Jan 5, 1981 - Sensitive Polyoma Virus: Superinfection Does Not Allow. Excision of the Resident Viral Genome. LOUIS DELBECCHI, DANIEL GENDRON, ...
Vol. 39, No. 1
JOURNAL OF VIROLOGY, July 1981, p. 196-206 0022-538X/81/070196-11$02.00/0
Inducible Permissive Cells Transformed by a TemperatureSensitive Polyoma Virus: Superinfection Does Not Allow Excision of the Resident Viral Genome LOUIS DELBECCHI, DANIEL GENDRON, AND PIERRE BOURGAUX* Departement de Microbiologie, Faculte de Medecine, Centre Hospitalier Universitaire, Universite de Sherbrooke, Sherbrooke, Quebec, Canada JIH 5N4 Received 5 January 1981/Accepted 7 April 1981
After exposure of mouse embryo cells to the early temperature-sensitive mutant tsP155 of polyoma virus (Py), a transformed cell line (Cyp line) that can be readily induced to synthesize Py by transfer to 330C was isolated at 390C (7). Virus production and synthesis of free viral DNA occurring after temperature shiftdown or superinfection with wild-type Py or both were studied in several clonal isolates of the Cyp cell line. Measurements of virus yields indicated that, although some could be induced more effectively than others, all cell clones behaved as highly permissive when subjected to superinfection. We analyzed the origin of free viral DNA accumulating in the superinfected cultures, taking advantage of (i) the unique physical properties of the low-molecular-weight DNA which, in the case of one of the Cyp clones, accumulates during temperature shiftdown, and (ii) the differences between resident and superinfecting viral genomes in their susceptibilities towards restriction endonucleases. At 330C, both viral genomes were found to accumulate in all clones studied whereas in the case of the clones with lower inducibility, the replication of the resident genome appeared to be enhanced by superinfection. At 390C, however, accumulation of the superinfecting genome was not accompanied by that of the resident genome, unless it had already been initiated before superinfection. These findings demonstrate that, when routinely cultivated at 390C, Cyp cells contain few viral DNA molecules readily available for autonomous replication and that, upon transfer to 330C, therefore, excision must first take place before the resident genome can accumulate as free viral DNA. Our findings also suggest that, unlike the P155 gene product provided by the resident viral genome upon induction, the allelic gene product supplied by the superinfecting genome may be less effective in triggering excision than in promoting replication.
Complementation tests have been used extensively to define the various genetic functions involved during the productive cycle of the two papovaviruses simian virus 40 (SV40) and Py. Although little attention has been paid to the actual mechanism underlying this phenomenon, it has been generally assumed that, during complementation between an early and a late temperature-sensitive (ts) mutant, the late mutant provides the early (tsa or tsA) mutant with the viral protein required for the initiation of DNA replication, presumably large T antigen (13, 23). The transcription of the amplified early mutant DNA would then result in the synthesis of wildtype late proteins required for the assembly of heat-stable virions. To the best of our knowledge, complementation of an integrated genome by a superinfecting genome within a transformed cell has not been 196
described for papovaviruses. Recently, however, isolated a transformed mouse cell line (Cyp line) that appeared suitable for the study of complementation between resident and superinfecting polyoma virus (Py) genomes (7). This cell line, which is transformed by the early tsP155 mutant of Py, carries the resident viral genome in an integrated form when routinely propagated at the restrictive temperature, 390C (9). However, Cyp cells synthesize large amounts of Py when either transferred to the nonrestrictive temperature, 330C, or superinfected at 390C with wild-type Py (7). These properties of Cyp cells could easily be interpreted in the light of current concepts about papovavirus DNA excision in transformed cells, a phenomenon that would require an active viral A/a gene product (2) to prime the replication of the integrated viral genome (4). Indeed, the defect in tsP155 we
VOL. 39, 1981
