Biomedical subjects

M Duc Dodon

Publications and source records attributed to M Duc Dodon.

At least 19 recordsLinked to original sources

PubMed2000

The herpes simplex virus 1 Us11 protein cooperates with suboptimal amounts of human immunodeficiency virus type 1 (HIV-1) Rev protein to rescue HIV-1 production.

The human immunodeficiency virus type 1 (HIV-1) RNA-binding Rev protein governs the expression of structural and enzymatic viral proteins at a posttranscriptional level. Binding of Rev to the stem-loop IIB (SLIIB) sequence of the Rev-response element (RRE) within unspliced and singly spliced viral mRNAs and to the nuclear export signal-binding receptor, hCRM1 (or exportin 1), is required for the export of these transcripts to the cytoplasm. We have previously shown that herpes simplex virus type 1 (HSV-1) RNA-binding Us11 protein is able to bind the RRE and substitute for Rev in inducing the expression of HIV-1 envelope glycoproteins. We show here that Us11 cannot substitute for Rev in rescuing a rev-deleted HIV-1 provirus. However, HIV-1 production is observed when Us11 is expressed with suboptimal amounts of Rev. An in vivo RNA-protein binding assay indicates that Us11 is unable to directly interact with the SLIIB RNA but can bind Rev assembled on that stem-loop structure. This association of US11 with Rev, which was confirmed by in vivo coimmunoprecipitation and GST-pulldown assays, therefore underlies a biological Us11-Rev cooperation. Furthermore this cooperation was shown to remain susceptible to the effect of leptomycin B, which blocks the binding of hCRM1 to the nuclear export signal of Rev. These observations performed with intron-containing constructs provide evidence that HSV-1 Us11 protein is not directly involved in the cytoplasmic accumulation of viral mRNAs but may be rather acting as an auxiliary protein, thus allowing this retroviral protein to fulfill the nuclear export of these transcripts and to rescue HIV-1 production.

Biological Transport↗

PubMed2000

Epstein-Barr virus EB2 protein exports unspliced RNA via a Crm-1-independent pathway.

Human herpesviruses encode posttranscriptional activators that are believed to up-regulate viral replication by facilitating early and late gene expression. We have reported previously that the Epstein-Barr virus protein EB2 (also called M or SM) promotes nuclear export of RNAs that are poor substrates for spliceosome assembly, an effect that closely resembles the human immunodeficiency virus type 1 Rev-dependent nuclear export of unspliced viral RNA. Here we present experimental data showing that EB2 efficiently promotes the nuclear export of unspliced RNA expressed from a Rev reporter construct. Site-directed mutagenesis as well as domain swapping experiments indicate that a leucine-rich region found in the EB2 protein, which matches the consensus sequence for the leucine-rich nuclear export signal, is not a nuclear export signal per se. Accordingly, leptomycin B (LMB), a specific Crm-1 inhibitor, impairs Rev- but not EB2-dependent nuclear export of unspliced RNA. Moreover, EB2 nucleocytoplasmic shuttling visualized by a heterokaryon assay is, unlike Rev shuttling, not affected by LMB. We also show that overexpression of an N-terminal deletion mutant of Nup214/can, a major nucleoporin of the nuclear pore complex involved in several aspects of nuclear transport, blocks both Rev- and EB2-dependent nuclear export of RNA. These results strongly suggest that EB2 nuclear export of unspliced RNA is mediated by a Crm-1-independent pathway.

3T3 Cells↗

PubMed1997

The human T-cell leukemia virus type 1 Rex regulatory protein exhibits an impaired functionality in human lymphoblastoid Jurkat T cells.

