Identification of a negative element in the human vimentin promoter: modulation by the human T-cell leukemia virus type I Tax protein.

The vimentin gene is a member of the intermediate filament multigene family and encodes a protein expressed, in vivo, in all mesenchymal derivatives and, in vitro, in cell types of various origin. We have previously demonstrated that the expression of this growth-regulated gene could be trans activated by the 40-kDa Tax protein of HTLV-I (human T-cell leukemia virus type I) and that responsiveness to this viral protein was mediated by the presence of an NF-kappa B binding site located between -241 and -210 bp upstream of the mRNA cap site (A. Lilienbaum, M. Duc Dodon, C. Alexandre, L. Gazzolo, and D. Paulin, J. Virol. 64:256-263, 1990). These previous assays, performed with deletion mutants of the vimentin promoter linked to the chloramphenicol acetyltransferase gene, also revealed the presence of an upstream negative region between -529 and -241 bp. Interestingly, the inhibitory activity exerted by this negative region was overcome after cotransfection of a Tax-expressing plasmid. In this study, we further characterize the vimentin negative element and define the effect of the Tax protein on the inhibitory activity of this element. We first demonstrate that a 187-bp domain (-424 to -237 bp) behaves as a negative region when placed upstream either of the NF-kappa B binding site of vimentin or of a heterologous enhancer such as that present in the desmin gene promoter. The negative effect can be further assigned to a 32-bp element which is indeed shown to repress the basal or induced activity of the NF-kappa B binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Comparative seroepidemiology of HTLV-I and HTLV-III in the French West Indies and some African countries.

The prevalence of antibodies detected by ELISA against human T-lymphotropic viruses, type I (HTLV-I) and type III (HTLV-III-LAV), is described in a comparative serosurvey in the French West Indies and African countries. The data confirm that the Caribbean basin is endemic for HTLV-I. In this region, HTLV-I antibody prevalence varied from 3.4% to 5.2% among blood donors and increased with age to reach a value of 33% among elderly people from the Dominica Island. In French Guyana, a South American country bordering the Caribbean sea, differences in antibody distribution across three ethnic groups (black Bonis, Indian Wayanas, and Hmongs from Asia) provide clues for investigation of the mode of HTLV-I transmission. Africa appears to be an endemic continent for HTLV-I and HTLV-III. For both viruses, the antibody prevalence exhibited an increasing gradient from northern to equatorial through Sudanic areas. These preliminary data by showing that Africa represents an endemic reservoir of HTLVs and, possibly, of other human retroviruses should stimulate further investigations on the natural history and the geographical origin of these viruses.

EGF promotes in vivo tumorigenic growth of primary chicken embryo fibroblasts expressing v-myc and enhances in vitro transformation by the v-erbA oncogene.

We report that the activation of the endogenous chicken EGF receptor leads to the tumorigenic growth in vivo of early passage chicken embryo fibroblasts (CEFs) that express a nonsarcomagenic oncogene, v-myc. To provide a continuous paracrine source of this growth factor in vivo, we employed irradiated Rat-1 cells which had been stably transfected with a synthetic cDNA to human EGF. Expression of another non-sarcomagenic nuclear oncogene, v-erbA, prones the CEFs to in vitro transformation by EGF, but does not cause EGF dependent tumorigenicity in vivo. The short period of incubation in the in vivo assay employed by our study (10 days), together with the genetic stability of primary chicken embryo fibroblasts, make it very likely that the reported alterations in cellular behaviour are a direct and primary effect of the expression of the relevant oncogenes and their cooperation with the EGF induced response. Dose response and ligand binding assays suggest that the EGF response is transmitted via the chicken c-erbB molecule, which by virtue of its preference for TGF-alfa is distinct from the mammalian EGF receptors studied so far. The level of expression of the endogenous chicken EGF receptor is within the same range as that reported for primary human fibroblasts (5-7 x 10(3) per cell). The cooperative effect of v-myc with chicken c-erbB probably takes place at a post receptor level, as its expression did not affect the steady state level or affinity for ligand of the chicken EGF receptor.

