HTLV-1-like particles and HTLV-1-related DNA sequences in an unambiguous case of Sèzary syndrome.

An unambiguous case of Sèzary syndrome associated with the presence of unusual retroviral infection markers is described. The blood smear showed 15% typical Sèzary cells but also rare atypical lymphocytes with convoluted nuclei, evocative of characteristic adult T-cell leukemia (ATL) flower cells. However, the patient did not present any clinical or biological manifestations of ATL, and human T-cell leukemia virus type 1 (HTLV-1) serology was consistently negative. After being cultured for 4 months, peripheral blood mononuclear cells (PBMC) produced typical type C retrovirus-like particles with budding forms strongly resembling HTLV-1 virions. The producer cells did not express HTLV-1-specific antigens detectable by indirect immunofluorescence (IIF). Southern blotting of uncultured PBMC DNA, submitted to digestion with the restriction enzymes PstI and SacI, and hybridized with a full genomic HTLV-1 probe, showed the presence of specific homologous sequences, absent in all of the healthy donor control PBMC DNAs. These HTLV-1-like sequences presented a restriction enzyme pattern distinct from that of the HTLV-1 prototype genome and of other HTLV-1 proviruses studied up to now. Polymerase chain reaction (PCR) with highly conserved HTLV-1 derived pol and env primers was consistently negative with the patient's DNA. All these results taken together suggest that our patient carries a retroviral agent partially homologous to, but probably different from HTLV-1. The possibility is discussed that this type of retroviral agent might be associated with a subtype of cutaneous T-cell lymphoma (CTCL) represented by a typical Sèzary syndrome with a very low percentage of ATL-like flower cells in the blood smear.

No evidence for HTLV-I infection in 24 cases of French and Portuguese mycosis fungoïdes and Sezary syndrome (as seen in France).

A survey in search of evidence for HTLV-I infection was conducted on French and Portuguese patients residing in France with a diagnosis of mycosis fungoïdes or Sezary syndrome. Methods used in this investigation included serological assays (ELISA, Western blot, particle agglutination, indirect immunofluorescence) and DNA molecular studies (Southern blot and polymerase chain reaction). Cultures of peripheral blood mononuclear cells were performed and checked by electron microscopy and reverse transcriptase assay. The results indicate that neither HTLV-I nor a closely related retrovirus are associated with mycosis fungoïde or Sezary syndrome in the cases studied.

Chronic myelopathies associated with human T-lymphotropic virus type I. A clinical, serologic, and immunovirologic study of ten patients in France.

Chronic myelopathy associated with human T-lymphotropic virus type I (HTLV-I) has been described in HTLV-I endemic areas. In Paris, 167 neurologic patients were screened for HTLV-I by enzyme-linked immunosorbent, indirect immunofluorescent, and Western blot assays. Ten of the 11 patients with positive results had a chronic spastic paraparesis with IgG oligoclonal bands and elevated HTLV-I antibody index. Two of them had been born and were living in France, without HTLV-I risk factors. Evoked potentials were abnormal in the nine tested patients and brain magnetic resonance images in three of seven patients. No improvement was observed with steroid treatment. A retrovirus similar to HTLV-I was isolated in five cases at different periods of the disease. Hypotheses of limited endemic areas in western countries are discussed. Early presence and persistence of HTLV-I suggest that it is the etiologic agent.

Paradoxical effects of recombinant mouse beta-interferon on different expression levels of intracisternal A particle genes.

We have studied the effect of mouse recombinant beta-Interferon on the expression of the intracisternal A particle (IAP) genes in a transformed mouse fibroblast cell line. Northern and immunoprecipitation analysis showed an increase in specific transcripts and polypeptides. Run-on experiments on isolated nuclei and transfection assays with an IAP long terminal repeat construct, coupled with the chloramphenicol acetyl transferase gene, showed no enhancement of the transcription rate following beta-IFN treatment. This suggests that the increase in IAP expression depends on a post-transcriptional event. Electron microscopic analysis revealed that in spite of the higher levels of IAP related polypeptides, the number of particles was significantly reduced by interferon.

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.

Study of a human heteroploid cell line cryptically infected by a murine oncornavirus.

This report concerns the establishment of a human cell line cryptically infected by a murine leukemia virus (MuLV). This cell line, named J112, is characterized by the presence of genetic viral information in a majority of cells as demonstrated by immunological and virological methods, and by the absence of detectable viral activity in the supernatant culture fluids. Nevertheless, electron microscopy demonstrated the presence of a small number of particles which have the morphology of immature type C particles. We discuss the importance of this experimental model in relationship with non-producing (NP) infections of human cells by oncogenic retroviruses.