The Rex protein of human T-cell leukemia virus type 1 (HTLV-1) intervenes in the posttranscriptional regulation of proviral gene expression. Its binding to the Rex response element (XRE) present in the 3' long terminal repeat ensures the coordinate cytoplasmic accumulation of spliced and unspliced forms of viral messengers. Consequently, synthesis of viral structural and enzymatic proteins is strictly dependent on the Rex posttranscriptional activity. Here we report that synthesis of HTLV-1 envelope glycoproteins by Jurkat T cells could be detected only when they were regulated in a Rex-independent manner. Indeed, Jurkat T cells transfected with a Rex-dependent env expression vector (encompassing both the env and pX open reading frames) do not produce significant levels of envelope glycoproteins despite the production of significant amounts of Rex protein. The analysis of levels and distribution patterns of the unspliced env and of the singly spliced tax/rex transcripts suggests that the failure in envelope glycoprotein synthesis may be ascribed to a deficiency of Rex in mediating the nucleocytoplasmic transport of unspliced env RNAs in these cells. Furthermore, despite the synthesis of regulatory proteins, HTLV-1 structural proteins were not detected in Jurkat T cells transfected with an HTLV-1 infectious provirus. Conversely, and as expected, structural proteins were produced by Jurkat cells transfected by a human immunodeficiency virus type 1 (HIV-1) infectious provirus. This phenotype appeared to be linked to a specific dysfunction of Rex, since the functionally equivalent Rev protein of HIV-1 was shown to be fully efficient in promoting the synthesis of HTLV-1 envelope glycoproteins in Jurkat cells. Therefore, it seems likely that the block to Rex function in these lymphoblastoid T cells is determined by inefficient Rex-XRE interactions. These observations suggest that the acquisition of this Rex-deficient phenotype by in vivo-infected HTLV-1 T cells may represent a critical event in the lymphoproliferation induced by this human retrovirus, leading to leukemia.

Gene Expression Regulation, Viral↗

PubMed1997

Transrepression of lck gene expression by human T-cell leukemia virus type 1-encoded p40tax.

To understand the mechanism of p56lck protein downregulation observed in human T cells infected by human T-cell leukemia virus type 1 (HTLV-1), we have investigated the ability of the 3' end of the HTLV-1 genome as well as that of the tax and rex genes to modulate p56lck protein expression and p56lck mRNA synthesis. By using Jurkat T cells stably transfected with constructs that expressed either the 3' end of the HTLV-1 genome (JK C11-pMTEX), the tax gene (JK52-Tax) or the rex gene (JK9-Rex), we found that the expression of p40tax (Tax) was sufficient to modulate p56lck protein expression. Similarly, we found that the expression of the mRNA which encoded p56lck was repressed in Jurkat T cells which expressed Tax. This downregulation was shown to be proportional to the amount of tax mRNA found in the transfected cells, as evidenced by experiments that used cells (JPX-9) stably transfected with a tax gene driven by a cadmium-inducible promoter. Furthermore, cadmium induction of Tax in JPX-9 cells transiently transfected with a construct containing the chloramphenicol acetyltransferase (CAT) gene under control of the lck distal promoter (lck DP-CAT) resulted in the downregulation of CAT gene expression. In contrast, cadmium induction of Tax in JPX-9 cells transiently transfected with a CAT construct driven by a lck DP with a deletion extending from position -259 to -253 (a sequence corresponding to a putative E-Box) did not modulate CAT gene expression, suggesting that the effect of Tax on p56lck is mediated through an E-Box binding protein.

Cell Line↗

PubMed1995

Human T cell leukaemia virus type I env gene-transfected HeLa cells display a decrease in cell fusion ability.

The envelope (env) gene of human T cell leukaemia virus type I (HTLV-I) was inserted into an expression vector, referred to as phMTenv, under the transcriptional control of the human metallothionein IIa gene promoter (hMT-IIa). When this vector was transiently transfected in HeLa cells treated with hMT-IIa inducers, formation of multinucleated cells was observed, indicating the expression of functional surface and transmembrane glycoproteins. Of several HeLa cell clones transfected with phMTenv together with a plasmid carrying the neomycin resistance gene and isolated after selection in G418-containing medium, env mRNA was detected in only two, in the presence of hMT-IIa inducers. Viral glycoproteins were found to be weakly expressed as detected in immunoprecipitation assays of 125I-surface-labelled cells. These env-transfected HeLa cell clones, although unable to form syncytia when cocultivated with untransfected control HeLa cells, retained the capacity to fuse with HTLV-I-producing C91PL T cells. However, a significant decrease in their fusogenic ability was observed, after treatment with hMT-IIa inducers. Under identical experimental conditions, control HeLa cell clones stably transformed with the same plasmid, but lacking the env gene, were still able to fuse with C91PL cells. These observations suggest that a post-transcriptional step in HTLV-I env expression is impaired, probably leading to the establishment of superinfection interference.

