Nucleotide sequences at host-proviral junctions for mouse mammary tumour virus

Proviruses cloned from rat cells infected with mouse mammary tumour virus, a B-type retrovirus regulated by glucocorticoid hormones, show the structural features of transposable elements: short inverted repeats conclude long direct repeats at the ends of viral DNA, and short sequences of cellular DNA are duplicated during integration and flank each provirus. The integrative mechanism joins a precise site in viral DNA to non-homologous sites in host DNA.     

Glucocorticoids regulate expression of dihydrofolate reductase cDNA in mouse mammary tumour virus chimaeric plasmids

Fusions between the mouse mammary tumour virus long terminal repeat and a mouse dihydrofolate reductase cDNA have been constructed in a SV40 vector. When these plasmids are transferred into recipient cells, the production of dihydrofolate reductase is regulated by glucocorticoid hormones. These results define a hormonally responsive region of the viral genome.     

Amplification and enhanced expression of the c-myc oncogene in mouse SEWA tumour cells

SEWA tumour cells are derived from an osteosarcoma induced in an A.SW mouse by infection with polyoma virus. Cytogenetic analyses have revealed three different characteristic chromosomal abnormalities diagnostic for the presence of amplified genes: 'double minutes' (DMs), homogeneously staining chromosomal regions (HSRs) and C-bandless chromosomes (CMs; for review see ref. 2). DMs may undergo fluctuation in number depending on the conditions in which the cells grow. Their number usually increases after injection of cells into a mouse and often is reduced to undetectable levels when the cells are explanted back into tissue culture; when the cells are re-introduced into the mouse, they again acquire multiple DMs. We show here that cells of SEWA lines carrying DMs, HSRs or CMs contain amplified copies of the proto-oncogene c-myc and enhanced levels of c-myc messenger RNA and c-myc protein. DMs or CMs are the sites of c-myc amplification in two different SEWA lines.     

APOBEC3 inhibits mouse mammary tumour virus replication in vivo

Genomes of all mammals encode apobec3 genes, which are thought to have a function in intrinsic cellular immunity to several viruses including human immunodeficiency virus type 1 (HIV-1). APOBEC3 (A3) proteins are packaged into virions and inhibit retroviral replication in newly infected cells, at least in part by deaminating cytidines on the negative strand DNA intermediates. However, the role of A3 in innate resistance to mouse retroviruses is not understood. Here we show that A3 functions during retroviral infection in vivo and provides partial protection to mice against infection with mouse mammary tumour virus (MMTV). Both mouse A3 and human A3G proteins interacted with the MMTV nucleocapsid in an RNA-dependent fashion and were packaged into virions. In addition, mouse A3-containing and human A3G-containing virions showed a marked decrease in titre. Last, A3(-/-) mice were more susceptible to MMTV infection, because virus spread was more rapid and extensive than in their wild-type littermates.     

Long-term proliferation of mouse primordial germ cells in culture.

Primordial germ cells (PGCs) are first identifiable as a population of about eight alkaline phosphatase-positive cells in the 7.0 days postcoitum mouse embryo. During the next 6 days of development they proliferate to give rise to the 25,000 cells that will establish the meiotic population. Steel factor is required for PGC survival both in vivo and in vitro and together with leukaemia inhibitory factor stimulates PGC proliferation in vitro. In feeder-dependent culture, PGCs will proliferate for up to 7 days, but their numbers eventually decline and their proliferative capacity is only a fraction of that seen in vivo. Here we report a further factor that stimulates PGC proliferation in vitro, basic fibroblast growth factor (bFGF). Furthermore, bFGF, in the presence of steel factor and leukaemia inhibitory factor, stimulates long-term proliferation of PGCs, leading to the derivation of large colonies of cells. These embryonic germ cells resemble embryonic stem cells, pluripotent cells derived from preimplantation embryos, or feeder-dependent embryonal carcinoma cells, pluripotent stem cells of PGC-derived tumours (teratomas and teratocarcinomas). To our knowledge, these results provide the first system for long-term culture of PGCs.     

Genetic retrieval of viral genome sequences from herpes simplex virus transformed cells

Oncogenic transformation of cultured cells by inactivated herpes simplex virus (HSV) types 1 and 2 has been demonstrated. Expression of HSV information in these transformed cells has been shown by immunofluorescence studies, detection of HSV neutralizing antibody in sera from tumour-bearing animals and by hybridization of HSV-specific RNA. Molecular hybridization studies of DNA from HSV-2 transformed hamster cells have detected up to 40% of the HSV genome present in several copies. Complementation of three HSV-2 temperature-sensitive mutants when superinfecting the RE1 rat embryo cell line (transformed by the HSV-2 temperature-sensitive mutant ts1) suggests that resident viral genes can be expressed. Brown et al. used a similar approach to detect HSV information latent in human ganglia. We report here retrieval of intertypic HSV recombinants from HSV transformed cells after superinfection with ts mutants of the alternative serotype of HSV. Restriction enzyme analysis which clearly differentiates between HSV-1 and HSV-2 DNA has demonstrated the isolation of recombinants spanning the genome and of virus indistinguishable from the original transforming virus.