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Integration and excision of polyoma virus genomes

Basilico C; Zouzias D; Della-Valle G; Gattoni S; Colantuoni V; Fenton R; Dailey L
PMID: 6253162
ISSN: 0091-7451
CID: 14459

Loss of integrated viral DNA sequences in polyomatransformed cells is associated with an active viral A function

Basilico C; Gattoni S; Zouzias D; Valle GD
Rat cells transformed by polyoma virus contain, in addition to integrated viral DNA, a small number of nonintegrated viral DNA molecules. The free viral DNA originates from the integrated form through a spontaneous induction of viral DNA replication in a minority of the cell population. Its presence is under the control of the viral A locus. To determine whether the induction of free viral DNA replication was accompanied by a loss of integrated viral DNA molecules in a phenomenon similar to the 'curing' of lysogenic bacteria, we selected for revertants arising in the transformed rat populations and determined whether these cells had lost integrated viral genomes. We further investigated whether the viral A function was necessary for 'curing' by determining the frequency of cured cells in populations of rat cells transformed by the ts-a mutant of polyoma virus following propagation at the permissive or nonpermissive temperature. A large proportion of the revertants isolated were negative or weakly positive when assayed by immunofluorescence for polyoma T antigen and were unable to produce infectious virus upon fusion with permissive mouse cells. The T antigen-negative, virus rescue-negative clones can be retransformed by superinfection and appear to have lost a considerable proportion of integrated viral DNA sequences. Restriction enzyme analysis of the integrated viral DNA sequences shows that the parental transformed lines contain tandem repeats of integrated viral molecules, and that this tandem arrangement is generally lost in the cured derivatives. While cells transformed by wild-type virus undergo 'curing' with about the same frequency at 33 degrees or 39 degrees C, cells transformed by the ts-a mutant contain a much higher frequency of cured cells after propagation at 33 degrees than at 39 degrees C. Our results indicate that in polyoma-transformed rat cells, loss of integrated viral DNA can occur at a rather high rate, producing (at least in some cases) cells which have reverted partially or completely to a normal phenotype. Loss of integrated viral DNA is never total and appears to involve an excision event. The polyoma A function (large T antigen) is necessary for such excision to occur. In the absence of a functional A gene product, the association of the viral DNA with the host DNA appears to be very stable
PMID: 225038
ISSN: 0092-8674
CID: 14460

T-antigen expression in proliferating and non-proliferating simian virus 40-transformed mouse cells

Zouzias D; Basilico C
Previous studies with simian virus 40-transformed mouse 3T3 cells which are temperature sensitive for the expression of the transformed phenotype (ts SV3T3 cells) have shown that T-antigen expression and viral DNA transcription are under cell cycle control. Using these ts SV3T3 cells, we studied the expression of the viral genome under proliferating and non-proliferating conditions, in the presence and absence of inhibitors of macromolecular synthesis and of the tumor promoter phorbol myristate acetate. ts SV3TE cells which are growth arrested at 39 degrees C by low serum concentration or saturation density accumulated in G1 and did not express T-antigen. When these cells were induced to proliferate, at either 32 or 39 degrees C, T-antigen synthesis preceded the entry of the cells into the S-phase and was not coupled to DNA replication. G1-arrested ts SV3T3 cells were induced to synthesize T-antigen by phorbol myristate acetate treatment, but T-antigen alone was not sufficient to induce cellular DNA synthesis. Isoleucine deprivation arrested growth of ts SV3T3 cells, but these cells, as well as normal 3T3, did not accumulate in G1 and continued to express T-antigen. The temperature-sensitive expression of the transformed phenotype in the ts SV3T3 cells does not appear to be due to a lack of transcription of specific regions of the integrated simian virus 40 genome at 39 degrees C
PMID: 225540
ISSN: 0022-538x
CID: 14461

Isolation and preliminary characterization of the small circular DNA present in African green monkey kidney (BSC-1) cells