Cell Fusion↗

PubMed1994

Critical involvement of human T cell leukaemia virus type I virions in mediating the viral mitogenic effect.

Human T cell leukaemia virus type I (HTLV-I) is a direct activator of human resting T lymphocytes. The present study was undertaken to delineate further the role of viral particles and to define the involvement of envelope glycoproteins in the induction of T cell mitogenic stimulation. Virus-producing cells treated with paraformaldehyde (PFA) were found to be unable to induce the formation of syncytia, but still able to trigger the proliferation of resting T cells. Likewise, PFA-treated virus particles were still mitogenic. These results suggest that the mitogenic event is triggered before the fusion of the envelope with the cell membrane. Furthermore, HTLV-I envelope-expressing cells obtained after infection of C8166/45 cells (HTLV-I-transformed, but defective in virion production) with an HTLV-I envelope recombinant vaccinia virus were unable to activate normal T cells. Human immuno-deficiency virus type 1 particles produced by C8166/45 cells were also devoid of mitogenic ability. However, when HTLV-I viral preparations were purified by chromatography, only the virion-containing fractions were found to be mitogenic for human resting T lymphocytes. This mitogenic activity was partially abolished by preincubating the purified virus with a monoclonal antibody directed to the surface envelope glycoprotein. Finally, treatment of HTLV-I-transformed cells by tunicamycin, an inhibitor of N-linked glycosylation, led to the production of virus particles with a decreased mitogenic activity. Collectively, these observations suggest that the HTLV-I mitogenic activity is triggered by the contact of HTLV-I virions with T cells.

Cell Division↗

PubMed1991

Molecular and cellular events at the onset of the lymphoproliferative process induced by HTLV-I (human T-cell leukemia virus, type I).

The human retrovirology opened its first chapter, 10 years ago, with the isolation and characterization of HTLV-I (Human T-cell leukemia virus, type I), a type C retrovirus, which was found to be etiologically linked first to adult T-cell leukemia and second to neurological disorders. Epidemiological data have indeed shown that patients who developed these diseases represent a small percentage of HTLV-I infected individuals living in restricted geographical areas. Experiments performed at molecular and cellular levels have revealed that HTLV-I plays an essential role in the initiation of the lymphoproliferative process. Indeed, viral particles deliver a mitogenic signal to human resting T lymphocytes. After proviral integration, two regulatory proteins--Tax and Rex--are controlling the replicative cycle of HTLV-I. The Tax protein trans-activates the proviral transcription and the Rex protein is essential in the synthesis of viral structural proteins. Furthermore, the Tax protein induces the transcription of several cellular genes involved in T-cell activation and proliferation. Studies are now aimed at identifying HTLV-I target cells among precursors of the T cell lineage and at unravelling the role of HTLV-I in the secondary events leading to leukemogenesis.

Gene Products, rex↗

PubMed1990

Polymerase chain reaction (PCR) amplification demonstrates the absence of human T-cell lymphotrophic virus (HTLV)-I specific pol sequences in peripheral T-cell lymphomas.

HTLV-I seronegative patients in nonendemic areas have been described with T-cell proliferations the DNA of which contains specific HTLV-I viral sequences. We have looked for the presence of HTLV-I DNA sequences in 27 HTLV-I seronegative patients with peripheral T-cell lymphomas, distinct from adult T-cell leukemia (ATL), and four HTLV-I seropositive patients, three with an ATL and one with a tropical spastic paraparesis. Using HTLV-I pol specific primers, the genomic DNA from peripheral blood mononuclear cells and lymph nodes massively infiltrated by tumor cells was analyzed by the enzymatic gene amplification procedure. In contrast to the peripheral blood lymphocytes from the four HTLV-I seropositive patients, the peripheral T-cell lymphoma samples did not harbor HTLV-I pol sequences. The data show that the detection of HTLV-I nucleotide sequences by the polymerase chain reaction correlates with serologic analysis in this series.

