Identification of a Cellular Repressor of Transcription of the Adenoviral Late IVa2 Gene That Is Unaltered in Activity in Infected Cells

Abstract

The gene encoding the adenovirus type 2 IVa(2) protein, a sequence-specific activator of transcription from the viral major late promoter, is itself transcribed only during the late phase of infection. We previously identified a cellular protein (IVa(2)-RF) that binds specifically to an intragenic sequence of the IVa(2) transcription unit. We now report that precise substitutions within the IVa(2)-RF-binding site that decreased binding affinity increased the efficiency of IVa(2) transcription in in vitro reactions containing IVa(2)-RF. Consistent with the conclusion that this cellular protein represses IVa(2) transcription, mutations that led to more efficient transcription in the presence of IVa(2)-RF were without effect in reactions lacking this cellular protein. No change in the concentration or activity of IVa(2)-RF could be detected in adenovirus-infected cells during the period in which the IVa(2) gene is transcribed. We therefore propose that restriction of IVa(2) transcription to the late phase is the result of titration of this cellular repressor as the number of copies of the IVa(2) promoter increases upon replication of the viral genome.

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

“…The data shown in Fig. 5A therefore establish unequivocally that viral DNA synthesis-dependent titration of a cellular transcriptional repressor is responsible for activation of IVa 2 transcription during the adenoviral infectious cycle, as predicted on the basis of biochemical analyses (31,32). Such a mechanism of temporal control of viral transcription is extremely efficient: no dedicated viral gene products are required to activate transcription of late genes, but rather the essential production of progeny viral genomes is coupled to the activity of specific viral promoters.…”

“…5B). These modest effects of the Rep mutations could therefore be a direct result of increased activity of the ML promoter, analogous to those previously observed in vitro (31). Experiments are in progress to distinguish such cis and trans mechanisms of stimulation of ML transcription by the Rep mutations.…”

“…We previously identified a number of substitution mutations that impair binding of the cellular transcriptional repressor to the Ad2 adenoviral IVa 2 promoter and increase the activity of this promoter in vitro (31). Two of these mutations, Rep6 and Rep7, which introduce one conservative amino acid substitution, and no change, respectively, into the overlapping coding sequence of the viral DNA polymerase (Fig.…”

“…Young, A. Timko, and S.J.F., unpublished observations). An initiator element spanning positions Ϫ4 to ϩ11 specifies recognition of the major initiation site (29)(30)(31), but efficient initiation from this site in vitro requires the intragenic sequence shown in Fig. 1 (29,30).…”

“…Superimposed on these IVa 2 promoter sequences is a binding site for a cellular repressor of transcription, termed IVa 2 -RF (31,32). Because adenovirus infection does not lead to inactivation of this repressor, we proposed that activation of IVa 2 transcription is the direct consequence of the increase in concentration of IVa 2 promoters as progeny viral genomes are synthesized (31). We have used transient expression assays to demonstrate that this repressor titration mechanism can regulate IVa 2 transcription in mammalian cells (33).…”