Genetic and biochemical analysis of transformation-competent, replication-defective simian virus 40 large T antigen mutants.
To study the role of the biochemical and physiological activities of simian virus 40 (SV40) large T antigen in the lytic and transformation processes, we have analyzed DNA replication-defective, transformation-competent T-antigen mutants. Here we describe two such mutants, C8/SV40 and T22/SV40, and also summarize the properties of all of the mutants in this collection. C8/SV40 and T22/SV40 were isolated from C8 and T22 cells (simian cell lines transformed with UV-irradiated SV40). Early regions encoding the defective T antigens were cloned into a plasmid vector to generate pC8 and pT22. The mutations responsible for the defects in viral DNA replication were localized by marker rescue, and subsequent DNA sequencing revealed missense and one nonsense mutation. The T22 mutation predicts a change of histidine to glutamine at residue 203. C8 has two mutations, one predicts lysine224 to glutamamic acid and the other changes the codon for glutamic acid660 to a stop codon; therefore, C8 T antigen lacks the 49 carboxy-terminal amino acids. pC8A and pC8B were constructed to contain the C8 mutations separately. Plasmids pT22, pC8, pC8A, and pC8B were able to transform primary rodent cell cultures. T22 T antigen is defective in binding to the SV40 origin. C8B (49-amino-acid truncation) is a host-range mutant defective in a late function in CV-1 but not BSC cells. Analysis of T antigens in mutant SV40-transformed mouse cells suggests that the replicative function of T antigen is important in generating SV40 DNA rearrangements that allow the expression of "100K" variant T antigens in the transformants.
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Gluzman
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D000818:Animals; D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor / Q000235:genetics*; D001483:Base Sequence; D002459:Cell Fusion; D002460:Cell Line; D002472:Cell Transformation, Viral; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D004261:DNA Replication; D005796:Genes; D005814:Genes, Viral; D007668:Kidney; D051379:Mice; D009154:Mutation; D010957:Plasmids; D011494:Protein Kinases / Q000235:genetics*; D013539:Simian virus 40 / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics*; D014779:Virus Replication
D000952:Antigens, Polyomavirus Transforming; D000957:Antigens, Viral, Tumor; D014764:Viral Proteins; D011494:Protein Kinases