Tissue and cell-specific expression of the p53-target genes: bax, fas, mdm2 and waf1/p21, before and following ionising irradiation in mice.

The mechanisms by which the p53 tumour suppressor protein would, in vivo, co-ordinate the adaptive response to genotoxic stress is poorly understood. p53 has been shown to transactivate several genes that could be involved in two main cellular responses, growth arrest and apoptosis. To get further insight into the tissue-specific regulation of p53 transcriptional activity, we performed an extensive study looking at the expression of four well characterized p53-responsive genes, before and after gamma-irradiation in p53 wild-type (p53+/+) and p53-deficient (p53-/-) mice. The waf1, bax, fas and mdm2 genes were chosen for their different potential roles in the cellular response to stress. Our data demonstrate the strict p53-dependence of mRNA up-regulation for bax, fas and mdm2 in irradiated tissues and confirm such findings for waf1. They further highlight complex levels of regulatory mechanisms that could lead, in vivo, to selective transcriptional activation of genes by p53. In addition, our results provide arguments for the involvement of p53 in the basal mRNA expression of the four genes in some organs. Finally, in situ expression of Bax and p21Waf-1 protein suggests, at least in lymphoid organs, a direct correlation between selective p53-target gene expression and a particular response of a cell to ionising radiation.

Long-term persistent infection of domestic rabbits by the human foamy virus.

Human foamy virus (HFV) belongs to the spumaretrovirus group of the Retroviridae taxonomic family. Attempts to associate HFV or other foamy viruses to a specific pathology still remain unsuccessful. However, viral gene expression as well as tissue-specific tropism in an in vivo context remain poorly analyzed. To address this issue, we have infected domestic rabbits with a single dose of HFV and followed them at the biological and molecular levels for 5 years. No apparent pathology was detectable in the infected animals which have developed a strong immunological response against major viral proteins. We found that HFV provirus in blood cells and several organs persisted predominantly in its defective form, delta HFV, suggesting that in vivo viral persistence could be related to homologous interference as was recently shown in vitro. This animal model might be useful for studying the in vivo targets of HFV and should also be convenient for testing therapeutic effects of antiretroviral drugs.

Inhibition of the in vitro infectivity and cytopathic effect of human foamy virus by dideoxynucleosides.

Human foamy virus (HFV) is a human retrovirus that has not been clearly associated with human disease. In this study, we tested the capacity of nucleoside derivatives to inhibit the infectivity and cytopathic effect of HFV in T-lymphoblastoid cells in vitro. H9 cells showed a dramatic cytopathic effect 3 weeks after exposure to HFV. At this time, viral infection was demonstrated by detection of viral antigens by immunofluorescence staining, release of reverse transcriptase activity (RT) in the supernatant, detection of typical viral particles by electron microscopy, and presence of proviral DNA by Southern blot analysis. H9 cells were pretreated with dideoxycytidine (ddC), dideoxyinosine (ddI), or azidothymidine (AZT) at various concentrations before HFV infection. ddC could not completely suppress viral replication at low concentrations, and inhibited cell proliferation at higher concentrations. ddI partially inhibited the formation of giant cells at 10 microM, with 95% inhibition of RT in the supernatant. AZT induced a complete inhibition of cytopathic effect at concentrations > or = 1 microM, with more than 95% inhibition of RT in the supernatant. Moreover, the synthesis of proviral DNA was completely suppressed by 10 microM AZT. These results show that AZT and ddI can inhibit HFV replication in vitro.

In vitro studies on interferon-inducing capacity and sensitivity to IFN of human foamy virus.

We demonstrate in this article that human foamy virus (HFV) fails to induce interferon (IFN) production in two different human tissue culture cell lines: U373-MG and AV3. We also show the effect of human alpha-, beta- and gamma IFN on the multiplication cycle of HFV. Treatment of cells with 100 IU/ml of any IFN led to strong inhibition of an HFV-induced cytopathic effect. This effect was associated with a significant diminution of reverse transcriptase activity in supernatant fluids of IFN-treated infected cultures, and a substantial decrease in viral particle production, as detected by electron microscopy. All these effects were accompanied by strong inhibition of both viral proteins and RNA synthesis, as well as almost total disappearance of free and integrated proviral DNA. In light of our data, human IFN action on HFV seems to be mediated by a mechanism which differs from that observed in the case of other retroviruses (type C and D for instance); however, it evokes that described for HIV.

Effects of serotonin and melanin on in vitro HIV-1 infection.

In this article, we describe the effect of indoleamines: serotonin (5-HT) and synthetic soluble melanin, on the multiplication of HIV-1 in T4 lymphocytic cell lines. The results show that viral production is increased when infected CEM-11 cells are incubated with 5-HT (10(-7) M and 10(-8) M) for 72 hours, whereas at higher doses (10(-3) M and 10(-4) M), there is an inhibition of viral multiplication. As well, when infected CEM cells were cultured in the presence of 5-HT at 10(-4) M, during 15 days, virus production, syncytia formation and cytolytic effect were drastically inhibited. Melanin also inhibits HIV-1 cytopathic effect on MT-2 cells, without cell toxicity, at concentrations of 0.2-10 micrograms/ml. Syncytium formation and cell lysis were also blocked by melanin at concentrations of 0.1 to 10 micrograms/ml, when uninfected MT-2 cells were mixed with HIV-1 chronically infected CEM-11 cells.

