Activation of intracisternal a particles by 5-azacytidine in mouse Ki-BALB cell line.

5-Azacytidine activates the production of intracisternal A particles (IAPs) in mouse Ki-BALB cell line as ascertained by electron microscopy scanning and numeration. Efficiency of the activation is higher than that obtained with iododeoxyuridine. The increase in particle production is concentration and time dependent. The results obtained in our system correlate the high IAP expression after drug treatment with a demethylation of IAP-related genes sequences.

Characterization of RNase H activity associated with reverse transcriptase in simian foamy virus type 1.

Spumavirinae or foamy viruses have been shown to have a characteristic RNA-dependent DNA polymerase activity. We demonstrate here the existence of an RNase H activity that copurifies with the 81-kilodalton monomeric polypeptide, which carries the RNA-dependent DNA polymerase activity of simian foamy virus type 1. RNase H degrades RNA hybrid substrates; however, it does not solubilize single-stranded RNAs. Inactivation assays with heat, high levels of bivalent cations, ethidium bromide, and sodium fluoride suggest that the RNase H catalytic site could be topologically independent from the DNA polymerase catalytic site.

Intracisternal A particles: RNA expression and DNA methylation in murine teratocarcinoma cell lines.

Two main intracisternal-A-particle-specific RNA species (29 to 30S and 35 to 36S) are variably expressed in particle-producing and particle-nonproducing murine teratocarcinoma cell lines. An analysis of DNA methylation patterns after hybridization with an intracisternal-A-particle-specific probe showed an apparent direct correlation between DNA methylation and RNA expression. However, when the methylation assay was performed on the excised intracisternal A particle genes in one of the particle-rich cell lines (PCC6), some undermethylated sequences were detectable. These results are consistent with the concept that only few of the genes are transcriptionally active.

Reverse transcriptase from simian foamy virus serotype 1: purification and characterization.

Chromatography on heparin-Sepharose, known for its affinity for nucleotide-binding polypeptides, was used to purify the viral RNA-dependent DNA polymerase (reverse transcriptase) from the core polypeptides of simian foamy virus type 1. This procedure allowed the recovery of highly purified enzyme with a high specific activity. The average molecular weight of this monomeric enzyme is 81,000 and is thus comparable to that found for other known primate retroviruses. Reverse transcriptase activity of simian foamy virus type 1 requires a ribonucleotide template as a primer or otherwise a DNA with 3'-OH ends. Other optimal conditions of activity are reviewed. Heat inactivation studies led to the concept of an enzyme with two loci, one specific for the substrate and the other for the template-primer.

Murine retrovirus genome directs the synthesis of gag protein precursor early after infection.

Incoming type C retroviral genomic 35S RNA is present in polysomes of undifferentiated and differentiated murine teratocarcinoma cell lines at 4 hours after infection. At the same time a 65,000 daltons viral specific protein is produced by the infected cells. These data present evidence that incoming viral RNA serves as messenger for the synthesis of gag protein precursor Pr65 early in the infectious cycle of ecotropic murine retrovirus.

Studies on the restriction of ecotropic murine retrovirus replication in mouse teratocarcinoma cells.

Retrovirus infection of cultured murine teratocarcinoma cells depends upon the state of differentiation. We have used two cell lines derived from a teratocarcinoma of mouse, strain 129. One, an undifferentiated pluripotential cell line (PCC4), is restrictive to viral infection, while the other, a differentiated myoblast-derived cell line (PCD1), is fully permissive to virus replication. We have shown that no virus RNA expression can be found in PCC4 cells 48 h post-infection and that no nucleic acid sequences can be found in an integrated form in PCC4 cells. However, the kinetics of formation of free proviral intermediates show that the three forms (I, II and III) of free virus DNA are synthesized in both PCC4 and PCD1. Free proviral DNA disappears gradually after 24 h in PCC4 cells while all forms increase in PCD1. These results suggest that the viral multiplication restriction occurs somewhere between the proviral DNA synthesis and integration of DNA in the cellular genome.

