Two conditional tsA mutant simian virus 40 T antigens display marked differences in thermal inactivation.
We have characterized the simian virus 40 (SV40) origin-containing DNA (ori-DNA) replication functions of two SV40 conditional mutant T antigens: tsA438 A-V (tsA58) and tsA357 R-K (tsA30). Both tsA mutant T antigens, immunopurified from recombinant baculovirus-infected insect cells, mediated replication of SV40 ori-DNA in vitro to similar extents as did wild-type T antigen in reactions at 33 degrees C. However, at 41 degrees C, the restrictive temperature, while tsA438 T antigen still generated substantial levels of replication products, tsA357 T antigen did not support any detectable DNA synthesis. Furthermore, preincubation for approximately fourfold-longer time periods at 41 degrees C was required to heat inactivate tsA438 T antigen than to heat inactivate tsA357 T antigen. Unexpectedly, results of analyses of the various DNA replication activities of the two mutant T antigens did not correlate with results from ori-DNA replication reactions. In particular, although tsA357 T antigen was incapable of mediating replication at 41 degrees C at all protein concentrations examined, it displayed either wild-type levels or only partial reductions of the several T-antigen replication-associated activities. These data suggest either that tsA357 T antigen is defective in an as yet unidentified replication function of T antigen or that the combination of its partial defects result in a protein that is unable to support replication. The data also show that two conditional mutant T antigens can be markedly different with respect to thermal sensitivity.
Reynisdóttir
I
I
Department of Biological Sciences, Columbia University, New York, New York 10027.
Prives
C
C
D000957:Antigens, Viral, Tumor / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D002474:Cell-Free System; D002874:Chromosome Mapping; D004265:DNA Helicases / Q000378:metabolism; D004261:DNA Replication; D004264:DNA Topoisomerases, Type I / Q000378:metabolism; D004279:DNA, Viral / Q000235:genetics / Q000378:metabolism; D006358:Hot Temperature; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D011489:Protein Denaturation; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013539:Simian virus 40 / Q000235:genetics* / Q000276:immunology
D000957:Antigens, Viral, Tumor; D004279:DNA, Viral; D011994:Recombinant Proteins; D004265:DNA Helicases; D004264:DNA Topoisomerases, Type I