Evidence that the major delayed-early DNA-binding proteins of herpesvirus saimiri are bound to DNA in vivo

Associations of herpesvirus saimiri-specific proteins with nuclear fractions from cultures of infected cells were probed by nuclease digestion, detergent extractions and immunofluorescence microscopy using monoclonal antibodies to virus polypeptides. Nuclease digestion selectively released delayed-early polypeptides with apparent mol. wt. of 110 000 (110K) and 51 000 (51K) from nuclei of infected cultures and the majority of each of these polypeptides partitioned with the insoluble fraction after detergent extraction of such nuclei. However, the nuclease mediated release of both these proteins was specifically reduced when nuclei were isolated from cultures in which virus DNA synthesis had been inhibited with phosphonoacetic acid (PAA). In addition, the 110K polypeptide partitioned into the soluble fraction when nuclei from PAA-treated cultures were extracted with detergent. Immunofluorescence microscopy revealed characteristic and distinctive subnuclear localizations of the 110K and 51K polypeptides in control cultures and these patterns of subnuclear accumulations were markedly altered in cultures treated with PAA. We conclude that the DNA-binding properties of the delayed-early 110K and 51K proteins of herpesvirus saimiri previously observed in vitro are likely to reflect their functions as DNA-binding proteins in vivo.