[Absence of the chemical activation of type C endogenous viruses in cells of 129/J mice]. / Absence d'activation chimique des cirus endogènes de type C dans les cellules de souris 129/J.

Production of Type C virus by Mouse cells generally activated by chemical agents does not occur in 129/J Mouse cells. No viral production, as tested by reverse transcriptase activity and electron microscopic methods can be detected in cells treated by chemical inducers. Immunofluorescence does not reveal the presence of viral specific proteins and viral RNA expression determined by molecular hybridization between cellular RNA and viral cDNA does not increase as it does in BALB/c cells under the action of the inducers studied.

Biochemical characterization of simian foamy virus type i.

Simian syncitium-forming ("foamy") virus type I (SFV1) was characterized biochemically. RNA was extracted from purified virus either with 0.1 per cent SDS or by the standard phenol-chloroform method. By both techniques a main component of 65-70S was found. Denaturation of the 65-70S RNA by heat resulted in a shift of the sedimentation coefficient mainly to a 30-35S component. Electrophoresis on a composite polyacrylamide gel demonstrated the existence of three minor RNA's: 8S, 5S and 4S respectively. PAGE-SDS analysis of disrupted purified virions enabled the separate migration of five viral proteins and the identification of two main proteins: a 30 kd polypeptide and a 70 kd polypeptide.

Biochemical characterization of endogenous type C virus information in differentiated and undifferentiated murine teratocarcinoma-derived cell lines.

Undifferentiated teratocarcinoma cells express sixfold-higher levels of endogenous xenotropic type C virus-related RNA than differentiated cells. Three species of polyadenylated viral RNA (35S, 24S, and 14S) have been identified in the undifferentiated teratocarcinoma cells. Paradoxically, neither viral particles nor viral proteins have been detected in these cells.

Restricted infectivity of ecotropic type C retroviruses in mouse teratocarcinoma cells: studies on viral DNA intermediates.

Replication of Gross strain N-tropic type C retrovirus was markedly restricted in a pluripotential undifferentiated embryonal cell line (PCC4) of murine teratocarcinoma, whereas the same virus could cause productive infection in a myoblast-derived differentiated line (PCD1) of the same tumor origin. To investigate the restriction mechanism, we compared the initial viral DNA formation in these two cell lines. Analyses by means of a modified Hirt extraction procedure and a modified Southern gel transfer method indicated that PCC4 and PCD1 cells supported the synthesis of viral DNA intermediates after inoculation of the Gross virus. In both cells, a linear DNA duplex (from III viral DNA) appeared at 4 hr, reached a maximal level at 8-9 hr, and declined rapidly thereafter, while two closed-circular supercoiled DNA duplexes (form I viral DNA) showed their appearance, increase and decline in the 8-24 hr period. During the period from 34 to 78 hr after virus inoculation, another burst of viral DNA synthesis occurred in PCD1 cells, presumably due to secondary virus infection, while at this period both form III and form I viral DNAs became undetectable in PCC4 cells. The Hirt supernatant DNAs prepared from PCD1 and PCC4 cells 10 hr after virus inoculation were equally infectious for NIH3T3 cells in a DNA transfection, although one positive result with PCD1 cells might suggest a difference between the two cell types in this aspect. These results indicate that restriction of type C retrovirus in undifferentiated embryonal carcinoma cells occurs at a step subsequent to formation and maturation of viral DNA intermediates.

Expression of retrovirus-associated 8S RNA in mammalian cells.

A comparative study of the expression of retrovirus-associated 8S RNA was made in different mammalian cells. This RNA is found in cultured cells from all mammalian species analysed but its expression varies. An increase of 8S RNA is observed in sarcoma virus-transformed cells as compared to control uninfected cells or cells infected with leukaemia virus. No increased quantities of 8S RNA were detected in cells transformed by SV40 or by methylcholanthrene. These data show that the level of 8S RNA was augmented following transformation by sarcoma viruses.

Fragility of attenuated Rauscher leukemia virus.