Immediate effects of reversible HTLV-1 tax function: T-cell activation and apoptosis.

The tax protein of Human T-cell leukemia virus type 1 (HTLV-1) is important for the transforming properties of this virus in vitro and is considered to be responsible for the early stages of leukemogenesis in infected hosts. To address the early consequences of HTLV-1 tax function, we have constructed fusion proteins containing tax sequence either aminoterminal (taxER) or carboxy-terminal (ERtax) of the hormone binding domain of the human estrogen receptor (ER). Addition of estrogen or the antagonist hydroxytamoxifen to Jurkat T-cells expressing these constructs led to the trans-activation or responsive promoters and upregulation of cell surface markers CD28, CD69 and CD5 but not CD25 (IL2R-alpha subunit) or B7 (ligand for CD28). Additional stimulation of the T-cell receptor CD3 complex, led to the upregulation of CD25. B7 was upregulated by concomittent activation of ERtax and CD3 or CD28 pathways. These events were in part reversible upon withdrawal of hormone and inactivation of ERtax. Severe inhibition of proliferation, and apoptosis was observed with cells which had been subjected to short term (3 days) activation of the tax fusion proteins and the CD3 complex. Induction of ERtax activity for longer than 3 days promoted cell death independently of CD3 stimulation. Co-stimulation through the CD28 cell surface molecule did not suppress induction of apoptosis.

Autocrine interferon-beta synthesis for gene therapy of HIV infection: increased resistance to HIV-1 in lymphocytes from healthy and HIV-infected individuals.

OBJECTIVE: To explore the possibility of gene therapy of HIV infection based on the multiple antiretroviral activities of interferon (IFN)-beta. DESIGN: We introduced into HIV target cells an IFN-beta gene placed under an expression control ensuring a low and constitutive expression, sufficient to confer a permanent antiviral state without impeding normal cell function. METHODS: We transformed, with an efficacy ranging from 20-55%, peripheral blood lymphocytes (PBL) derived from healthy, seronegative donors, and from asymptomatic HIV-infected individuals by the HMB-KbHuIFN beta retroviral vector carrying the human IFN-beta coding sequence driven by a fragment of the murine H-2Kb gene promoter. RESULTS: The replication rate of the IFN-beta-expressing cells was no different from that of untransformed controls during the 21-day period of in vitro observation. When IFN-beta-transformed, purified CD4+ lymphocytes from healthy donors were HIV-1LAI-infected, virus replication was inhibited and most of the cells survived, in contrast to untransformed CD4+ cells which were all destroyed 12 days after infection. Protection of CD4+ cells from the same donors was also observed in suspensions of IFN-beta-transformed total PBL that were infected with HIV-1LAI. In IFN-beta-transformed PBL from four HIV-infected donors, endogenous HIV replication was decreased and 28-69% of the CD4+ cells survived at the end of the 21 days in culture. In the untransformed control PBL suspensions, all CD4+ cells were destroyed. In long-term experiments, HIV-infected, IFN-beta-transformed cell populations of the lymphocytic CEM and the promonocytic U937 line were kept in culture for 60 days, during which time they remained resistant to HIV infection. CONCLUSION: These results indicate that further exploration of autocrine IFN-beta production for somatic cell gene therapy of HIV infection is warranted.

Organization and expression of intermediate filaments in epithelial cells expressing the HTLV-I Tax protein.

