Nucleotide sequence changes in polyoma virus A gene mutants

By using a DNA fragment immunoassay, the binding of simian virus 40 (SV40) and polyomavirus (Py) large tumor (T) antigens to regulatory regions at both viral origins of replication was examined. Although both Py T antigen and SV40 T antigen bind to multiple discrete regions on their proper origins and the reciprocal origin, several striking difrerences were observed. Py T antigen bound efficiently to three regions on Py DNA centered around an MboII site at nucleotide 45 (region A), a Bgll site at nucleotide 92 (region B), and another MboII site at nucleotide 132 (region C). Region A is adjacent to the viral replication origin, and region C coincides with the major early mRNA cap site. Weak binding by Py T antigen to the origin palindrome centered at nucleotide 3 also was observed. SV40 T antigen binds strongly to Py regions A and B but only weakly to region C. This weak binding on region C was surprising because this region contains four tandem repeats of GPuGGC, the canonical pentanucleotide sequence thought to be involved in specific binding by T antigens. On SV40 DNA, SV40 T antigen displayed its characteristic hierarchy of affinities, binding most efficiently to site 1 and less efficiently to site 2. Binding to site 3 was undetectable under these conditions. In contrast, Py T antigen, despite an overall relative reduction of affinity for SV40 DNA, binds equally to fragments containing each of the three SV40 binding sites. Py T antigen, but not SV40 T antigen, also bound specifically to a region of human Alu DNA which bears a remarkable homology to SV40 site 1. However, both tumor antigens fail to precipitate DNA from the same region which has two direct repeats of GAGGC. These results indicate that despite similarities in protein structure and DNA sequence, requirements of the two T antigens for pentanucleotide configuration and neighboring sequence environment are different. * Corresponding author. MATERIALS AND METHODS Cells and viruses. Cos 7 cells are SV40-transformed monkey CV-1 cells (8). 3T6 cells were infected with Py wild-type strain LL 3. CV-1 cells were infected with SV40 wild-type strain 776 (33). Preparation of cell extracts for DNA binding. Extracts from 107 cells were made as follows. A 150-mm plate of cells was 532

mentioning

confidence: 99%

Simian virus 40 and polyomavirus large tumor antigens have different requirements for high-affinity sequence-specific DNA binding

Scheller¹,

Prives²

1985

“…Genetic analysis of the early region of the viral genome defined two complementation groups: host-range, transformation negative (hr-t) mutants (2,3), and temperature-sensitive early (tsA) mutants, which at the restrictive temperature are defective for viral DNA replication and have a strongly reduced capacity to transform cells (9, 10). hr-t mutations map in the intron region of IT and change mT and sT in the same way (3), whereas tsA mutations cause single amino acid changes in the carboxy-terminal region of IT (7,40). A third class of viable early mutants (mlT) has deletions between 89 and 95 map units and therefore has altered mT and IT ( Fig.…”

mentioning

confidence: 99%

Small and middle T antigens contribute to lytic and abortive polyomavirus infection

Türler¹,

Salomon²

1985

Using three different polyomavirus hr-t mutants and two polyomavirus mlT mutants, we studied induction of S-phase by mutants and wild-type virus in quiescent mouse kidney cells, mouse 3T6 cells, and FR 3T3 cells. At different times after infection, we measured the proportion of T-antigen-positive cells, the incorporation of [3H]thymidine, the proportion of DNA-synthesizing cells, and the increase in total DNA, RNA, and protein content of the cultures. In permissive mouse cells, we also determined the amount of viral DNA and the proportion of viral capsid-producing cells. In polyomavirus hr-t mutant-infected cultures, onset of host DNA replication was delayed by several hours, and a smaller proportion of T-antigen-positive cells entered S-phase than in wild-type-infected cultures. Of the two polyomavirus mlT mutants studied, dl-23 behaved similarly to wild-type virus in many, but not all, parameters tested. The poorly replicating but well-transforming mutant dl-8 was able to induce S-phase, and (in permissive cells) progeny virus production, in only about one-third of the T-antigen-positive cells. From our experiments, we conclude that mutations affecting small and middle T-antigen cause a reduction in the proportion of cells responding to virus infection and a prolongation of the early phase, i.e., the period before cells enter S-phase. In hr-t mutant-infected mouse 3T6 cells, production of viral DNA was <10% of that in wild-type-infected cultures; low hr-t progeny production in 3T6 cells was therefore largely due to poor viral DNA replication.

“…To confirm that the RNAs we have analyzed originate from stalled RNA polymerases, we examined the temperature dependence of their synthesis by using a temperature-sensitive virus, AT3ts25E (7). This strain has a substitution of cysteine for glycine in the carboxy terminus of the large T antigen (16,47). This mutation renders the protein thermolabile at the nonpermissive temperature (39ЊC).…”

Section: Fig 4 Effect Of Ribonuclease Treatment On the Elongation Rmentioning

confidence: 99%

RNA footprint mapping of RNA polymerase II molecules stalled in the intergenic region of polyomavirus DNA

Brabant

Acheson

1995

RNA polymerase II molecules that transcribe the late strand of the 5.3-kb circular polyomavirus genome stall just upstream of the DNA replication origin, in a region containing multiple binding sites for polyomavirus large T antigen. Stalling of RNA polymerases depends on the presence of functional large T antigen and on the integrity of large T antigen binding site A. To gain insight into the interaction between DNA-bound large T antigen and RNA polymerase II, we mapped the position of stalled RNA polymerases by analyzing nascent RNA chains associated with these polymerases. Elongation of RNA in vitro, followed by hybridization with a nested set of DNA fragments extending progressively farther into the stalling region, allowed localization of the 3 end of the nascent RNA to a position 5 to 10 nucleotides upstream of binding site A. Ribonuclease treatment of nascent RNAs on viral transcription complexes, followed by in vitro elongation and hybridization, allowed localization of the distal end of stalled RNA polymerases to a position 40 nucleotides upstream of binding site A. This RNA footprint shows that elongating RNA polymerases stall at a site very close to the position of DNA-bound large T antigen and that they protect approximately 30 nucleotides of nascent RNA against ribonuclease digestion.