A nonlethal mutation in large T antigen of polyomavirus which affects viral DNA synthesis

Section: Resultssupporting

confidence: 63%

Polyomavirus tumor induction in mice: effects of polymorphisms of VP1 and large T antigen

Freund

Calderone

Dawe

et al. 1991

“…(Table 1, experiment 2). We assume that this effect is due to complementation by the B2 parent of a large-T-antigen mutation which was very recently discovered in strain RA (19). Similar examples of complementation have been documented previously between large-T-antigen and middle-T-antigen mutants (10).…”

Section: Resultssupporting

confidence: 69%

“…The pseudo-wild-type strain NG59RA (RA) was obtained by marker rescue (7) of the middle-T-antigen defect of hr-t mutant NG59 (3). Differences between RA and A2 in the enhancer-origin region (24), as well as a large-T-antigen mutation in RA (19), have been noted previously. RA and A2 also differ in plaque morphology: the former is a small-plaque virus with a heat-stable hemagglutination pattern (6), while the latter is a large-plaque virus.…”

Section: Methodssupporting

confidence: 63%

“…We considered the possibility that the underrepresentation of hr-t mutant B2 in transformants derived from mixed infections with strain RA is specific to this particular pair of genomes. This possibility needs to be considered, since RA and B2 differ at multiple sites: the origin-enhancer (K. Higgins, K. Friderici, M. C. Chen, and M. M. Fluck, unpublished data), the large-Tantigen coding region (between EcoRI and NsiI) (19), and the capsid region (6;Friderici and Fluck,unpublished data). To analyze the role of differences in the origin-enhancer region (encompassing binding sites for large T antigen and transcription factors), strain RA(eB2) was constructed, in which the small BglI-BclI fragment comprising the originenhancer region from strain RA was replaced by the equivalent region from mutant B2.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Low probability of double integration in transformation of nonpermissive cells by polyomavirus

Amalfitano

Friderici

et al. 1990

Self Cite

The fate of polyomavirus genomes in stable transformants was examined in experiments in which rat or hamster cells were infected with mixed viral populations containing either two distinguishable wild types or a wild type plus a transformation-deficient mutant. The results demonstrate that in 90% of the cases in either situation, a single polyomavirus parental genome became integrated into the host genome. Both parents in the mixed infection were present in the same cells and both persisted in the infected cells until transformants appeared, eliminating the possibility that one virus would exclude the other in the early steps of the infection process. We believe that these results can be generalized to transformation events derived from normal single infections. Thus, contrary to previous results for the number of integration sites determined by restriction endonuclease and blot hybridization analysis, it appears that the probability for more than one integration event in transformation by polyomavirus is low (1 in 10). A reinterpretation of previous data is proposed.

“…First, their constructs were derived from a different viral strain. Compared to strain A2, strain 59RA has a number of base substitutions in the intergenic region (11,26), as well as a mutation in the large T-antigen gene which results in a replication defect in rat F-111 cells (31). Second, the polyomavirus sequences used by Kern et al included about one-half of the early coding region of the viral genome, making them capable of expressing small and middle T antigens and the amino-terminal half of large T antigen.…”

Section: Discussionmentioning

confidence: 99%

Replication-dependent transactivation of the polyomavirus late promoter

Cahill

Roome

Carmichael

1990

When a plasmid containing the wild-type polyomavirus intergenic regulatory region fused to the bacterial cat gene was introduced into mouse NIH 3T3 cells along with a plasmid coding for the early viral proteins (T antigens), chloramphenicol transacetylase enzyme activity and mRNA levels were increased about 10-fold over levels observed in the absence of early proteins. To investigate this transactivation phenomenon further, 11 specific deletion mutant derivatives of the wild-type parent plasmid were constructed and studied. One mutant (NAL) with a minimal level of chloramphenicol transacetylase expression in the absence of T antigens was capable of being transactivated more than 40-fold. A number of other mutants, however, had little capacity for transactivation. Each of these mutants had in common a defect in large T-antigen-mediated DNA replication. Interestingly, one of the transactivation-defective mutants showed a basal late promoter activity fivefold higher than that of wild type and replicated in mouse cells in the absence of large T antigen. Subsequently, a small deletion abolishing viral DNA replication was introduced into those mutants capable of transactivation. The effect of the second deletion was to eliminate both replication and transactivation. Finally, wild-type and mutant constructs were transfected into Fisher rat F-111 cells in the presence or absence of early proteins. No transactivation or replication was ever observed in these cells. We concluded from these studies that the observed transactivation of the polyomavirus late promoter by one or more of the viral early proteins was due to either higher template concentration resulting from DNA replication or replication-associated changes in template conformation.