presumably resides in a function concerned with the initiation of DNA replication (11, 12, 13), as we have observed that [3S]methionine-labeled large T antigen is thermolabile in mouse 3T6 cells acutely infected with tsP155 (C. Galup, unpublished data). We thus undertook to investigate whether superinfection of Cyp cells with wild-type virus would activate the replication of the resident ts viral genome. Such a study, however, requires that free superinfecting and resident genomes be distinguishable in some way. From the original Cyp line, derived from a single focus of transformed cells, various clones were isolated that differ widely in their ability to synthesize virus and free viral DNA after temperature shiftdown (22). In all clones, except one (C12/al), the free viral DNA synthesized at 330C comprises primarily genome-size P155 DNA, which displays a characteristic susceptibility to certain restriction endonucleases (22). In C12/al cells, the DNA accumulating at 330C consists predominantly of RmI, a Py-mouse hybrid molecule of 4.5 x 10 daltons (22). The Cyp system thus offers several opportunities to determine the origin of the viral DNA synthesized during superinfection by wild-type Py. The data reported here indicate that both the resident ts genome and the superinfecting wild-type genome can replicate at the same time in Cyp cell cultures, and that under certain conditions, the wild-type genome will enhance the replication of the ts genome, as it does in acutely coinfected 3T6 cells. Regardless of the wild-type Py used, superinfection, however, does not seem to be followed by excision of the resident genome. Throughout the paper, we mean by excision, the mechanism whereby the integrated viral genome is made available for replication, and by replication, the amplification of already excised, or at any rate free, viral DNA molecules. Accumulation is assumed to result from both excision and replication, except in the case of the superinfecting genome where it would result from replication only. MATERLALS AND METHODS Cells. The isolation, cultivation, and properties of the Cyp cell line and of its clones C10, Cll, C12, C13, and C12/al have already been described (7, 22). Virus. The A2 large-plaque and P16 small-plaque
variants (14) of wild-type polyoma virus (Py) and the temperature-sensitive mutant tsP155 and ts-1260 (11, 12), all kindly provided by Walter Eckhart, were propagated in secondary cultures from whole mouse embryos, as described previously (5, 6), from an initial multiplicity of infection (MOI) of 0.1 PFU per cell. Revertant R (tsP155)A, which was isolated in large amounts from a culture of Cyp-C13 cells maintained at 390C (see below), was found to plaque at 33 and 390C with the same relative efficiency as wild-type Py.
EXCISION OF POLYOMA GENOMES
197
It was thus used in superinfection experiments before any cloning by plaque purification. Superinfection (monolayers). Subconfluent monolayers of Cyp cells in 100-mm plastic petri dishes were infected (1 ml of virus in Tris-saline) or mockinfected (1 ml of Tris-saline) for 1 h at 390C. The monolayers were then covered with nutrient medium and incubated at 390C. At 5, 24, or 48 h later, the cells were trypsinized, washed by centrifugation, suspended in nutrient medium, and seeded in 100-mm petri dishes (1.5 x 10' cells per plate). These were incubated at either 39 or 330C until the time of virus or DNA extraction.
Supernfection (suspension). Cells from confluent monolayers were trypsinized, washed, and counted. The cell suspension was then adjusted to 1 x 10' cells per ml and distributed between small siliconized glass vials containing magnets. Finally, virus was added to obtain the selected MOI. Virus adsorption was performed for 1 h at 390C on a magnetic stirrer. The cells were distributed into 60-mm petri dishes (5 x 105 cells each) and incubated at either 39 or 330C until the time of harvest. Virus extraction and titration by hemagglutination. Virus was extracted from the cell debris with receptor-destroying enzyme and assayed by hemagglutination as already described (5, 10). Extraction of viral DNA from Cyp cells. Free viral DNA was selectively extracted by the Hirt procedure (17), and the Hirt supernatant was extracted once with phenol-chloroform (50:50) and once with chloroform-isoamyl alcohol (24:1). The DNA was then precipitated with ethyl alcohol, collected by centrifugation in a Beckman Microfuge B (5 min, 400), and finally suspended in 0.2 ml of 1 mM EDTA-10 mM Tris-hydrochloride (pH 7.9; TE buffer). Marker DNAs were prepared by essentially the same procedure, but further purified by banding in ethidium bromide-cesium chloride solution (6). Endonuclease treatment. The low-molecularweight DNA from 105 to 2 x 105 cells was treated for 4 h at 370C with 4 U of HhaI (New England Biolabs, Beverly, Mass.) in 20 pl of 50 mM NaCl-7 mM MgC127 mM Tris-hydrochloride (pH 7.4). The samples were then directly loaded on the gel.
