A deletion in the simian virus 40 large T antigen impairs lytic replication in monkey cells in vivo but enhances DNA replication in vitro: new complementation function of T antigen.
We describe a new complementation function within the simian virus 40 (SV40) A gene. This function is required for viral DNA replication and virus production in vivo but, surprisingly, does not affect any of the intrinsic enzymatic functions of T antigen directly required for in vitro DNA replication. Other well-characterized SV40 T-antigen mutants, whether expressed stably from integrated genomes or in cotransfection experiments, complement these mutants for in vivo DNA replication and plaque formation. These new SV40 mutants were isolated and cloned from human cells which stably carry the viral DNA. The alteration in the large-T-antigen gene was shown by marker rescue and nucleotide sequence analysis to be a deletion of 322 bp spanning the splice-donor site of the first exon, creating a 14-amino-acid deletion in the large T antigen. The mutant gene was expressed in H293 human cells from an adenovirus vector, and the protein was purified by immunoaffinity chromatography. The mutant protein directs greater levels of DNA replication in vitro than does the wild-type protein. Moreover, the mutant protein reduces the lag time for in vitro DNA synthesis and can be diluted to lower levels than wild-type T antigen and still promote good replication, which is in clear contrast to the in vivo situation. These biochemical features of the protein are independent of the source of the cellular replication factors (i.e., HeLa, H293, COS 7, or CV1 cells) and the cells from which the T antigens were purified. The mutant T antigen does not transform Rat-2 cells. Several different models which might reconcile the differences observed in vivo and in vitro are outlined. We propose that the function of T antigen affected prepares cells for SV40 replication by activation of a limiting cellular replication factor. Furthermore, a link between the induction of a cellular replication factor and transformation by SV40 is discussed.
Maulbecker
C
C
Laboratory of Chemical Biodynamics, Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
Mohr
I
I
Gluzman
Y
Y
Bartholomew
J
J
Botchan
M
M
D000595:Amino Acid Sequence; D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000737:chemistry / Q000502:physiology*; D002460:Cell Line; D002472:Cell Transformation, Viral / Q000235:genetics; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D004261:DNA Replication; D005091:Exons; D005816:Genetic Complementation Test; D000882:Haplorhini; D007700:Kinetics; D008969:Molecular Sequence Data; D010957:Plasmids; D013539:Simian virus 40 / Q000235:genetics / Q000502:physiology*; D014779:Virus Replication
D000952:Antigens, Polyomavirus Transforming