[Role of retroviruses in human cancerogenesis]. / Rôle des rétrovirus dans la cancérogenèse humaine.

Retroviruses are viral agents which are natural and experimental inductors of leukemias and solid tumors among numerous animal species. In man, they are implicated as ethiological agents of a specific type of leukemia, adult-T cell leukemia. Thus is for retrovirus HTLV-I. Another retrovirus, HIV, implicated in AIDS is capable of leading to the formation of several types of opportunistic tumors, such as non Hodgkin lymphomas and Kaposi's sarcoma.

Transient immunosuppressive effect induced in rabbits and mice by the human spumaretrovirus prototype HFV (human foamy virus).

Spumaviruses (foamy viruses) constitute one of the three retroviral genera isolated from man. Although spumaviruses have not been clearly linked to a given pathology in humans and other infected species, it is well established that they lead in vivo to chronic infections without detectable viral expression. We thought it of interest to investigate certain aspects of the pathology induced in laboratory animals by human foamy virus (HFV). In this work, we demonstrate that HFV infection of rabbits and mice gives rise to a transient immunosuppressive effect, as evaluated in vitro by lymphocyte transformation tests. This phenomenon occurs shortly after viral inoculation, at around 4-5 days, and regresses within thirty days.

Comparative studies on IL-6 production status in HTLV-1 chronically infected cell lines derived from different HTLV-1 associated pathologies.

We have comparatively studied the qualitative and quantitative status of Interleukin 6 (IL-6) production in several HTLV-1 chronically infected T-cell lines derived from patients suffering either of adult-T-cell leukemia/lymphoma or of tropical spastic paraparesis (TSP). Two other HTLV-1 chronically infected cell lines, coming from healthy seropositive carriers, were also analyzed. Our results demonstrate that, independently of the origin, all these T cell lines, as compared to uninfected HTLV-1 T cell ones, which were always IL-6 non producers, synthesize and yield comparable amounts of IL-6 synthesis, he nosological origin of infecting viral strains does not seem to play a differential role on IL-6 qualitative or quantitative production parameters.

Human foamy virus polypeptides: identification of env and bel gene products.

Human foamy virus (HFV) proteins were identified in human cells cultured in vitro by immunoprecipitation and immunoblotting with specific antisera. Among several viral polypeptides, four glycoproteins of approximately 160, 130, 70, and 48 kDa were identified in HFV-infected cells. gp130 was shown to represent the intracellular env precursor, and gp70 and gp48 were shown to represent the external and transmembrane env proteins, respectively. The nature of gp160, which shares sequences with the env, bel1, and bel2 proteins, is not yet resolved. In addition, a p62 identified with bel1- and bel2-specific antisera likely corresponds to the bet gene product.

Human spumaretrovirus-related sequences in the DNA of leukocytes from patients with Graves disease.

Viruses, and more particularly retroviruses, have been postulated to play a role in the pathogenesis of autoimmune diseases. In a search for spumaretrovirus infection markers, we screened a group of 29 patients with Graves disease and a representative healthy population (23 subjects) as a control. Southern blot hybridization under stringent conditions, of patients' DNA extracted from peripheral blood lymphocytes, with a spumaretrovirus-specific genomic probe derived from the human spumaretrovirus (HSRV) prototype, gave a positive signal in 10 cases. Moreover, by PCR, HSRV-related sequences were detected in the DNA of 19 patients (66%). Positive DNA samples in Southern blots were also positive in PCR for all regions tested (gag, bel1, bel2, long terminal repeat). Amplified (gag and bel2) products were cloned and sequenced; they showed high homology with HSRV. On the other hand, all 23 control subjects were negative by both procedures. Sera from both populations were examined for the presence of antibodies reactive with antigens of the spumaretrovirus family. These sera were negative by several immunodetection techniques: ELISA, indirect immunofluorescence, serum neutralization, and Western blotting. These results strongly suggest the existence of an association between Graves disease and the presence of HSRV-related infection markers.

Circulating interferon in cytomegalovirus infected bone-marrow-transplant recipients and in infants with congenital cytomegalovirus disease.

In a study concerning five CMV-infected bone-marrow-transplant recipients, five congenital CMV diseases and appropriate controls, presence of high levels of circulating interferon (IFN) was demonstrated exclusively during the course of CMV disease. This interferon was predominantly "immune" or gamma interferon (gamma-IFN). These results suggest that during CMV disease the interferon compartment of the immune response is modified.

Further characterization of the gapped DNA intermediates of human spumavirus: evidence for a dual initiation of plus-strand DNA synthesis.

