| The adenovirus genome encodes a protein, called IVa2, that is needed for maximally efficient transcription from the viral major late (ML) promoter. Expression of the major late transcription unit is essential for the synthesis of the majority of viral late proteins. IVa2 protein is a sequence-specific DNA-binding protein that also promotes the assembly of progeny virus particles. Previous observations have established that a IVa2 protein dimer, termed DEF-B, binds specifically to one intragenic ML promoter sequence necessary for late phase-specific stimulation of ML transcription. However, binding of at least one additional infected cell-specific protein, termed DEF-A, to intragenic ML promoter sequences correlates with activation of transcription from the promoter. Using an assay for the DNA-binding activity of DEF-A, I have identified this protein by using conventional purification methods, purification of FLAG-tagged IVa2 protein-containing complexes, and transient synthesis of viral late proteins. The results of these experiments established that the viral L4 33KDa protein is the only component of DEF-A, and the IVa2 and L4 33 kDa proteins are necessary and sufficient for formation of all previously described complexes in the intragenic control region of the ML promoter. Futhermore, the L4 33 kDa protein binds to the ML promoter with the same specificity characteristic of DEF-A and stimulates transcription from the ML promoter in transient expression assays. Subsequently, I have constructed viruses with mutations in the downstream element (DE) region that prevent protein binding to the site. As the L4 33 kDa protein has been implicated in activation of MLP and also splicing of late-phase-specific mRNA, I sought to investigate the effects of these DE mutations on ML transcription and late-phase-specific splicing. However, data indicated that binding of IVa2 and L4 33-kDa proteins to adenovirus major late DE is not essential for stimulation of primary major late transcription in the context of infected HeLa cells. Furthermore, binding of L4 33-kDa protein to the DE site is not required for late-phase-specific preferential splicing of IIIa mRNA in virally infected HeLa cells. |