Intermediate filaments (IF) represent major components of the cytoskeletal network. These proteins which are differentially expressed according to the cell type, constitute a dynamic structure which not only contributes to the cell architecture but also defines its state of differentiation. Furthermore, numerous observations have shown that the IF network is altered in cells transformed by tumorigenic viruses. We have previously demonstrated that HTLV-I (human T-cell leukemia virus type I) transformed T cells were characterized by a high level of vimentin transcripts and that the HTLV-I Tax regulatory protein was able to transactivate the vimentin promoter transfected into Jurkat and HeLa cells. To enlarge the scope of this study, we investigated the effects of the Tax protein on the expression and organization of IF of epithelial cells in which the IF network is composed of vimentin and cytokeratin. To this aim, we have developed a model of epithelial cells (HeLa) stably expressing the tax sequences which were introduced by using retrovirus-mediated gene transfer. Half of the Tax expressing HeLa clones were loosely adherent to the culture surface and were displaying remarkable morphological alterations, as ascertained by the presence of round-shaped or spindle-shaped cells. In these cells, expression of this viral protein correlated to a pronounced disruption in the distribution of both the vimentin and the cytokeratin networks, as shown by immunofluorescence and ultrastructural analysis. Indeed, vimentin filaments appeared to be concentrated in discrete spots throughout the cytoplasm, while the cytokeratin filaments appeared to form a dense ring around the nucleus. More importantly, mRNA and protein analysis indicate an enhanced expression of the cytokeratin 7 gene.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Malondialdehyde production from spermine by homogenates of normal and transformed cells.

The oxidation of spermine in vitro by a mixture of polyamine oxidase and diamine oxidase from pig kidney gives rise to malondialdehyde via 3-aminopropanol as the intermediate. Conversely, with spermidine, under similar experimental conditions, no evidence could be obtained for malondialdehyde formation within the limits of sensitivity of the assay (2.0 nmol). The activities of both these enzymes show about a 2-fold increase in normal rat kidney cells (LA31 NRK) transformed by the temperature sensitive mutant of Rous sarcoma virus (LA31) and incubated at the non permissive temperature (39 degrees C) compared to the activities in LA31 NRK at the permissive temperature (33 degrees C). These same enzymatic activities show no temperature dependent changes in normal rat kidney cells (NRK) or in these same cells infected by the wild type virus (NRK B77). In extracts derived from Friend erythroleukemic cells induced to differentiate by dimethyl sulfoxide or hexamethylene bis acetamide, spermine oxidation takes place more efficiently than in non induced cells. A rise in diamine oxidase activity is seen in LA31 NRK (39 degrees C) 12 h after the temperature shift, whereas morphological manifestations of normalcy are seen only at 48 h. The Km of diamine oxidase is 10(-6) M for putrescine and 10(-3) M for 3-aminopropanol. A possible mechanism involving the well documented acetylation of putrescine [23,26] is proposed for diverting intracellular putrescine away from cytosolic diamine oxidase and towards intramitochondrial monoamine oxidase.

Immunohistochemical study of normal skin of HIV-1-infected patients shows no evidence of infection of epidermal Langerhans cells by HIV.

Langerhans cells (LC) are dendritic epidermal antigen-presenting cells expressing the surface molecule CD4, which renders them theoretical cellular targets for direct infection by the human immunodeficiency virus (HIV). To date, somewhat conflicting results have been reported concerning the in vivo infection of LC by HIV as well as the numerical alteration of these cells in the course of HIV infection. In the present work we studied clinically normal skin of a group of 44 HIV-1-seropositive patients classified according to the Centers for Disease Control (CDC) stages II (n = 14), III (n = 9), and IV (n = 21). Monoclonal antibodies (MAb) to HIV p18, p24, and gp120 and to HLA-DR and CD1a antigens (specific for LC) were applied on frozen skin sections using an amplification biotin-streptavidin-fluorescein technique. The MAb to HIV p18 cross-reacted with a cytoplasmic antigen of epidermal basal keratinocytes also present on HIV-seronegative skin specimens. No other reactivity was observed with any of the three anti-HIV MAb. The quantitative study showed that no significant correlations could be established between the number of LC (evaluated independently by HLA-DR and CD1a antigens) and the number of peripheral blood CD4+ve lymphocytes or the CDC disease stage. These results cast some doubt on the previously reported in vivo infection and numerical decrease in LC in HIV infection. The precise involvement of LC in HIV infection awaits further investigation.

HTLV-I maternal transmission in Martinique, using serology and polymerase chain reaction.

