Embryonal carcinoma (EC) cells are nonpermissive for retrovirus replication. Restriction of retroviral expression in EC cells was studied by using DNA transfection techniques. To investigate the activity of the Moloney murine leukemia virus (M-MuLV) enhancer and promoter sequences, the M-MuLV long terminal repeat and the defined long terminal repeat deletions were linked to neo structural gene sequences that encode resistance to the neomycin analog G418. Transient expression data and drug resistance frequencies support the findings that the M-MuLV enhancer is not active in EC cells but that promoter sequences are functional. In addition, a proviral DNA fragment that encodes the leader RNA sequence of a M-MuLV recombinant retrovirus was found to restrict expression specifically in EC cells. Deletion analysis of the leader fragment localized the inhibitory sequences to a region that spans the M-MuLV tRNA primer binding site. It is not known whether restriction occurs at a transcriptional or posttranscriptional level, but steady-state RNA levels in transient expression assays were significantly reduced.

A negative regulatory element (NRE) spanning the tRNA primer-binding site (PBS) of Moloney murine leukemia virus (M-MuLV) mediates repression of M-MuLV expression specifically in embryonal carcinoma (EC) cells. We precisely defined the element by base-pair mutagenesis to an 18-base-pair segment of the tRNA PBS and showed that the element also restricted expression when moved upstream of the long terminal repeat.A DNA-binding activity specific for the M-MuLV NRE was detected in vitro by using crude EC nuclear extracts in exonuclease m protection assays. Binding was strongly correlated with repression in EC cells. Mutations within the NRE that relieved repression disrupted binding activity. Also, nuclear extracts prepared from permissive, differentiated EC cell cultures showed reduced binding activity for the NRE. These results indicate the presence of a stem cell-specific repressor that extinguishes M-MuLV expression via the NRE at the tRNA PBS.Moloney murine leukemia virus (M-MuLV) does not productively infect early embryonic stem cells (22) or murine embryonal carcinoma (EC) cells (17,40,42). EC cells share many properties with early embryonic stem cells (24) and are a convenient cell culture system with which to study MMuLV restriction. The block in the viral life cycle occurs at the stage of viral RNA accumulation, subsequent to proviral DNA integration (11,41). At least two cis-acting elements are responsible for the block in RNA expression: the MMuLV enhancer and an M-MuLV intragenic element. The M-MuLV enhancer is inactive in EC cells (29,30), possibly due to an enhancer-specific repressor (19) or the lack of specific positive enhancer-binding factors (36). The intragenic element spans the M-MuLV proline tRNA primerbinding site (PBS) and inhibits RNA expression from MMuLV recombinants specifically in EC cells (13,30,47). Competition assays indicate that the inhibition is mediated by a trans-acting factor. The mechanism of action is probably at the transcriptional level, since inhibition is independent of the orientation of the intragenic element (31). In addition, a point mutation in the tRNA PBS of an M-MuLV host range mutant, termed B2, allows virus replication (4) and expression (47) in EC cells.In this report, we precisely define the borders of the intragenic inhibitory element, identify the element as a transcriptional silencer sequence, and correlate in vitro factor-binding activity at the tRNA PBS to EC-specific repression.MATERUILS AND METHODS Cell lines. The PC13 (2), F9 (5), and C2 cells were grown in Dulbecco modified Eagle medium supplemented with 10% fetal bovine serum and antibiotics. The F9 and PC13 EC cells were grown on gelatin-coated plasticware.Recombinant constructions. The mutagenized recombinants used to map the negative regulatory element (NRE) were derived by synthesis of the indicated oligonucleotides with a SaI 4-base-pair (bp) overhang and a BgilI restriction * Corresponding author. site at the 5' and 3' termini, respectively. The annealed oligonucleotides were ligated with MLV.4...

