Isolation and characterization of polyoma virus mutants which grow in ...

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Isolation and characterization ofpolyoma virus mutants which grow in murine embryonal carcinoma and trophoblast cells. Kenichi Tanaka, Kamal Chowdhuryl, ...

The EMBO Journal Vol. I No. 12

pp. 1521-1527, 1982

Isolation and characterization of polyoma virus mutants which grow in murine embryonal carcinoma and trophoblast cells Kenichi Tanaka, Kamal Chowdhuryl, Kenneth S.S. Chang, Mark Israel', and Yoshiaki Itol* Laboratory of Cell Biology, National Cancer Institute, and 'Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20205, USA Communicated by B. Griffin Received on 22 September 1982

Mouse trophoblast cell lines established from cultured midterm placenta and a cell line obtained from cultured blastocyst resemble trophectoderm cells. These cells are resistant to infection by wild-type polyoma virus. We have isolated six polyoma virus mutants capable of growing in trophoblast cell lines. Restriction enzyme analyses and marker rescue experiments revealed that the genetic changes necessary for the growth of these mutants (PyTr mutants) in trophoblast cells were located in a regulatory region of the genome between the origin of viral DNA replication and the region encoding the viral structural proteins. PyTr mutants are, therefore, similar to PyEC mutants, described by others, which are able to grow in embryonal carcinoma cell lines such as F9 or PCC4. The nucleotide sequence of two independently obtained PyTr mutants has an identical 26-bp deletion from nucleotide 5131 to 5156. This deleted region is replaced by either the sequence GGGA or by viral DNA sequences that flank this deletion. PyECF9 mutants grow well in trophoblast and trophectoderm cells, but PyTr mutants do not grow in F9 or PCC4 cells. Key words: polyoma virus mutants/embryonal carcinoma cells/trophoblast cells/trophectoderm cells/DNA sequence Introduction Mouse embryonal carcinoma (EC) cells, the stem cells of teratocarcinomas, are restrictive for growth or gene expression of polyoma virus, SV40, and murine retroviruses (Swartzendruber and Lehman, 1975; Topp et al., 1977; Peries et al., 1977; Teich et al., 1977; Gautsch, 1980), and lose these restrictions when they are allowed to differentiate. The lytic cycle of polyoma virus in EC cells is blocked after penetration of viral particles but before synthesis of T-antigens (Boccara and Kelly, 1978). Polyoma virus mutants capable of growing in EC cell lines, F9 and PCC4, have been isolated (PyEC mutants). The region of the viral genome altered in these mutants has been identified between the origin of viral DNA replication and the initiation codon for a viral coat protein (Katinka et al., 1980, 1981; Sekikawa and Levine, 1981; Fujimura et al., 1981a). This region of the genome is considered important in regulating viral RNA transcription and viral DNA replication (Herbomel et al., 1981; Tyndall et al., 1981 de Villiers and Schaffner, 1981; Fujimura et al., 198 lb; Fujimura and Linney, 1982). These observations have suggested that the mechanism regulating differential expression of wildtype and PyEC mutants in EC cells may reflect more general cellular regulatory processes specific to stages of cellular differentiation. *To whom reprint requests should be sent.

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At the 8-cell stage in early murine embryogenesis, each individual cell is totipotent (Johnson et al., 1977). At about the 32-cell stage, the pre-implantation blastocyst begins to form, and the first morphologically detectable differentiation occurs. At this time, the external cell layer forms the trophectoderm from which placental trophoblast is derived. The internal cells remain multipotent; the latter are called the inner cell mass which will develop into the embryo and extraembryonal endoderm (for review, see Martin, 1975, 1980; Jacob, 1977). EC cells resemble cells of the inner cell mass in several respects. EC cells share antigens present on the cells of the inner cell mass, are multipotent, and participate in the formation of chimeric mice when they are injected into the blastocyst, thereby mixing with the inner cell mass (Jacob, 1977). Permanent cell lines have recently been established from cultured midterm placentas of mice and shown to produce transplantable tumors. These cell lines, designated as trophoblast cell lines (Log et al., 1981), resemble trophectoderm cells and have some characteristics of undifferentiated cells (Tanaka et al., 1982). The cultured trophoblast cells are unable to support the growth of wild-type polyoma virus. We have isolated mutant viruses capable of growing in trophoblast cells (PyTr mutants). Mutations in the PyTr viruses are in the same region of the viral genome as the previously described mutations of PyEC mutants. We have compared the nucleotide sequence changes in PyEC and PyTr mutants and have studied the growth characteristics of these viruses in several embryonic cell lines of the mouse. These studies provide insight into the hypothesis that cells of different stages of differentiation possess a different mechanism for the control of gene expression.

Results Properties of trophoblast cells We sought to establish mouse cell lines of primitive origin which were non-permissive for the growth of wild-type polyoma virus, thus allowing the isolation of virus mutants which would replicate in these cells. Cell lines established from mid-term placenta of five strains of mice were found to possess a number of phenotypic similarities to trophoblast and its more primitive precursor, trophectoderm. All five trophoblast cell lines were devoid of stage-specific embryonal antigen, SSEA-1 (Table I), which has been shown to be present specifically in the 8-cell to morula stages, the inner cell mass and all EC cells (Solter and Knowles, 1978). In the control shown in Table I, although the antigen was detected in a majority of F9 cells, the number of antigen-positive cells decreased significantly after 7 days of culture in the presence of retinoic acid, which induces F9 cells to differentiate into endoderm-like cells (Strickland and Mahdavi, 1978). Cytoskeletal proteins Endo A (mol. wt. 55 000) and Endo B (mol. wt. 50 000), which are thought to be present only in extraembryonic endoderm cells and trophectoderm (Oshima, 1981, 1982), could be immunoprecipitated from lines of trophoblast cells obtained from BALB/c and C57BL/6 mice but not from F9 cells or BALB/c 3T3 cells (in preparation). 1521

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Table I. Expression of SSEA-1 antigen Cell line

F9 (stem cells) F9a (differentiated) Trophoblast cell BALB/c C57BL/6 CBF1

SJL/J CF-1 Trophectoderm cell CF-1 BALB/c 3T3

Percent immunofluorescence positive cells

95 10

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