We have investigated HTLV-I and HTLV-II infection in children born to HTLV-I-seropositive or indeterminate Western blot mothers in Martinique by using the polymerase chain reaction (PCR). Only HTLV-I and no HTLV-II-positive samples were found in this study. All the samples from HTLV-I-seropositive children and adults were PCR positive, whereas the four HIV-I-seropositive and Western blot HTLV-I-negative mothers and their eight children were all PCR negative. Therefore, PCR and serology were in complete agreement in these patients. However, two of the six mothers who were first indeterminate by Western blot, and who later became seronegative, were found positive by PCR. Of the 27 children (ages 2-12 years), born to HTLV-I-seropositive and PCR-positive mothers, 2 were seropositive and PCR positive, 5 were seronegative and PCR positive with 2 primer pairs in gag and pol, and 4 were seronegative and PCR positive with only 1 of the primer pairs. In contrast to an initial rate of transmission of 7% estimated by serology we found a rate of transmission of 28 to 41% (whether or not children who were positive with only one of the primer pairs were included). Thus, our study confirms that PCR is useful in detecting HTLV-I infection in children before seroconversion and underlines the potential lack of sensitivity of serology to detect contaminating HTLV-I blood units in endemic areas.

Detection of HTLV-I (human T-cell lymphotropic virus, type I) proviral DNA in leukemic cells from a French patient with Sezary syndrome.

Human T-lymphotropic type I (HTLV-I) proviral sequences were detected in leukemic cells of a patient living in Marseilles (south of France) and suffering from Sezary syndrome. He did not have any travel history outside France and did not receive blood transfusion or hepatitis B vaccination. This case of HTLV-I positive Sezary syndrome is the first one described outside the known endemic regions for HTLV-I. Moreover, this patient was found to be negative for viral antibodies. This observation should therefore stimulate new and thorough analysis of the association of this human retrovirus with leukemia and lymphoma in the Mediterranean region, both by seroepidemiological and molecular biology techniques.

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.

Expression of the Tat protein of HIV1 in human promonocytic U937 cells.

Numerous studies have shown that, upon HIV1 infection, human promonocytic U937 cells were induced to differentiate, as indicated, for example, by increased expression of adhesion molecules. One of the viral proteins involved in this process might be the Tat protein. Indeed, this viral protein, which is essential for productive infection, has also been shown to display growth-stimulating properties and immunomodulatory activities. In order to apprehend the role of the HIV1 tat gene in inducing the differentiation of HIV1-infected U937 cells, we have successfully introduced this gene into U937 cells by infecting them with retroviral particles transducing tat. The effect of the Tat protein constitutively expressed by these cells upon their differentiation was then evaluated by looking for the expression of the c-fos and of the c-fms proto-oncogenes which are linked to the differentiation of myelomonoblastic cells. Northern blot analysis revealed in these cells, an increase in the transcription of these two proto-oncogenes, and this increase was amplified after treatment with phorbol myristate acetate. No such increase was observed in control U937 cells. These results indicate that, among HIV1 gene products, the Tat protein appears to trigger monocytic differentiation, and suggests that this viral protein directs progenitors of the monocyte/macrophage lineage towards a differentiation stage in which production of viral antigens and virions might be more efficient.

Target cells infected by avian erythroblastosis virus differentiate and become transformed.

Transformation in vitro of bone marrow cells by avian erythroblastosis virus (AEV) gives rise to rapidly growing cells of erythroid nature. Target cells of neoplastic transformation by AEV are recruited among the early progenitors of the erythroid lineage, the burst-forming units-erythroid (BFU-E). They express a brain-related antigen at a high level and an immature antigen at a low level. We show that AEV-transformed cells express low levels of the brain antigen and high levels of the immature antigen. Their response to specific factors regulating the erythroid differentiation indicates that they are very sensitive to erythropoietin. Furthermore, cells transformed by a temperature-sensitive mutant of AEV differentiate into hemoglobin-synthesizing cells 4 days after being shifted to the nonpermissive temperature. All these properties are similar to those of late progenitors of the erythroid lineage, the colony-forming units-erythroid (CFU-E). These results indicate that the AEV-transformed cells are blocked in their differentiation at the CFU-E stage.