An intragenic region spanning the tRNA primer binding site of a Moloney murine leukemia virus recombinant retrovirus was found to restrict expression specifically in embryonal carcinoma (EC) cells. When the inhibitory domain was present, the levels of steady-state RNA synthesized from integrated recombinant templates in stable cotransformation assays were reduced 20-fold in EC cells but not in C2 myoblast cells. Transient-cotransfection assays showed that repression of a template containing the EC-specific inhibitory component was relieved by an excess of specific competitor DNA. In addition, repression mediated by the inhibitory component was orientation independent. This evidence demonstrates the presence of a saturable, transacting negative regulatory factor(s) in EC cells and suggests that the interaction of the factor(s) with the intragenic inhibitory component occurs at the DNA level. * Corresponding author. mediated by the tPBS domain was not known. In this report, we characterize more fully the EC-specific inhibitory activity in this region and indicate by competition assays that expression is repressed by a transacting factor(s) that interacts with the leader element. MATERIALS AND METHODS Cell lines. The mouse F9 and PC13 EC cells were generously provided by E. Adamson (La Jolla Cancer Research Foundation, La Jolla, Calif.) and B. Hogan (Nationtv Institute for Medical Research, London, United Kingdom), respectively. All cell lines werz grown in Dulbecco modified Eagle medium (GIBCO Laboratories, Grand Island, N.Y.) supplemented with 10% fetal bovine serum (GIBCO) and antibiotics. The EC cell lines were propagated on gelatincoated plasticware. Genomic DNA isolation. High-molecular-weight DNA was isolated by proteinase K digestion and organic extraction (29) from a total cell lysate prepared from a monolayer of 107 cells. Cytoplasmic RNA isolation. Cells M ere harvested by trypsinization, and total cytoplasmic RNA was isolated as described previously (25). In some preparations, the RNA samples were enriched for poly(A)+ species by oligo(dT)cellulose chromatography (2). DNA transfections. All transfections were done with plasmid DNA purified on two sequential cesium chloride equilibrium gradients. The transfection experiments were done as described below except for the competition assays, which are described in a figure legend (see Fig. 3). (i) Transient expression. The PC13, F9, and L cells were seeded at 1 x 106 cells per plate (diameter, 10 cm), and the C2 cells were seeded at 5 x 105 cells per plate. Generally, at 6 to 8 h after seeding, 5 ,ug of plasmid DNA was added per plate in the presence of Ca3(PO4)2 (45) for 15 to 16 h. The medium was replaced, and either the cells were harvested after an additional 30-h incubation period or G418 (GIBCO) was added for a 2-week period to determine frequencies of G418 resistance. Comparisons in expression levels were only made between concurrent transfections. (ii) Stable cotransformation. Cotransformants were obtained by transfection with two unlinked plasmids co...

Embryonal carcinoma (EC) cells are nonpermissive for retrovirus replication. Restriction of retroviral expression in EC cells was studied by using DNA transfection techniques. To investigate the activity of the Moloney murine leukemia virus (M-MuLV)enhancer and promoter sequences, the M-MuLV long terminal repeat and the defined long terminal repeat deletions were linked to neo structural gene sequences that encode resistance to the neomycin analog G418. Transient expression data and drug resistance frequencies support the findings that the M-MuLV enhancer is not active in EC cells but that promoter sequences are functional. In addition, a proviral DNA fragment that encodes the leader RNA sequence of a M-MuLV recombinant retrovirus was found to restrict expression specifically in EC cells. Deletion analysis of the leader fragment localized the inhibitory sequences to a region that spans the M-MuLV tRNA primer binding site. It is not known whether restriction occurs at a transcriptional or posttranscriptional level, but steady-state RNA levels in transient expression assays were significantly reduced.

A negative regulatory element (NRE) spanning the tRNA primer-binding site (PBS) of Moloney murine leukemia virus (M-MuLV) mediates repression of M-MuLV expression specifically in embryonal carcinoma (EC) cells. We precisely defined the element by base-pair mutagenesis to an 18-base-pair segment of the tRNA PBS and showed that the element also restricted expression when moved upstream of the long terminal repeat. A DNA-binding activity specific for the M-MuLV NRE was detected in vitro by using crude EC nuclear extracts in exonuclease III protection assays. Binding was strongly correlated with repression in EC cells. Mutations within the NRE that relieved repression disrupted binding activity. Also, nuclear extracts prepared from permissive, differentiated EC cell cultures showed reduced binding activity for the NRE. These results indicate the presence of a stem cell-specific repressor that extinguishes M-MuLV expression via the NRE at the tRNA PBS.