Human foamy virus capsid formation requires an interaction domain in the N terminus of Gag.

Retroviral Gag expression is sufficient for capsid assembly, which occurs through interaction between distinct Gag domains. Human foamy virus (HFV) capsids assemble within the cytoplasm, although their budding, which mainly occurs in the endoplasmic reticulum, requires the presence of homologous Env. Yet little is known about the molecular basis of HFV Gag precursor assembly. Using fusions between HFV Gag and a nuclear reporter protein, we have identified a strong interaction domain in the N terminus of HFV Gag which is predicted to contain a conserved coiled-coil motif. Deletion within this region in an HFV provirus abolishes viral production through inhibition of capsid assembly.

Isolation and characterization of an equine foamy virus.

Foamy viruses (FVs) are complex retroviruses which have been isolated from different animal species including nonhuman primates, cattle, and cats. Here, we report the isolation and characterization of a new FV isolated from blood samples of horses. Similar to other FVs, the equine foamy virus (EFV) exhibits a highly characteristic ultrastructure and induces syncytium formation and subsequent cell lysis on a large number of cell lines. Molecular cloning of EFV reveals that the general organization is that of other known FVs, whereas sequence similarity with its bovine FV counterpart is only 40%. Interestingly, EFV buds exclusively from the plasma membrane and not from the endoplasmic reticulum (ER), as previously shown for other FVs. The absence of the ER retrieval dilysine motif in EFV Env is likely responsible for this unexpected sorting pathway.

Effects of the binding of a dextran derivative on fibroblast growth factor 2: secondary structure and receptor-binding studies.

CMDB (carboxymethyldextran-benzylamide) are dextrans statistically substituted with carboxymethyl and benzylamide groups which can mimick some of the biological properties of heparin. It has previously been shown that CMDB inhibit autocrine growth of breast tumor cells (Bagheri-Yarmand et al., Biochem. Biophys. Res. Commun. 239: 424-428, 1997) and selectively displace fibroblast growth factor 2 (FGF-2) from its receptor. Here, we used circular dichroism and fluorescence anisotropy measurements to show that the conformation of FGF-2 was significantly altered upon its binding to CMDB and to short CMDB fragments prepared within this study. CMDB and fragments formed a stable 1:1 complex with FGF-2, with affinities being estimated as 20+/-10 nM from fluorescence anisotropy analysis. No such a complex was formed with insulin-like growth factor (IGF-1) or epidermal growth factor (EGF). CMDB competed with the FGF-2 receptor for binding to FGF-2 but did not disturb the binding of IGF-1 and EGF to their receptors. Thus, our results highlight the selectivity of CMDB and their fragments towards FGF-2. Heparin, however, competes with CMDB and their fragments for binding to FGF-2. The carboxymethyl and benzylamide groups of these molecules likely interact directly with a heparin-binding region of FGF-2. The resulting change in conformation disturbs the binding of FGF-2 to its receptor and consecutively its mitogenic activity.

Low-molecular-weight dextran derivatives (f-CMDB) enter the nucleus and are better cell-growth inhibitors compared with parent CMDB polymers.

Carboxymethyldextrans-benzylamide (CMDB) are dextran derivatives that are statistically substituted with carboxymethyl and benzylamide groups. These molecules display a variety of biological effects, one of which is their inhibitory activity against mammary tumor cell growth, both in vitro and in vivo. We and others have previously shown that the effects of CMDB on cell growth are related to their ability to interact with the growth factor FGF-2. The binding modifies the conformation of FGF-2, leading to the suppression of its mitogenic activity. Here, the method previously reported to fragment natural polysaccharide fucans has been applied to CMDB (80,000 g/mol). f-CMDB (fragmented CMDB) of molecular weights from 6000 to 20,000 g/mol were found to be more potent inhibitors of MCF7 mammary tumor cell growth than high-molecular-weight CMDB. Confocal microscopy experiments using CMDB and f-CMDB labeled with the fluorophore DTAF (5-([4,6-dichlorotriazine-2-yl]amino) fluorescein) indicate that only low-molecular-weight f-CMDB penetrate into the nucleus of MCF7 cells. It is thus assumed that the better inhibitory properties demonstrated by f-CMDB, compared with CMDB, are related to their better ability to penetrate the nucleus and interact with nuclear targets, including topoisomerase II. The DNA relaxation properties of the latter are inhibited in vitro by both CMDB and f-CMDB. These findings could help us to develop models of low-molecular-weight oligosaccharide derivatives exhibiting better antiproliferative and antitumor properties.

Isolation and characterization of infectious full-length DNA clones of chimpanzee foamy viruses SFV6 and SFV7: evidence for a Taf-dependent internal promoter.

We have cloned complete viral genomes directly from Hirt supernatant DNAs of simian foamy virus types 6 and 7 (SFV6 and SFV7) -infected cells. These clones were shown to be infectious by transfection into cells and subsequent infection of susceptible cells either by cocultivation or by passage of cell-free supernatants. The presence of virus particles, suggested by a typical cytopathic effect, was confirmed by electron microscopy. These viruses were characterized at different levels of the replication cycle. The proviral genomes revealed a taf deletion comparable to that previously described in the human foamy virus (HFV) bel1 gene. Analysis of viral RNAs revealed similar patterns of transcripts for SFV6- and SFV7-infected cells, with predominant expression of accessory genes. Characteristic major viral polypeptides were identified by radioimmunoprecipitation for both isolates. Sequences homologous to the gene encoding Taf and to a potential internal promoter were identified in the infectious clones and subcloned into expression vectors. Their functional properties were tested by transfection assays, which provided evidence for the presence of a Taf-dependent internal promoter in both SFV6 and SFV7 isolates.

