In vivo characterization of the poly(ADP-ribosylation) of SV40 chromatin and large T antigen by immunofractionation

Abstract

We have confirmed the poly(ADP-ribosylation) of large T antigen of SV40 by using antibodies to both large T antigen and poly(ADP-ribose) and consequently have begun to characterize how this post-translational nuclear modification of the viral protein modulates its biological functions. SV40 minichromosomal subpopulation containing replicative intermediate DNA was shown to have a significantly higher affinity for anti-poly(ADP-Rib)-Sepharose than viral chromatin fractions containing mature minichromosomal DNA. An anti-large T-Sepharose column was used to isolate T antigen from crude extracts by two different approaches: (1) large T antigen was labeled with [35S]methionine in vivo and the infected cell extract was immunofractionated to isolate large T antigen and (2) large T antigen from infected cell extracts was immunofractionated followed by immunostaining. Using these techniques, 1-10% of the total T antigen from infected cells was found to be poly(ADP-ribosylated). Minichromosome preparations per se were also subjected to immunofractionation on anti-large T-Sepharose. The high level of retention of poly(ADP-ribosylated) species of minichromosomes on this matrix suggested that this post-translational modification of viral chromatin may be related to those steps in viral replication and transcription under regulation by large T antigen.