Adenovirus vector systems permitting regulated protein expression and their use for in vivo splicing studies

Abstract: We have constructed two adenovirus-based gene expression vector systems permitting regulated protein expression. They are based on the tetracycline-regulated Tet-ON- and the progesterone antagonist RU 486-regulated gene expression systems, which were rescued into E1-deficient adenovirus vectors. The vectors function in a number of cell types representing a broad species-variety and the regulation of protein expression was shown to be tightly controlled in cells not permissive for virus replication. Furthermore, the adenovirus-Tet-ON system was shown to perform in mice after intramuscular administration.The novel adenovirus-vector systems were then used to study the effects of overexpression of selected proteins on adenovirus replication during a lytic infection, with focus on regulation of adenovirus alternative splicing. Expression of adenovirus transcription units is to a large extent temporally regulated at the level of alternative pre-mRNA splicing, where viral splice site usage shifts from proximal to distal splice site selection as infection proceeds. This makes adenovirus an appropriate model for mechanistic studies of regulated splicing. We show that overexpression of the essential host cell splicing factor ASF/SF2 inhibits this shift by promoting usage of proximal splice sites. As a consequence, the virus displayed a markedly inhibited growth. Interestingly, mRNA expression from the adenovirus major late promoter was almost completely lost as a consequence of ASF/SF2 overexpression. Collectively, the cellular splicing factor ASF/SF2 prevents adenovirus from entering the late phase of infection. This strongly argues for a need for the virus to block the splicing enhancer activity of ASF/SF2 for establishment of a lytic infection. Further, from analysis of the strict inhibition of late region 1 late pre-mRNA splicing we propose that the temporal regulation of alternative splicing is merely a consequence of fitness rather than profoundly deleterious effects of an unregulated expression. During our studies we noted that in 293 cells, which are used for growth of E1-deficient Ad vectors, an unwanted background reporter gene expression was evident in our vector systems. We therefore introduced an additional regulatory element, functioning as a transcriptional road-block, and showed that this methodological innovation represents a way to overcome the potentially deleterious effects of background reporter gene expression. This modified viral vector system should make it possible to reconstruct recombinant viruses expressing highly toxic proteins.In conclusion, this work presents a new in vivo model system to study proteins involved in RNA splicing and other gene regulatory mechanisms.