Hairpins in a DNA site for topoisomerase II studied by 1H- and 31P-NMR.

1H- and 31P-NMR and UV-absorption studies were carried out with the oligonucleotide strands d(AGCT-TATC-ATC-GATAAGCT) (-ATC-) and d(AGCTTATC-GAT-GATAAGCT) (-GAT-) contained in the strongest and salt resistant cleavage site for topoisomerase II in pBR322 DNA. We found that the two oligonucleotides were stabilized under a hairpin structure characterized by a eight base pair stem and a three base loop at low DNA and salt concentrations. In such experimental conditions, only the -GAT- oligonucleotide displayed a partial homoduplex structure in slow equilibrium with its folded structure. Temperature dependencies of imino protons showed that the partial homoduplex of -GAT- melted at a lower temperature than the hairpin structure. It was suggested that the appearance of the partial homoduplex in -GAT- is related to the formation of two stabilizing (G.T) mismatched base pairs in the central loop of this structure. Finally, it was inferred from the dispersion of chemical shifts in the 31P-NMR spectra that the distortions affecting the backbone of the hairpin loop are larger in the case of -ATC- compared with -GAT-. At the same time NOEs proved that the base stacking was stronger within the loop of the -ATC- hairpin.

Biological and molecular studies on Syrian hamster intracisternal R-type particles.

Retrovirus-like intracisternal R-type particles (IRP) are structures present in Syrian hamster (Mesocricetus auratus) cells cultured in vitro where they appear either spontaneously or under chemical induction conditions. We have tested several chemical inducers and ten different cell lines, looking for the best IRP induction conditions. BHK21 cl. 13 showed the highest inducibility one day after a 24 h treatment with 1 microgram/ml of 5-aza-2'-deoxycytidine. Using detergent treatments and sucrose gradients, we obtained semi-purified IRP cores. A 7.2 kb RNA associated with the core fraction was revealed by hybridization with total Syrian hamster genomic DNA, but not with Syrian hamster intracisternal A particle (IAP) specific probes. This suggests that the IRP genes are distinct from IAP ones.

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.

2,4-Dimethyl-6 H-pyrido[3,2-b]carbazole, an isomer of the antitumor alkaloid ellipticine via the Combes-Beyer reaction.

The synthesis of 2,4-dimethyl-6 H-pyrido[3,2-b]carbazole (2a) and of its 9-methoxy (2b) and 9-hydroxy (2c) derivatives via the Combes-Beyer reaction is described. Pyridocarbazole 2a has been obtained by two different routes. In the course of the synthesis of 2b either a partial or a total demethoxylation has been observed depending on the route employed.

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.

Effect of distortions in the phosphate backbone conformation of six related octanucleotide duplexes on CD and 31P NMR spectra.

We examined the structural properties of six octanucleotide duplexes d(TGACGTCA), d(ACTGCAGT), d(CTTCGAAG), d(CATCGATG), d(GTACGTAC), and d(CATGCATG). Circular dichroism (CD) and 2D 31P and 1H NMR spectroscopies were used in conjunction. Although of the B-DNA type, it was possible to arrange CD spectra into two families, A and B. Family A resembled poly(dG-dC) with a positive signal at approximately 280 nm and a negative one at approximately 260 nm, while family B resembled poly(dA-dT) with a positive signal at approximately 270 nm and a negative one at approximately 250 nm. All 31P resonances were assigned through constant-time heteronuclear 31P-1H correlated spectra. J(H3'-P) coupling constants related to dihedral angeles epsilon (C4'-C3'-O3'-P) were determined from 1H-31P J-resolved selective proton-flip 2D experiments. A good correlation was observed between 31P chemical shifts and coupling constants for all oligonucleotides. The patterns of these two parameters vs the base position along the sequences were almost similar. They were confronted with CD spectra. The results indicated that the position and magnitude of the signals were mainly affected by the CpG and ApT steps whose 31P chemical shifts were the farthest away from the mean 31P chemical shift value. This is in keeping with greater rigidity at these steps and should explain the influence of the local order on the shape of the CD spectra. Lastly, both UV absorption and 31P chemical shifts vs temperature provided normal temperature melting (Tm) values for all of the octanucleotide duplexes except for d(CTTCGAAG), for which the Tm was approximately 10 degrees C lower compared to its counterpart d(CATCGATG). The decrease in the thermal stability of this octanucleotide duplex was imputed to its contained TT and AA repeats, which might be able to induce correlated base destacking and phosphate group distortion in the oligonucleotide and especially on the intermediate CpG. We demonstrate that the CpG step displayed 31P NMR properties similar to those found in mismatched nucleotides exclusively in the d(CTTCGAAG) duplex.

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.

[Large-scale production of amastigotes by a human monoblastoid cell line]. / Production massive d'amastigotes par une lignée de cellules monoblastoides humanines.

In this study, a human monoblastoid cell line (TPH-1) was tested in vitro for the production of Leishmania amastigotes. The number of TPH1 cells increased with time and 6 days after promastigote infection the percentage of infected cells was around 45%. Pre-treatment of TPH1 cells with retinoic acid induced the cells to differentiate into unreplicating macrophage-like cells. Ninety per cent was parasitized 6 days after promastigote infection; the number of amastigotes quintuplied during this period of time; this result was irrespective of the Leishmania species used for experiments. Viable and infective parasites were obtained from treated and nontreated cells. TPH1 cells merit further consideration for research concerning new molecules active against Leishmania.

[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.

[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.