Some biochemical properties of virulent (RRL+) and attenuated (RCL-) Rauscher leukemia virus were compared. It was shown that the reverse transcriptase (RT) activity of 'aged' as well as 'fresh' RCL- virions was 25--30% of that found for RRL+. The thermal sensitivity of the RT was the same for both viruses. A 60--70S RNA could be extracted from 'aged' RRL+, while no high molecular weight RNA was obtained from 'aged' RCL-. After centrifuging in sucrose gradient, most of the RT activity and 3H-labeled RNA of 'aged' RRL+ was recovered at 1.14--1.16 g/cm3, while for 'aged' RCL- no labeled RNA, and at most 10% of the original RT activity were found in the same zone. The fragility seemed to increase in the course of aging, since 'fresh' RCL- banded at 1.14--1.16 g/cm3 as did 'fresh' RRL+. Also, 3H-labeled viral RNA was found in the viral bands, from which 60--70S RNA could be extracted. Molecular hybridizations showed that 20% of the nucleic acid sequences related to Rauscher leukemia virus found in the RNA of RRL+-infected cells were missing in the RNA of RCL--infected cells.

[Murine type C endogenous virus expression in murine 129 teratocarcinoma cell line]. / Etude de l'expression des virus endogènes murins de type C dans les lignées cellulaires dérivées du tératocarcinome de la Souris 129.

Murine type C viral information is detectable in the cellular genome of both differentiated and undifferentiated cell lines derived from 129 Mouse teratocarcinoma. Cytoplasmic RNA expression which is negligible in differentiated cells, is significantly higher in multipotential undifferentiated cells. Furthermore it was observed that in vitro differentiation of multipotent cells leads to a decrease of this expression.

[Response of an undifferentiated and nullipotential cell line derived from an A/Heston mouse teratocarcinoma to infection with type C ecotropic murine viruses]. / Réponse à l'infection par des virus écotropiques de type C de la Souris d'une lignée cellulaire indifférenciée et nullipotentielle dérivée d'un tératocarcinome de la Souris A/Heston.

The PCC6 cell line, derived from an A/Heston Mouse Teratocarcinoma, and composed of nullipotential embryonic carcinoma cells (ECC), is completely resistant to infection with type C murine ecotropic viruses. It reacts as other cell lines previously studied which are derived from a 129 Mouse teratocarcinoma and composed of multipotential ECC.

[Multiplication of murine C-type viruses in mouse teratocarcinoma cell lines]. / Etude de la multiplication des virus murins de type C dans des lignées cellulaires dérivées du tératocarcinome de la souris.

The host-virus interactions of several murine C type viruses with cell lines established in vitro from a mouse teratocarcinoma were studied. The cells used in this study were the multipotential stem cells, or embryonal carcinoma cells, and the differentiated cells derived from a same tumor.

[High poly A content of the native genome of murine oncornaviruses : presence of 65-70 S RNA lacking polyadenylic sequences]. / Richesse en poly A du génome natif des oncornavirus murins : présence de RNA 65-70 S dépourvu de séquences polyadenylées

High molecular weight Poly (A) minus RNA was isolated from native genome of murine oncornaviruses. The Poly (A) content of viral genomes seems to depend more on cellular multiplication than viral maturation.

Effect of murine leukaemia virus age on its 8S RNA components.

The effect of virion age on the two A and B 8 S RNA conformational isomers was investigated by electrophoresis on polyacrylamide gels. The relative proportions of A and B 8 S RNA components of the immature virus (rapid harvest, 30 min) are intermediate between those previously found for the mature virus and for the host cell. These results suggest that a change of 8S RNA conformation occurs during virus maturation.

Low-molecular-weight RNAs of murine sarcoma virus: comparative studies of free and 70S RNA-associated components.

Several low-molecular-weight RNAs were released from mouse leukemia sarcoma virus 70S RNA under conditions of thermal denaturation. One of them, the 70S-associated 8S RNA, exhibits a number of structural properties characteristic of a previously described free viral 8S RNA. This similarity was revealed by polyacrylamide gel electrophoresis and nucleotide composition.