DNA synthesis-dependent relief of repression of transcription from the adenovirus type 2 IVa₂ promoter by a cellular protein

The promoter of the human adenovirus type 2 IVa₂ gene, which becomes active only during the late phase of infection, is built largely from sequences spanning, and downstream of, the sites of initiation of transcription. These sequences comprise an initiator, an intragenic sequence necessary for efficient transcription from the promoter by RNA polymerase II, and an intragenic binding site for a cellular repressor of IVa₂ transcription. The properties of the latter protein, which is termed IVa ₂-RF, suggested that it might account for the viral DNA synthesis-dependent activation of IVa₂ transcription during the adenoviral productive cycle. Here we report the results of experiments to assess the contributions of DNA template concentration and IVa₂-RF binding to the activity of the IVa₂ promoter using a transient expression system. When a IVa₂-EGFP reporter gene was introduced into HeLa cells, in which IVa₂-RF was identified, no EFGP synthesis could be detected. In contrast, in IVa₂-RF-containing cells in which the plasmid carrying the chimeric gene replicated, synthesis of both the EGFP protein and the IVa₂-EGFP mRNA was readily detected. A vector mutation that blocked plasmid replication reduced IVa₂ promoter activity to undetectable levels. In contrast, a IVa₂ promoter substitution that impaired binding of IVa₂-RF increased IVa ₂ promoter activity under all conditions examined. Furthermore, introduction of DNA containing the IV-RF binding site with the chimeric reporter genes resulted in increased transcription from the IVa₂ promoter in the absence of plasmid replication. These properties are consistent with the hypothesis that the relative concentration of the IVa₂ promoter and of the cellular repressor that binds to it governs transcription from this adenoviral promoter.