Base Sequence↗

PubMed1990

Effect of human T-cell leukemia virus type I tax protein on activation of the human vimentin gene.

We report that the expression of the vimentin gene, a cytoskeletal growth-regulated gene, is activated in trans by the Tax (p40x) transactivator protein encoded by the human T-cell leukemia virus type I. Expression of the Tax protein activates a number of cellular genes, such as those coding for the alpha chain of the high-affinity interleukin-2 receptor and interleukin-2. These findings indicate that the Tax protein is involved in the unregulated T-cell growth associated with human T-cell leukemia virus type I infection. Higher levels of vimentin mRNA were expressed in two human T-cell leukemia virus type I-transformed T cell lines, C91/PL and C81-66/45, when compared with that in Jurkat T cells. We demonstrate that this activation is conferred by the vimentin upstream flanking sequences. Indeed, enhanced activity was detected when constructs with the vimentin promoter linked to the chloramphenicol acetyltransferase gene were transfected in HeLa cells and in two cell lines of hematopoietic origin (Jurkat T lymphoblastoid cells and U937 promonocytic cells) together with a Tax expression plasmid. By introducing a series of deletions in the vimentin promoter, we further restrict these sequences to 30 base pairs, located between 241 and 210 base pairs upstream of the mRNA cap site. A 40-base-pair oligonucleotide containing this regulatory region proved sufficient to confer Tax inducibility upon a heterologous promoter linked to chloramphenicol acetyltransferase. Importantly, this segment includes an 11-base-pair promoter segment that has homology with the binding site for the NF-kappa B transactivating factor. Our findings indicate that constitutive expression of the vimentin gene under the control of the Tax protein may be relevant in understanding the progression of the lymphoproliferative process associated with human T-cell leukemia virus type I infection.

Base Sequence↗

PubMed1989

Restriction of HIV-1 replication in promonocytic cells: a role for IFN-alpha.

A comparative study of the replication kinetics of human immunodeficiency virus type 1 (HIV-1) was performed in the promonocytic U937 cells and in the T lymphoblastoid H9 cells. If a productive HIV-1 infection of both cell types could be established, the time which elapses before most of the cells could express viral proteins is always proportionally longer for U937 cells than for H9 cells. Indeed, when U937 cells are infected with HIV-1, this nonproductive phase is followed by a lag phase during which the percentage of virus-producing cells is slowly increasing when compared to H9 cells. The restriction of HIV-1 replication in U937 cells might be consecutive to the lower adsorption of viral particles to these cells, even though the same percentage of U937 and H9 cells was expressing the CD4 molecule. Furthermore, we demonstrate that HIV-1 replication in U937 cells is mainly restricted by endogenous IFN-alpha. Indeed, addition of anti-IFN-alpha antibodies at the time of infection, during the nonproductive phase of the viral replication cycle, or during the lag phase leads to an earlier expression of viral proteins and/or to a rapid increase in the percentage of virus-producing cells. Likewise, the treatment of cultures of HIV-1 chronically infected U937 cells with the same antibodies induces an increased production of viral particles. Thus, IFN-alpha appears to be involved in the persistence of HIV-1 in the monocytes/macrophages of infected individuals.

Antigens, Differentiation, T-Lymphocyte↗

PubMed1989

Comparative analysis of the HTLV-I Rex and HIV-1 Rev trans-regulatory proteins and their RNA response elements.

