Identification of a novel downstream bingind protein implicated in late-phase-specific activation of the adenovirus major late promoter

Abstract The adenovirus major late promotor (MLP) is induced to very high levels after the onset of the viral DNA replication. Previous studies have identified sequence elements located downstream of the MLP startsite (DE1, between +85 and +98; DE2, between +100 and +120) implicated, together with the upstream promoter element, in this late-phase-specific transcriptional activation. One protein (DEF, now renamed DEF-A), induced during the late phase of viral infection, has been identified and shown to bind to the DE1 element (Jansen-Durr et al., 1989, J. Virol. 63, 5124-5132). Here we report about a distinct late-phase-specific protein (DEF-B) and its interactions with DEF-A. DNA-binding studies reveal that DEF-B interacts with the 5' part of DE2 (DE2b), whereas DEF-A, besides its interaction with DE1, also binds to the 3' portion of DE2 (DE2a), but with a lower affinity than for DE1. Furthermore, when added together, DEF-A and DEF-B cooperatively assemble onto the DE2 element as a heteromeric complex which is substantially more stable than the complexes formed by each protein alone. Using an in vivo transcriptional assay of the MLP, we show that DEF-A and DEF-B both have intrinsic transactivating properties.