Viral DNA synthesis-dependent titration of a cellular repressor activates transcription of the human adenovirus type 2 IVa2 gene

Synthesis of progeny DNA genomes in cells infected by human subgroup C adenoviruses leads to several changes in viral gene expression. These changes include transcription from previously silent, late promoters, such as the IVa2 promoter, and a large increase in the efficiency of major-late (ML) transcription. Some of these changes appear to take place sequentially, because the product of the IVa2 gene has been implicated in stimulation of ML transcription. Our previous biochemical studies suggested that IVa2 transcription is regulated by viral DNA synthesis-dependent relief of transcriptional repression by a cellular protein that we termed IVa2-RF. To test the relevance of such a repressor-titration mechanism during the viral infectious cycle, we introduced into the endogenous IVa2 promoter two mutations that impair in vitro-binding Of IVa2-RF, but introduce no change (Rep7) or one conservative amino acid substitution (Rep6) into the overlapping coding sequence for the viral DNA polymerase. The results of run-on transcription assays indicated that both mutations induced earlier-than-normal and more efficient IVa2 transcription. Both mutations were also observed to result in modest increases in the efficiency of viral DNA synthesis. However, measurement of the concentration of IVa2 transcripts as a function Of IVa2 template concentration demonstrated that the Rep mutations increased by up to 60-fold the efficiency with which IVa2 templates were used during the initial period of the late phase of infection, as predicted by the repressor titration hypothesis. These mutations also increased the efficiency of ML transcription in infected cells.