[Effect of 5-azacytidine on the expression of intracisternal A and epsilon particles in various mouse cell lines]. / Action de la 5-azacytidine sur l'expression des particules intracisternales A et epsilon dans différentes lignées cellulaires de Souris.

5-Azacytidine treatment of several Mouse cell lines cultured in vitro induces the synthesis of a high number of intracisternal A particles. This induction could be related to the demethylation effect of the 5-Azacytidine. On the contrary, the epsilon intracisternal particles are not activated by 5-Azacytidine.

Effect of human interferon on type D retroviruses multiplication in chronically infected cell lines.

A study was made on the effect of human interferon (HuIFN) on the synthesis and release of type D viruses (MPMV and SMRV) by chronically infected human lines. Interferon at concentrations of 50 IU/ml lead to an inhibition of the extracellular virus production by about 50%-80% as measured by virus-associated reverse transcriptase activity, by metabolic labeling of the virus with (3H) uridine or (3H) amino acids, or by electron microscopy counting of particles. The profiles of intracellular viral proteins, as visualized by radioimmunoprecipitation and electrophoresis, were not different in control or IFN-treated cells and the yield of intracellular protein p27 stayed unchanged or was slightly increased. Electron microscopy of thin sections taken from the control and IFN-treated cells revealed no difference in any subcellular structures including that of the viruses. However, electron microscopic examination of IFN-treated cells showed an increased number of either budding particles and intracytoplasmic precursors (two- to five-fold) or mature virus particles (four- to ten-fold) on the cell surface of the IFN-treated cells. These results indicate that IFN inhibits further developments of MPMV and SMRV replication as was described for type C and B retroviruses.

Unintegrated HTLV-I proviral DNA in cell lines and uncultured peripheral blood mononuclear cells from tropical spastic paraparesis (TSP/HAM) and adult T-cell leukemia (ATL) patients.

Ultrastructural studies on cell cultures derived from TSP/HAM and ATL patients, show the presence of large quantities of HTLV-I viral particles in extracellular spaces and budding at the cytoplasmic membrane. In addition, mature enveloped particles and images of endopinocytosis of virions are seen in the cytoplasm vacuoles suggesting the existence of a reinfection phenomenon. In this context, we decided to investigate some features of the replicative cycle, in particular the synthesis of unintegrated proviral forms. To increase the sensitivity of detection, we applied a procedure which combines the electrophoretic separation of closed circular forms and PCR amplification. By this procedure we produced evidence for the existence of supercoiled HTLV-I DNA in established cell lines from TSP/HAM and ATL and in patients peripheral blood mononuclear cells. These HTLV-I unintegrated proviral forms may play an important role in the physiopathology of HTLV-I associated diseases. Preliminary results of AZT/interferon treatment in ALT patients are largely superior to chemotherapy. The therapeutic effect of AZT, it known inhibitor of reverse transcriptase, may be through its inhibition of the synthesis of HTLV-I unintegrated proviral DNA.

Isolation and characterization of a new simian foamy virus serotype from lymphocytes of a Papio cynocephalus baboon.

A new type of syncytium-forming (foamy) virus was isolated from peripheral lymphocytes of a Papio cynocephalus baboon after cocultivation with a rhesus monkey cell line. The isolate was characterized by cytopathogenic effect, host range, electron microscopy, reverse transcriptase activity and physical and chemical properties. Seroneutralization tests indicate that the new isolate is antigenically distinct from the other nine known simian foamy virus serotypes.

Simultaneous activation by 5-azacytidine of intracisternal R particles and murine intracisternal-A particle related sequences in Syrian hamster cells.

5-Azacytidine treatment of Syrian hamster (Mesocricetus auratus) cells, BHK21-cl.13, and primary embryo fibroblasts, activates the production of intracisternal type R particles (IRP) as ascertained by electron microscopy scanning and counting. Efficiency of the activation is dose- and time of treatment-dependent. The transcription of mouse intracisternal A particle (IAP) related sequences, which have been described in Syrian hamster genomic DNA, is increased at the same time. This correlates with demethylation of hamster IAP related genes, as shown by restriction of high molecular weight DNA from 5-azacytidine treated cells with methylation sensitive enzymes, and subsequent electrophoresis and molecular hybridization of the fragments with specific DNA probes. In 5-azacytidine treated cells derived from other hamster species (Cricetulus griseus and C. migratorius), intracisternal R particles were not induced, nor were mouse IAP-related RNAs detected.

