FUNCTIONAL-CHARACTERIZATION OF THE MAJOR LATE PROMOTER OF MOUSE ADENOVIRUS TYPE-1

During the late phase of adenovirus infection, the major late promoter (MLP) controls the regulated expression of the genes that encode most viral structural proteins. Recently, the region of the genome of mous e adenovirus type 1 (MAV-1), predicted to contain the MLP, was sequenc ed and compared to that of the human virus MLP. The general organizati on of the transcriptional elements of the putative MAV-1 MLP is simila r to that of the human virus counterpart, with some interesting differ ences. We wished to investigate the function of the predicted MLP of M AV-1 and to determine the significance of the differences found in the MAV-1 MLP. To test the activity of the predicted MLP of MAV-1, both N orthern blot and primer extension analyses were performed on intracell ular RNA isolated from cells infected with MAV-1. The results show tha t late RNA can be detected 48 hr postinfection and increases up to 6 d ays p.i. Primer extension analysis revealed that the major start sites of transcription are 28 and 31 nt downstream of the first T residue o f the predicted TATA box. To analyze the functional significance of th e predicted transcriptional elements, a transient transfection system, using the firefly luciferase gene controlled by the MAV-1 MLP sequenc e, was established. The predicted MLP sequence was capable of directin g luciferase gene expression, to a level some 60% of that of the human virus MLP. Mutations were created in the inverted CAAT box, the SP1 s ite, and the TATA box, either singly or in combination. Each single-el ement mutation causes a marked reduction in luciferase gene expression , with the SP1 mutation showing the greatest effect. Double mutations were even more deficient, suggesting a level of functional redundancy among the various transcriptional elements. Finally, the putative SP1- binding site was examined by gel mobility shift assay and shown to int eract with purified SP1 protein specifically, supporting the functiona l significance of this transcriptional element. These findings contrib ute to a better understanding of gene expression in MAV-1 and to its d evelopment as an appropriate model for the study of the molecular basi s of pathogenesis in a natural host animal. (C) 1997 Academic Press.