Electrophoresis. Electrophoresis was performed through 1% agarose vertical slab gels (20 by 10 cm) in E buffer (40 mM Tris-5 mM sodium acetate-i mM EDTA; pH 7.9) at 15 to 20 V, until the marker dye (bromophenol blue) reached the bottom of the gel. After electrophoresis, the gels were stained for 30 mi in E buffer containing 1 ug of ethidium bromide per ml, rinsed in distilled water, and photographed under UV light (254 nm) with a Polaroid camera. Blotting and annealing of DNA fragments. The method used was that of Southern (21). In general, annealing was perforned with 0.25 x 10W to 1 x 106 cpm of 32P-labeled probe. Radioactive probes were prepared by the procedure of Maniatis et al. (19) with Py DNA cloned in pBR322 (3) as template (9). Autoradiography and quantitation of DNA.
Blots were autoradiographed at -800C with the help of intensifying screens (Cronex Par Speed, DuPont). In several experiments, both the ethidium bromidestained gel and the resulting autoradiogram were
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scanned with a Corning 750 microdensitometer, equipped with visible and UV light sources and an integrator. Comparison of the scans thus obtained clearly demonstrated that the amount of radioactivity indicating the presence of closed circular DNA was consistently less than what would have been expected from the amount of-DNA measured in the gel by fluorescence. With that reservation, however, the intensity of the bands in the autoradiograms appeared to be a good indication of the amount of viral DNA present. Marker viral DNAs were introduced in known amounts in every gel, and several exposures of each blot were made. For all experiments, it was thus possible to evaluate the sensitivity with which free viral DNA could be detected. In all experiments, this sensitivity was such that free viral DNA synthesis would have been detected if 0.1%, or less, of the cells in the cultures had been undergoing a productive cycle.
RESULTS
Permissivity of Cyp
cells.
Earlier experi-
ments indicated that cells of the Cyp line and mouse embryo cells yield comparable amounts of virus at 390C after exposure to wild-type Py
(7). In this work, we have extended these observations to clones of Cyp cells isolated as colonies growing on plastic, like clones C10, C11, C12, C13 (7), and to clone C12/al, isolated as a colony after plating C12 cells in agar medium (22). Table 1 illustrates the production of viral hemagglutinin by cultures from these clones, under restrictive and nonrestrictive conditions, with and without superinfection. At 390C in the absence of superinfection, virus production was very low for all five clones. On one occasion, however, cultures of C13 cells displayed a cytopathic effect and yielded large amounts of virus
J. VIROL.
at 390C. Virus isolated from one such culture behaved exactly like wild-type virus when titrated for infectivity at 33 and 390C (L. Delbecchi, unpublished data). This virus was thus designated R (tsP155)A and used later in superinfection experiments (see below). As to the virus occasionally detectable in small amounts in Cyp cells maintained at 390C, it displayed in infectivity assays the thermosensitivity characteristic of partial revertants (L. Delbecchi, unpublished
data). The amounts of virus produced at 330C in the absence of superinfection varied from clone to clone; C12 and C13 were generally good producers, C11 was an average producer, and C10 was a poor producer. These differences correlated well with the amounts of viral DNA synthesized at 330C by cultures from the various clones (22), but not with the ability of the cells themselves to produce infectious centers when plated over monolayers of mouse embryo cells: all clones were identical in this respect (7). This is what one would expect if the resident viral genome could be activated with the same efficiency, but did not accumulate to the same extent, in every clone. Although they responded distinctively to temperature shiftdown, cultures from the five clones generally produced comparable amounts of virus after superinfection, whether performed at 39 or 330C. Thus, all clones appeared equally permissive, even though they were not equally inducible. Virus production was generally low at 330C for mock-infected C12/al cells. We should recall here that C12/al cells are unique in that they synthesize predominantly RmI rather than ge-
TABLE 1. Synthesis of viral hemagglutinin by Cyp cells with and without superinfectiona Hemagglutinating absorption units per 100-mm petri dish Cellsb
Mock infection at: 39°C
C10 C1l C12
a a a a C13 a a c C12/al a b c MEd a
330C
Infection with P16 at:
390C
330C
Infection with A2 at:
390C
330C