DeLap RJ; Rush MG; Zouzias D; Khan S
PMID: 107539
ISSN: 0147-619x
CID: 17081

Regulation of viral functions in simian virus 40-transformed cells

Zouzias D; Basilico C
To define the relationship between simian virus 40 (SV40)-specific T-antigen and cell growth and to look for regulatory mechanisms that might control T-antigen synthesis in transformed cells, we studied the expression of T-antigen and the viral transcription in SV40-transformed cells that were exponentially growing or arrested in the G1-phase of the cell cycle. We took advantage of the behavior of two lines of SV40-transformed mouse 3T3 cells (ts SV3T3), which, although transformed by wild-type SV40, are temperature sensitive for the expression of the transformed phenotype. At 32 degrees C, ts SV3T3 cells behave like standard transformants, whereas at 39 degrees C, they become arrested in G1 after reaching saturatio n density or under serum starvation. At 32 degrees C or growing at 39 degrees C, ts SV3T3 were 100% T-antigen positive and contained virus-specific mRNA. However, after G1 arrest at 39 degrees C, most of the cells became T-antigen negative. This seems to be caused by a lack of transcription of the integrated viral DNA, since these cells contain no appreciable amounts of SV40-specific RNA. Induction of proliferation in resting, T-antigen-negative ts SV3T3 cultures results in the reappearance of T-antigen a few hours before the cells enter DNA synthesis. These results suggest that transcription of the viral genome and T-antigen expression in SV40-transformed cells is subjected to a cell cycle control
PMID: 219356
ISSN: 0083-1921
CID: 14467

Nonintegrated viral DNA in rat cells doubly transformed by SV40 and polyoma virus

Prasad I; Zouzias D; Basilico C
PMID: 206012
ISSN: 0042-6822
CID: 14468


Zouzias, DC; Basilico, C
ISSN: 0014-9446
CID: 29910

State of the viral DNA in rat cells transformed by polyma virus. II. Identification of the cells containing nonintegrated viral DNA and the effect of viral mutations

Zouzias D; Prasad I; Basilico C
F2408 rat cells transformed by polyoma virus contained integrated and nonintegrated viral DNA. The presence of nonintegrated viral DNA is under control of the A early viral function. Polyoma ts-a-transformed rat cells lose the free viral DNA when growth at the nonpermissive temperature (40 degrees C), but they reexpress it 1 to 3 days after they are shifted back to the permissive temperature. In contrast, rat cells transformed by a late viral mutant, ts-8, contain free viral DNA at both permissive and nonpermissive temperatures. Treatment of the transformed rat cells with mitomycin C produces a large increase in the quantity of free viral DNA and some production of infectious virus. Experiments of in situ hybridization, with 3H-labeled polyoma complementary RNA as a probe, show that only a minority (approximately 0.1%) of the transformed cells contain nonintegrated viral DNA at any given time. These results suggest that the presence of free viral DNA in polyoma-transformed rat cells is caused by a spontaneous induction of viral DNA replication, occurring with low but constant probability in the transformed cell population, and that the free viral DNA molecules originate from the integrated ones, probably through a phenomenon of excision and limited replication
PMID: 198573
ISSN: 0022-538x
CID: 14469

Symmetric transcription of simian virus 40 DNA in the nuclei of transformed mouse cells

Yanagi K; Zouzias D; Rush MG
PMID: 199162
ISSN: 0006-291x
CID: 17082

Regulation of viral transciption and tumor antigen expression in cells transformed by simian virus 40

Basilico C; Zouzias D
We have studied the expression of simian virus 40 (SV40) specific tumor antigen (T-antigen) and viral RNA in SV40-transformed mouse 3T3 cells that are temperature-sensitive for the expression of the transformed phenotype (ts SV3T3). Although transformed by wild-type SV40, ts SV3T3 cells at 32 degrees behave like standard transformants, while at 39 degrees they became arrested in G1 after reaching saturation density or under conditions of serum starvation. ts SV3T3 cells at 32 degrees or exponentially growing at 39 degrees are uniformly T-antigen positive. However, after G1 arrest at 39 degrees the majority of the cells becomes T-antigen negative. Induction of proliferation in the resting cultures results in the reappearance of T-antigen in most of the cells, concomitant with the induction of DNA synthesis. The reason for the disappearance of T-antigen from ts SV3T3 cells arrested in G1 seems to reside in a transcriptional control operating on the integrated viral DNA, since these cells contain no appreciable amounts of SV40 specific RNA. Viral RNA can be easily detected in cells growint at 32 degrees or at 39 degrees. The results suggest that transcription of the viral genome in SV40-transformed cells is cell-cycle-dependent
PMID: 180530
ISSN: 0027-8424
CID: 14474