Studies on in vitro interferon induction capacity and interferon sensitivity of simian foamy viruses.

We demonstrate that Simian Foamy viruses (SFV) types 1, 2, 4 and 10 do not induce Interferon (IFN) production in mouse and primate (simian and human) cell lines, but that their cytopathogenic effect is blocked by this viral inhibitor. The mechanisms of action of IFN seems to be different from that of other Retroviridae. No trapping of virions appears in treated cells examined by ectron microscopy. Moreover, neither precursor nor mature virus particles were observed in infected cultures submitted to IFN treatment.

[C-type virus particles in lymphocytes cultured from cows with persistant lymphocytosis. Preliminary results on French cattle stock]. / Présence de particules virales de type C dans des cultures de lymphocytes de bovins présentant une lymphocytose persistante, résultats préliminaires concernant le cheptel bovin francais

C type virus particles were shown in cultures of lymphocytes originating from cows with persistent lymphocytosis. These observations on French cattle are in complete accordance with data previously obtained in the USA. Viral replication is highly stimulated by the addition of phytohemagglutinin in the culture medium. Several morphological and biological properties, specific to the bovine virus differentiate this virus from C type viruses of other species.

[Attempt at activating mouse intracisternal type A particles by iododeoxyuridine and dexamethasone]. / Essai d'activation des particules intracisternales de type A de la Souris par l'iododéoxyuridine et la dexaméthasone.

In this study we have compared the effects of Iododeoxyridine (IUdR) and dexamethasone on different Mouse cell lines to determine any differences in their actions on intracisternal A-type particles (IAP). Among the nine cell lines studied only four responded to IUdR stimulation by a subsequent augmentation of IAP. No similar activating effect was detected with dexamethasome treatment.

Purification and characterization of simian foamy virus type I structural core polypeptides.

The present study concerns the purification and partial characterization of simian foamy virus type 1 (SFV 1) structural core polypeptides. The obtention of SFV 1 cores separated from envelope components after viral disrupture was verified by electron microscopy (EM), density gradient, and polyacrylamide gel electrophoresis (PAGE). Multistep purification by column chromatography, verified by PAGE, enabled us to separate the structural core polypeptides from the 80,000 molecular weight reverse transcriptase. Two species of structural core polypeptides were identified with apparent molecular weights of 51 and 15 kd. By affinity chromatography on a double-stranded DNA-cellulose column, the main internal protein, p51, was shown to be composed of a major 30 kd protein and a minor 19 kd polypeptide, which binds to double stranded DNA. The p15 internal protein was shown to have a ribonucleotide binding nature.

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.

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.

[Biological and biochemical characterization of a new isolate of baboon Papio papio endogenous type C virus]. / Caractérisation biologique et biochimique d'un nouvel isolat du virus endogène de type C du babouin Papio papio.

A new strain of Baboon Papio papio endogeneous type C virus was isolated from the testis of an animal of this species. The strain was characterized biochemically and biologically. It presents a host-range different from that of the prototype strain of the same virus.

The innate resistance of CBA mice to endogenous murine leukaemia virus infection.

The incidence of lymphomata in CBA mice is low and furthermore is unaltered by transplantation at the early blastocyst stage and being born from the lymphoma-prone AKR. The number of C-type murine leukaemia virus particles in CBA derived in this manner and milk-fostered by AKR mice in no way differs from normal CBA. The results suggest that the oncogenic Gross virus does not pass through either the transplacental or transmammary routes, or alternatively that viral replication in the CBA was in some way inhibited. Both possibilities have still to be distinguished.

Characterization of a human lymphoblastoid cell line permanently modified by simian foamy virus type 10.

Simian Spumavirinae serotype, SFV10, of a Papio cynocephalus baboon, was used to infect a human lymphoblastoid cell line, LV2. Permanent growth and morphological alterations of infected cells occurred, even though no viral particles were detected. Evidence for the presence of viral genomes in the modified cell line is provided indirectly from immunological studies and induction experiments followed by coculture procedures. The permanently modified cell line obtained (LV2-FB10) is an interesting model for the investigation of the possible integration of foamy viruses into the host genomes.