maedi virus-induced inflammatory lesions. Viral RNA expression was confined to macrophages, but only in tissues showing inflammatory lesions. A persistent and high level of Ia antigen expression was seen in macrophage-like cells in the inflammatory lesions, and the amounts of viral RNA and Ia expression were closely correlated. A small subpopulation of macrophages contained both viral RNA and Ia antigen, and these were found in greatest number in the lung. In vitro experiments showed that a lentivirus-induced interferon (LV-IFN) could induce Ia antigens in normal sheep spleen and lymph node cells as well as in a transformed sheep macrophage cell line. Ia antigen expression in macrophages was transient in the absence of a continuing IFN stimulus and persisted for at least 2 wk in the presence of LV-IFN. LV-IFN also restricted viral replication in macrophages. It is suggested that LV-IFN induced by the inflammatory cells in visna-maedi lesions may induce Ia antigen expression in macrophages, thereby indirectly causing the lymphoproliferative response and restricted virus replication.

Animals↗