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

Abstract

New polyomavirus mutants (PyEC-C) selected on LT1 cells and exhibiting a strong cytopathic effect in all embryonal carcinoma (EC) cell lines tested have been isolated. They were derived by a sequence duplication event from a new multiadapted mutant isolated in PCC4 cells. A quantitative analysis of viral DNA replication and transcription in 3T6 and EC cell lines was performed to compare PyEC-C mutants and PyEC mutants previously isolated on F9 or PCC4 cell lines. Analysis of the results indicated that PyEC-C mutants were more efficient in all EC cell lines tested than all other PyEC mutants; on the contrary, they were less adapted to 3T6 cells than wild-type polyomavirus. In both 3T6 and EC cells, uncoupling between early transcription and viral DNA replication was observed; different viruses were shown to replicate with the same efficiency, while their levels of early transcripts differed by two orders of magnitude. Attempts to correlate the genome structure of the mutants with their biological properties indicate that duplication of protein-binding sequences is not the only event responsible for their phenotype. PyEC mutants were also analyzed with respect to their interactions with early mouse embryos and embryonal stem (ES) cell lines derived from the inner cell mass of blastocysts. They showed different degrees of expression in ES cells and preimplantation embryos. ES cells were most efficiently infected and lysed by mutants which exhibit both a multiadapted and a lytic phenotype in EC cells. Preimplantation embryos were not permissive to any PyEC mutants. However, EC-multiadapted mutants were infectious in blastocysts after two days of in vitro culture.

Analysis of transcription factors binding to the duplicated PEA1 and PEA3 sites that are required for polyomavirus mutant expression in PCC4 embryonic carcinoma cells

“…Cells and viruses. All cell lines were cultivated as previously described (28). wt A2 PyV was obtained from M. Fried (Imperial Cancer Research Fund, London, England), and the wt evlO01 strain was obtained from G. Magnusson (Uppsala Biomedical Center, Uppsala, Sweden).…”

“…wt A2 PyV was obtained from M. Fried (Imperial Cancer Research Fund, London, England), and the wt evlO01 strain was obtained from G. Magnusson (Uppsala Biomedical Center, Uppsala, Sweden). Isolation of all Py EC-PCC4 mutants was performed by chronic infection of PCC4 cells with either wt A2 (mutants P500, P5000, A, 204, and 97) or wt evlO01 (mutants Ml to M5) (28,29). Mutants M112 and M206 have been renamed Ml and M2.…”

“…Plasmid pPyC306 LacZ, a derivative of pCH110 (Pharmacia) containing the ,B-galactosidase gene under the control of the early enhancer/ promoter sequences of the Py EC-C306 mutant, was used as an internal control. The activities in each cell line were expressed relative to the corresponding wt A2 luciferase activity (which is half of the wt ev1001 luciferase activity, as shown by Melin et al [28]) and normalized by the 3-galactosidase activity of the cotransfected gene to assess transfection efficiencies. Cell extracts were prepared 48 h after seeding of the cells and stored at -20°C.…”

“…The molecular mechanisms involved in the viral restriction are different in F9 and PCC4 cell lines, since PCC4 mutation does not lead to adaptation to F9 cells. However, some Py EC-PCC4 mutants such as the Ml mutant are able to express their early antigens after infection of F9 cells (28). How can one explain the high activity of Ml in all cell lines and in particular in PCC4 cells?…”

Specific Transcription Factors Stimulate Simian Virus 40 and Polyomavirus Origins of DNA Replication

“…The purpose of auxiliary components may be to regulate DNA replication by determining when initiation occurs. For example, the polyomavirus (Py) on functions only in those mouse cell types that can activate its enhancer (6,12,58,74). In mammalian chromosomes, active genes are replicated early during S phase while quiescent genes are replicated late (82), and initiation of replication may require specific transcription factors (76) that bind DNA sites where replication begins (11).…”