The need for more convenient and versatile cloning vectors for the expression of foreign genes in eukaryotic cells has prompted us to develop a viral vector system that can accomodate and express a large range of foreign gene sequences. Our system is designed to allow specific selection of recombinant viruses that express high levels of foreign gene products under the control of viral transcription promoters. In the past three years, we have developed a number of strategies for expressing high levels of the SV40 tumor antigen in a mammalian viral vector system. We succeeded in obtaining overproduction of the SV40 A gene mRNA by constructing recombinant viruses in which the A gene was under the transcriptional control of the adenovirus major late promoter. However, we discovered that efficient translation of T mRNA appeared to require specific leader sequences to be attached to the 5' end of the T antigen mRNA. This finding let us to hypothesize that efficient initiation of translation in our host vector system requires specific cis-acting sequence elements that are commonly found at the 5' ends of adenovirus late mRNAs. Here we propose to identify the translational control signals in the leader sequences of late viral mRNAs and to explore the relationship between the function of the tripartite leader sequence and the selective translation of mRNAs in virus-infected host cells. We intend to further explore our ability to position foreign DNA within the adnovirus genome by homologous recombination. In addition to constructing and isolating recombinant viruses, we will also apply a battery of techniques to characterize the genome, transcript and protein structures of the genes expressed various adenovirus recombinants. Finally, we propose to generalize the expression system by constructing cloning vectors that contain the SV40 helper function gene fused to other foreign genes in order to select for expression of other genes. In particular, we plan to construct a series of recombinant vectors designed to express the tumor antigen of polyoma virus. Our interest in the biochemical properties of polyoma T antigen stems from studies which indicate that although the polyoma A gene encodes a polypeptide that is analogous in structure and function to the SV40 T antigen, there are significant differences between the arrangement of regulatory sequences that influence the T antigen mediated transcription and viral DNA synthesis in polyoma and SV40.