The Rex proteins of types I and II human T-cell leukemia viruses (HTLV-I, HTLV-II) are required for expression of the viral structural gene products, gag and env and, thus, are essential for the replication of these pathogenic retroviruses. The action of Rex is sequence specific, requiring the presence of a cis-acting Rex response element located in the 3' long terminal repeat. This element corresponds to a predicted RNA secondary structure and functions in an orientation-dependent but position-independent manner. Rex acts through this response element to stimulate the nuclear export of the unspliced or singly spliced viral mRNA species encoding the virion structural proteins that are normally excluded from the cytoplasm. Although the Rex proteins of HTLV-I and HTLV-II can also function via the related Rev response element present in the env gene of the type I human immunodeficiency virus (HIV-1), the analogous HIV-1 Rev protein is unable to act on the HTLV-I Rex response element. This nonreciprocal pattern of genetic complementation by Rex and Rev suggests that these viral trans-regulators may interact directly with their RNA response elements.

Base Sequence↗

PubMed1987

Loss of interleukin-2 requirement for the generation of T colonies defines an early event of human T-lymphotropic virus type I infection.

Accessory cells and/or soluble factors, together with interleukin 2 (IL2), are required for the proliferation and differentiation of phytohemagglutinin (PHA)-activated T lymphocytes. Human T-lymphotropic virus, type I (HTLV-I), a human retrovirus isolated from patients with adult T cell leukemia, can transform T cells in vitro. We investigated the role of HTLV-I-transformed T cell lines as accessory cells in promoting the growth of T colony-forming cells. We found that T cells isolated by E rosetting and then activated with PHA, when seeded with as few as 5 X 10(3) irradiated HTLV-I-producing cells, could generate colonies in the absence of IL2. We analyzed further the effects of HTLV-I virions on T colony formation. Infection of T cells with semipurified HTLV-I viral particles promoted colony formation, in the absence of IL2, of accessory cells or soluble factors. The same results were obtained either with monocyte-depleted T lymphocytes, or with T4 or T8 lymphocytes. Furthermore, T lymphocytes in the presence of heat-inactivated HTLV-I (devoid of replicative potential) could form colonies independently of IL2. Finally, experiments with sera positive for HTLV-I antibodies (to abolish binding of viral particles to cellular receptors) indicated that HTLV-I promoted IL2-independent colony formation, only by "touching" T colony-forming cells. These results taken together demonstrate that the loss of the exogenous IL2 (and other growth-helping factors) requirement defines an early event of HTLV-I infection. The results also suggest that viral attachment to T cells possibly supplies an accessory function triggering autocrine secretion of IL2 by these cells.

Cell Division↗

PubMed1985

Type-I and type-III HTLV antibodies in hospitalized and out-patient Zairians.

Sera from 182 Zairians (99 females and 83 males), aged 5 to 71 years, including maternity and child care consultants, out-patients suffering from minor injuries and patients hospitalized for tuberculosis, malaria or trauma, were analyzed for specific antibodies to HTLV-I and HTLV-III. Following pre-screening by the ELISA technique, reactive sera were further analyzed for specificity to HTLV-I or HTLV-III antigens by competition and/or Western blotting experiments. African sera, possibly because they have higher immunoglobulin levels than US and European sera, are highly reactive in ELISA systems and confirmatory assays are essential to rule out false-positive results. Confirmed antibody prevalence for HTLV-I was 13.2% (II females and 13 males) and increased with age, suggesting continuous exposure to the virus throughout life. Confirmed antibody prevalence for HTLV-III was 6.0% (8 females and 3 males) and showed a peak age range between 21 and 40 years, suggesting heterosexual transmission. Individuals positive for HTLV-I antibodies were not the same as individuals positive for HTLV-III antibodies, suggesting that infection with one virus did not increase susceptibility to infection by the other virus. Further investigations of the epidemiology and of the immunovirology of HTLV-III in Zaire, in relation to acquired immuno-deficiency syndrome (AIDS)-associated pathologies, should enlighten the question of the significant percentage of HTLV-III-infected individuals who do not manifest symptoms of AIDS.

Acquired Immunodeficiency Syndrome↗

PubMed1985

Analysis of effects of lipopolysaccharide and interferon on murine macrophages: modulation of elastase secretion in vitro.

