Linker insertion mutants of simian virus 40 large T antigen that show trans-dominant interference with wild-type large T antigen map to multiple sites within the T-antigen gene.
Linker insertion mutants affecting the simian virus 40 (SV40) large tumor (T) antigen were constructed by inserting a 12-base-pair oligonucleotide linker into restriction endonuclease cleavage sites located within the early region of SV40. One mutant, with the insertion at amino acid 5, was viable in CV-1p and BSC-1 cells, indicating that sequences very close to the amino terminus of large T could be altered without affecting the lytic infection cycle of SV40. All other mutants affecting large T were not viable. In complementation assays between the linker insertion mutants and either a late-gene mutant, dlBC865, or a host range/helper function (hr/hf) mutant, dlA2475, delayed complementation was seen with the 6 of the 10 nonviable mutants. Of these 10 mutants, 5 formed plaques 3 to 4 days later than in control complementations, while complementation by one of the mutants, inA2827, with an insertion at amino acid 520, was delayed more than 1 week. Most mutants which showed delayed complementation replicated less well in Cos-1 cells than did a control mutant, dlA1209, which produced no T antigen. The replication of inA2827(aa520) was reduced by more than 90%. Similar interference with viral DNA replication was seen when CV-1, HeLa, or 293 cells were cotransfected with an origin-defective plasmid encoding wild-type large T antigen and with inA2827(aa520). Only one of the mutant T antigens, inA2807(aa303), was unstable. These results indicate that some of the mutant T antigens interfered with functions of wild-type T required for viral DNA replication. However, not all of the mutants which showed delayed complementation also showed interference with viral DNA replication. This indicates that mutant large T antigens may interfere trans dominantly with multiple activities of wild-type large T antigen.
Zhu
J Y
JY
Department of Biochemistry, Dartmouth Medical School, Hanover, New Hampshire 03756.
Cole
C N
CN
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D001483:Base Sequence; D002460:Cell Line; D004261:DNA Replication; D004251:DNA Transposable Elements; D004279:DNA, Viral / Q000235:genetics; D005799:Genes, Dominant; D005814:Genes, Viral; D005816:Genetic Complementation Test; D007700:Kinetics; D008969:Molecular Sequence Data; D009154:Mutation; D015345:Oligonucleotide Probes; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics* / Q000276:immunology; D010948:Viral Plaque Assay; D015678:Viral Structural Proteins / Q000235:genetics*
D000952:Antigens, Polyomavirus Transforming; D004251:DNA Transposable Elements; D004279:DNA, Viral; D015345:Oligonucleotide Probes; D015678:Viral Structural Proteins