We recently reported the presence of linear duplex DNA intermediates with a gap in the middle of the molecules in the replicative cycle of human (HSRV) and simian (SFV1) spumaviruses. The polypurine tract (PPT), at the 5' boundary of the 3' long terminal repeat, was found to be duplicated in the gap region. By molecular analysis of HSRV proviral DNA with region- and strand-specific probes, we have now determined that the gap is located on plus-strand DNA and that it is 120 bases long with the 3' end mapping at the duplicated PPT site. Kinetic analysis of proviral DNA provided evidence that the gap did not result from processing of a complete, full-length DNA molecule. These data strongly suggest that plus-strand DNA synthesis is initiated at both PPT sites.

Replication of HIV-1 in a wide variety of animal cells following phenotypic mixing with murine retroviruses.

Human T cells co-infected with the human immunodeficiency virus type 1 (HIV-1) and the xenotropic or dual-tropic mouse type C virus (MuLV) give rise, by phenotypic mixing, to progeny virus that can transfer HIV-1 into a wide variety of mammalian and avian cells. Differences in the extent of HIV-1 replication in these animal cells can be observed. Replication is best in human cells, but occurs substantially in cells from many animal species including mink, horse, and bush wallaby. Virus production in murine and avian cells is very limited. These results confirm that the major block to HIV-1 infection of animal cells is at the cellular surface but that intracellular regulation of viral replication is also involved. Moreover, an enhancement of HIV-1 cytopathic effects can be seen in human cells co-infected by MuLV. All these data suggest phenotypically mixed viruses might be useful for developing an animal model system for studying AIDS, and that the pathological expression of HIV-1 could be modified by the presence in cells of other retroviruses. They also indicate a potential mechanism by which HIV strains can be generated with an increased ability to spread in nature.

Effects of human recombinant alpha and gamma and of highly purified natural beta interferons on simian Spumavirinae prototype (simian foamy virus 1) multiplication in human cells.

The present study demonstrates the inhibitory effect of human recombinant interferons (r-Hu-IFN) alpha and gamma, and that of highly purified natural human interferon beta on the replication of simian foamy virus type 1 (SFV1) in human AV3-cell cultures. All IFN led to strong inhibition of the SFV1 cytopathic effect. Electron microscopy showed a 70 to 95% decrease in viral particles. Significant inhibition of virus-associated reverse transcriptase activity was found in supernatant fluids of infected IFN-treated cultures. Metabolic labelling of the virus confirmed the inhibition of extracellular release of SFV1. PAGE analysis of immunoprecipitates indicated a reduction in viral-specific protein bands. Altogether, these results indicate that the mechanism of inhibition of Spumavirinae infection by interferon differs from that described for the other Retroviridae, and particularly for types B, C and D viruses. Our data is of therapeutic interest since Spumavirinae have been linked to pathological processes such as de Quervain thyroiditis.

In vitro induction of early mouse embryo intracisternal particles (epsilon particles) in cultured cell lines.

The experimental induction of epsilon particles, retrovirus-like structures corresponding to the small IA particles of the mouse, was studied by electron microscopy in rodent-cultured cell lines. Among the chemicals tested, only IdUr was shown to be an effective inducer, but not cycloheximide, puromycin , deoxy-fluorouracil or 5-azacytidine. However, only two mouse-derived cell lines: Ki-BALB and FG 10, among 27 cell lines of mouse, rat and mink origins tested, expressed epsilon particles upon IdUr treatment. Epsilon particles thus respond to chemical inducers very differently in comparison with large IAP. Moreover, the addition of interferon previously shown to attenuate IAP production, had no effect on that of epsilon particles.

Effect of recombinant human alpha A interferon on Visna virus multiplication in acutely infected ovine cells.

We have studied the effects of recombinant human alpha A interferon (IFN) on the multiplication of Visna retrovirus in ovine cells. Pretreatment with IFN followed by continuous IFN treatment, drastically blocked the Visna virus induced cytopathogenic effect and the emergency of neoformed virions. Thus, virus yield and virus dependent reverse transcriptase activity were highly reduced in supernatant fluids. All these effects were accompanied by a strong inhibition of viral protein synthesis. In the light of these data, human IFN action on Visna retrovirus seems to be determined by a mechanism of action distinct from that described in the case of other Retroviridae.

Electron microscopic characterisation of retrovirus and retrovirus-like particles induced by demethylating agents (5-azacytidine and 5-azadeoxycytidine) in Syrian hamster (Mesocricetus auratus) cells.

In a framework of a study on retrovirus expression in Syrian hamster (mesocricetus auratus), three cell lines were examined by electron microscopy. As we had previously demonstrated the efficacy of demethylating drug in activating the formation of retroviral particles, we used 5-azacytidine to activate endogenous retrovirus expression. Proceeding to a more detailed survey we were able to observe various types of retrovirions. All of them were present in the different activated cell lines. They were: intracisternal R-type particles (IRP), intracisternal A-type particles (IAP), intracytoplasmic A-type particle (ICAP), intramitochondrial A-type particle (IMAP) and M-432 virus-like particles. Some of these particles have never been described before in Syrian hamster cells. The significance of their presence is discussed here.