SV40 T-antigen binding to site II is functionally separated from binding to site I.

During lytic infection SV40 T antigen binds specifically to three different regions of the SV40 DNA to initiate viral DNA replication and to regulate early and late transcription. We have used the recently described plasmids pKB1, containing a 23-bp oligonucleotide coding for site I, pdl1085 containing sites II and III together with SV40 specific flanking sequences, and as a control pATC, a plasmid which contains all three binding sites (D. Müller et al. (1987), Virology 161, 81-91) to analyze the differential binding of T antigen to these individual binding sites in the course of an SV40 infection. We found that shortly after infection the amount of bound DNA increased with the concentration of T antigen reaching a steady-state level at about 20 hr after infection. In comparison to binding at site I, binding to site II appeared with a delay of about 8-9 hr corresponding to the onset of viral DNA replication. The correlation between binding of T antigen to site II and the SV40 DNA replication could be further corroborated by using T antigen from the heat-sensitive mutant tsA58 which completely failed to bind to site II at nonpermissive temperature but exhibited a residual binding to site I. This reduced binding to site I proved insufficient for the proper functioning of autorepression. Our results support the hypothesis that distinctly different subclasses of T-antigen binding to site I or site II may exist.