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

Abstract

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 IV a2 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 IV a2 gene has been implicated in stimulation of ML transcription. Our previous biochemical studies suggested that IV a2 transcription is regulated by viral DNA synthesis-dependent relief of transcriptional repression by a cellular protein that we termed IV a2-RF. To test the relevance of such a repressor-titration mechanism during the viral infectious cycle, we introduced into the endogenous IV a2 promoter two mutations that impair in vitrobinding of IV a2-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 earlierthan-normal and more efficient IV a2 transcription. Both mutations were also observed to result in modest increases in the efficiency of viral DNA synthesis. However, measurement of the concentration of IV a2 transcripts as a function of IVa2 template concentration demonstrated that the Rep mutations increased by up to 60-fold the efficiency with which IV a2 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.late transcription ͉ repressor titration T he infectious cycles of animal viruses with DNA genomes are characterized by transcription of viral genes in a stereotyped temporal sequence, regardless of whether transcription is carried out by cellular or viral DNA-dependent RNA polymerases. The complexity of such temporal regulation of transcription increases with viral genome size. Nevertheless, transcription of viral late genes encoding structural proteins invariably depends on synthesis of progeny viral DNA genomes in the infected cell (reviewed in ref. 1). In some cases, the activities of a single viral protein control both viral DNA synthesis and late gene transcription. For example, the simian virus 40 early protein large T antigen is not only the origin-recognition protein required for initiation of viral DNA synthesis but also an activator of late transcription (see refs. 2-5). The transition from the early to the late transcriptional program is considerably more intricate in cells infected by DNA viruses with larger genomes, such as human adenoviruses.Synthesis of viral DNA in cells infected by human subgroup C adenoviruses, such as adenovirus type 2 or 5 (Ad2 or Ad5), results in transcription by cellular RNA polymerase II from three previously inactive viral promoters, those of the IX and IVa 2 genes and the E2 late promoter (reviewed in ref. 6) Such off-to-on swit...

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“…For simian virus 40, the large T antigen plays an essential role in the activation of viral promoters (5,27), and viral DNA replication attenuates in trans the repressor of viral late promoters (58,67). For adenovirus, both viral trans-acting factors (23,40) and viral DNA replication (53,54) are required for its late gene expression. Viral DNA replication facilitates late gene expression directly (53) or indirectly by promoting the expression of viral trans-acting factors (23) or recruitment of cellular transcription factors to late promoters (54).…”

The Adenovirus L4 33-Kilodalton Protein Binds to Intragenic Sequences of the Major Late Promoter Required for Late Phase-Specific Stimulation of Transcription

“…Transcription of the IVa 2 gene is activated upon viral DNA synthesis-dependent titration of a cellular repressor (10,27,36), making the IVa 2 protein available soon after the onset of the late phase. In contrast, the proposition that a late protein encoded within the ML transcription unit participates in ML transcription appears paradoxical: the L4 33-kDa protein cannot be made until the late phase-specific switches to transcription of the complete ML transcription unit and utilization of all polyadenylation sites (see the introduction) have taken place.…”

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