RNA viraux des cellules infectées par un retrovirus murin : (II) influence de la toyocamycine sur le métabolisme des RNA viraux

Abstract

Nous avons précédemment montré que la Toyocamycine (analogue de l'adénosine), ajotée à des cultures de cellules produisant le virus de Friend provoque une diminution de la production virale et une perte de l'infectiosité. On observe également que le RNA 60–70 S des virions incorpore de la Toyocamycine. Dans ce travail, nous avons examiné l'état des RNA viraux intracellulaires. 16 heures après le début d'un traitement par une dose cytostatique de Toyocamycine (0.2 g/ml), on trouve par hybridation moléculaire que les cellules contiennent les 3/4 des RNA viraux présents dans les cellules non traitées. Ces RNA se répartissent en trois espèces majeures 70S, 35S et 20–24S. Lorsque les hybrides cDNA-RNA sont analysés sur une colonne d'hydroxyapatite, le cDNA viral est hybridé à 100 p. cent avec les RNA contenant la Toyocamycine. Par contre, l'analyse des mêmes hybrides par la nucléase S 1 montre que 90 p. cent du même cDNA est protégé par des concentrations équivalentes de RNA. Cette différence entre les deux méthodes d'hybridation suggère que la Toyocamycine déstabilise localement les régions où elle a été incorporée et les rend sensibles à la nucléase S 1 . Enfin, la teneur en Poly (A) des RNA viraux n'est pas significativement modifiée. Tous ces faits indiquent que l'inactivation des virus résults de l'incorporation de la Toyocamycine dans de RNA viral. The adenosine analogue Toyocamycin (4 amino-5-cyano 7 B-D ribofuranosyl-pyrrolo 2-3-d pyrimidine) is known to incorporate into the RNA species of mammalian cells. However, structural changes owing to the incorporation of the drug prevent the maturation of preribosomal 45S RNA precursor into 28 and 18S ribosomal RNA. The drug does not impede the transcription of other cellular RNA species such as transfer RNA, 5S ribosomal RNA, other small RNA species and does not prevent occurence of rapidly labeled RNA in cytoplasmic polysomes. We have previously shown that Toyocamycin depresses the production of a murine retrovirus by a chronically infected cell line (Eveline cells producing the Friend virus complex). Although possessing a 70S RNA and an exogenous reverse transcriptase activity the viruses released under Toyocamycin treatment are not infectious and lack endogenous reverse transcriptase activity. This can be explained by the incorporation of Toyocamycin in the viral RNA, resulting in alteration of transcriptional and translational capacities. In this work we examined the effect of Toyocamycin on intracellular viral RNA synthesis. Molecular hybridization techniques using a cDNA probe prepared with purified murine virus revealed that after treatment for 16 hours with a cytostatic concentration of Toyocamycin (0,2 g/ml), cells were still containing 75 per cent of the viral RNA present in untreated cells. Hybrids between the cellular RNA species and viral cDNA were analysed by chromatography on hydroxyapatite or by S 1 nuclease treatment. It was observed that the cDNA was totally hybridized when the first method was used, whereas 90 per cent only were protected against digestion by S 1 nuclease. The difference observed between the two methods suggests that the hybrid regions containing the analogue are somewhat destabilized and become more sensitive to the S 1 nuclease action. The intracellular viral RNA species are distributed into three main classes sedimenting at 70S, 35S and 20-24S. This profile does not show great differences with that of untreated infected cells. It is assumed that the 70S RNA comes from viruses budding at the plasmic membrane and from intracellular vacuolar particles. When the Toyocamycin-containing RNA is heated at 65°C before loading onto the sucrose gradient, most of the hybrids formed with viral cDNA are located in a peak sedimenting at 20S–24S. This result can be compared to the one observed with the 70S RNA isolated from extracellular virions synthezised in the presence of Toyocamycin, as it is dissociated after heating into 35S and 20–24 S species. It is not yet clearly established whether the 20–24S RNA detected by hybridization with the viral cDNA is a subgenomic fraction of viral RNA rather than a degradation product of 35S due to the incorporation of toyocamycin. Further experiments are currently performed to test the translational capacities of these Toyocamycin-containing viral RNA.