Macrophages are able to produce and secrete elastase in response to a variety of agents which induce macrophage differentiation. In this study, we compared elastase levels in macrophage cultures derived from lipopolysaccharide (LPS)-responsive and LPS-hyporesponsive mice. After the in vivo administration of fluid thioglycolate, both types of macrophages exhibited increased secretion of elastase, and further enhancement was observed after in vitro stimulation with colchicine or phorbol myristic acetate or ingestion of latex beads. In contrast, phenol- and water-extracted, protein-free preparations of LPS (Ph-LPS) markedly inhibited elastase secretion below basal levels in LPS-responsive macrophages. The LPS-induced inhibition was reversible with polymyxin B and was not observed in Ph-LPS-stimulated C3H/HeJ (LPS-hyporesponsive) macrophage cultures. Stimulation of either LPS-responsive or -hyporesponsive macrophage cultures with interferon (IFN) also resulted in a significant reduction in elastase secretion below basal levels. LPS-induced inhibition of elastase secretion could be reversed and elastase secretion could be augmented in the presence of an antibody directed against IFN-alpha/beta. These findings suggest that LPS induces the production of both elastase and IFN, and that the latter product acts to suppress secretion of the proteinase.

Animals↗

PubMed1984

Involvement of hyposialylated immunoglobulins in the inactivation of alpha 1-proteinase inhibitor.

The elastase inhibitory capacity of alpha 1-proteinase inhibitor (alpha 1-PI) was measured, using a direct and reproducible method, with phagocytic cells maintained in the tissue culture plate through the assay. The oxidative inactivation of alpha 1-PI is known to be mediated by the action of myeloperoxidase (MPO). The fact that hyposialylated IgG (hs IgG) induce the release of MPO prompted us to investigate the effects of such hs IgG on the inhibitory capacity of alpha 1-PI. The results show that 1-PI inactivation was observed only when phagocytic cells were activated by aggregated hs IgG, and not by unaggregated hs IgG. These observations indicate that hyposialylation should be completed by aggregation to perpetuate the oxidative reactions characteristic of inflammatory diseases.

Blood Proteins↗

PubMed1984

Viral neuraminidase and cellular ectosialyltransferase in human lymphoblastoid cells infected with influenza virus.

In human lymphoblastoid cells, infected with an influenza virus, Fowl Plague Virus (FPV), glycoproteins (such as secreted IgM) are hyposialylated, through the action of viral neuraminidase. In this study, the modulation of the cellular ectosialyltransferase activity during viral infection was investigated. This activity was detectable in FPV-infected cells, was shown to be 2.5-fold higher than that of uninfected cells, and to be able to restore, at least partially, the level of sialylation of the cell surface acceptors.

Cell Line↗

PubMed1983

Asialylated immunoglobulins and rheumatoid factors.

A covalently coupled IgG-latex reagent was instrumental in isolating rheumatoid factors (RF) from normal immunoglobulins in the sera of patients with rheumatoid arthritis. It was found that RF contained hyposialylated immunoglobulins of both IgG and IgM classes. This biochemical alteration might be relevant to understanding the etiopathogenesis of the disease.

Aged↗

PubMed1982

Secretion of immunoglobulins by human lymphocytes after infection with influenza virus.

The biosynthesis of IgM by the Epstein-Barr virus-negative RAMOS lymphoblastoid cell line infected with an influenza A virus, fowl plague virus Dobson strain (FPV-B), was investigated. The results show that FPV infection of RAMOS cells slightly inhibited overall cellular protein synthesis only at 24 h after infection, despite the synthesis of FPV-specific proteins. However, even at this time, the synthesis and secretion of IgM were not affected by virus infection. Secreted IgM contained a reduced amount of sialic acid. The quantity of the asialylated IgM increased proportionally to the amount of enzymically active neuraminidase, suggesting that the asialylation of IgM is due to the action of virus neuraminidase. No such asialylated IgM was observed in RAMOS cells infected with measles virus, which does not possess neuraminidase. These results, together with a previous observation of ours that asialylated immunoglobulins acquire an altered antigenicity, suggest that the modulation of enzyme activities in B lymphocytes in response to an exogenous aggression may lead to disturbances in the structure and in the antigenic properties of immunoglobulins.

Cell Line↗