Superinfection rescue of an integrated defective polyomavirus genome

Abstract

We have characterized the viral sequences integrated in a polyomavirus-transformed mouse cell line, Py-3T3 (clone Py-6), and followed their excision and packaging upon superinfection. The polyomavirus sequences contained in Py-6 cells are present as a single insert of nonidentical tandem copies which includes, in addition to a normal middle T-antigen-coding region, some very rearranged sequences. Infection of Py-6 cells with polyomavirus strains encoding a normal large T antigen leads to the reproducible recovery in the resulting viral stock of specific defective viral genomes. The defective genomes contain a wild-type coding region for middle and small T antigens and intact viral origin and enhancer sequences. The remainder of the viral genome is rearranged or lost, so that there is no capacity to code for large T antigen or viral capsid proteins. The recovered defective sequences are also found integrated in Py-6 genomic DNA. Presumably, in infections of Py-6 cells, large T antigen, provided by the superinfecting virus, amplifies and excises the integrated viral sequences. The superinfecting helper virus must also produce viral capsids for packaging of the defective viral DNA and thus provides a means to shuttle the defective sequences from the mouse cells into other hosts, such as rat cells. In the latter host, the defective sequences are able to induce transformation.

Properties of cells transformed by the middle T-antigen-coding region of polyomavirus

“…Rearranged sequences hybridizing to fragments 3, 2, and 1 are present in all clones. The rearrangements faithfully reflect the structure of the infecting defective virus described in detail elsewhere (12). In some clones, the integrated viral sequences are present as head-to-tail tandem repeats of the FIG.…”

“…3), the large T antigen carboxy-terminal fragment (HpaII-2) is missing. As shown for the parental defective virus (12), sequences hybridizing to fragment HpaII-2 are present in two small fragments ( Fig. 3), which also hybridize to fragment 3 (data not shown).…”

“…A set of 10 independent transformed clones of Fischer rat cells selected for focus formation were isolated and characterized. These clonal cell lines were transformed by a defective virus rescued from the cell line Py-3T3 (3), which contains an intact middle and small T-antigen-coding region as well as scrambled segments of the remainder of the polyomavirus genome (12). The designation Fdf was chosen for these cell lines to mark the defective origin of the viral sequences and the F-111 parental cell origin; the numbers refer to independently derived clones.…”

“…We have recently isolated a series of independently transformed Fischer rat clonal cell lines from the established cell line F-111 that have integrated only the middle and small T-antigen-coding region of the viral genome. These cell lines, designated Fdf, were obtained from viral infections with a defective polyomavirus containing a scrambled genome except for a functional middle and small T-antigencoding region (12). We examined the production of viral proteins in these Fdf cell lines; none of the lines tested produced detectable levels of either normal-sized T antigen or fragments of large T antigen.…”

“…The derivation of the set of transformed clonal cell lines under study, resulting from viral infections of the established Fischer rat cell line F-111 (10), and the description of the viral sequences integrated in those lines are reported in detail in the accompanying paper (12). In summary, these cells were transformed by polyomavirus sequences rescued from the Py-3T3 cell line (designated Py-6), which are defective or scrambled except for a functional middle and small T-antigen region (12). The 10 cell lines studied are designated Fdf-1, -10, -11, -13/14, -15, -16, -21, -23, -26, and -27/28.…”