Characterization of the T antigen content of simian virus 40 viral transcription complexes.

Abstract

Simian virus 40 large tumor antigen (T-antigen) is known to be involved in both the repression of early viral RNA synthesis and the activation of late RNA synthesis. However no direct evidence indicating that T-antigen is associated with transcriptionally active minichromosomes has been presented. Therefore the T-antigen content of transcriptionally active minichromosomes was specifically investigated. Both monoclonal and polyclonal antibodies directed against T-antigen can immunoprecipitate between 10 and 30% of the viral late transcription complexes that were initiated in vivo and can be extended in vitro. Characterization of these in vitro extendable transcription complexes indicates that 70% contain RNA polymerase II molecules located well within the late genes and that 30% of the in vitro transcription activity is derived from RNA polymerases located within 100 base pairs of the late RNA initiation sites. T-antigen associated transcription complexes are preferentially deficient in promoter proximal RNA polymerases, suggesting that T-antigen is associated with transcription complexes that are actively producing mRNA in vivo. Immunoprecipitation analysis of viral transcription complexes labeled in vivo confirms that T-antigen is preferentially associated with transcription complexes which are actively producing mRNA in vivo. Determination of where T-antigen is associated with in vitro extendable transcription complexes indicates that (i) T-antigen is not bound to transcriptionally active minichromosomes at its high affinity binding sites located at the origin of replication and (ii) T-antigen is associated with transcriptionally active minichromosomes at multiple points located over the 3$\sp\prime$ end of the late genes. These results indicate that a direct association of T-antigen with its origin binding sites is not required to repress early RNA synthesis from late transcription complexes and that T-antigen may activate late transcription by a mechanism involving a direct association of T-antigen with transcription complexes as opposed to, or in addition to, T-antigen activating late transcription in an indirect manner.