T-antigen-independent replication of polyomavirus DNA in murine embryonal carcinoma cells.

Abstract

Expression of wild-type polyomavirus (Py) is restricted in murine embryonal carcinoma (EC) cells. The block appears to be located at the level of early transcription. Since no T antigen is produced, we investigated the fate of viral DNA upon infection of these cells; we showed that wild-type Py DNA replicates efficiently in all EC cells, probably via a T-antigen-independent mechanism. Furthermore, we studied, at permissive and restrictive temperatures, the replication of tsa (thermosensitive for T antigen) viral DNA of an in vitro-constructed deletion mutant lacking part of the early region coding sequences and of a double mutant carrying both the tsa mutation and the PyEC F9 mutation (allowing expression of early and late viral functions in EC cells does replicate in EC cells, although less efficiently than the DNA of PyEC mutants. This replication proceeds via a Tantigen-independent mechanism as confirmed by the behavior of a Py tsa mutant, which produces a thermosensitive large T antigen, of a deletion mutant which lacks part of the large T protein-coding sequence, and of a double mutant carrying both the tsa and the PyEC F9 mutations. We also show that the ability to replicate Py DNA in the absence of T antigen is lost when EC cells are allowed to differentiate. Finally, we discuss the involvement of both viral DNA sequences and cellular proteins in this phenomenon. MATERIALS AND METHODSCell cultures and virus strains. NIH 3T6 mouse cells and the EC cell lines PCC4-aza-1, F9, and LT1 (12,16,21) were grown in Dulbecco modified Eagle medium supplemented with 10% fetal calf serum (GIBCO Diagnostics, Madison, Wis.). F9 cells were grown on gelatin-coated (0.1%) dishes. All virus strains were propagated by infecting secondary mouse embryo cells at a multiplicity of 0.01 PFU per cell. The wild-type Py used in these studies was the large plaque strain A2 (10). The nucleotide numbers refer to the Py DNA sequence as reported by Soeda et al. (19). The PyEC mutant PyF9-1 has been previously described (13

Relationship of eukaryotic DNA replication to committed gene expression: general theory for gene control.

“…Instead, infection of these various undifferentiated cell types with wild-type polyomavirus will usually result in persistent maintenance of low levels of episomal viral DNAs (for early references, see reference 221). These cells include inner cells of a mouse blastocyst, various lines of undifferentiated pluripotent embryonal carcinoma (EC) cells (13,73,122,146), normal embryo fibroblasts (221), undifferentiated myoblasts (110), undifferentiated erythroleukemia cells (86,89), and neuroblastoma cells (89,234). In all these cases, terminal differentiation of various cells leads to high-level wildtype polyomavirus DNA replication (replication with trophectoderm differentiation is shown in Fig.…”

Simian virus 40 T antigen is required for viral excision from chromosomes.

“…One study by Lania et al (29) reached the opposite conclusion regarding the involvement of T antigen in excision from polyoma-transformed rat cell lines fused to permissive mouse cells. However, the presence of polyoma T antigen increased the rate of excision 100-fold, and there is now some evidence that, at least in certain polyoma-transformed cell lines, wt large T antigen may not be absolutely required in large amounts to initiate viral replication (30).…”

Host range specificity of polyomavirus EC mutants in mouse embryonal carcinoma and embryonal stem cells and preimplantation embryos

“…One-fourth of the DNA extracted at each time point was digested with BamHI and run on a 1.4% agarose gel. The gels were stained with ethidium bromide, transferred to nitrocellulose filters, and hybridized with an in vitro 32P-labeled PyV DNA probe as described previously (1 RNAs were denatured by incubation with 1 M glyoxal (44) without dimethyl sulfoxide. Samples (5,10, and 15 ,ug) of RNAs isolated from each virus-infected EC line were transferred in duplicate onto a reinforced nitrocellulose mem-brane by using a Minifold II apparatus (Schleicher & Schuell).…”