The regulatory rep protein of adeno-associated virus binds to sequences within the c-H-ras promoter

The large rep gene products (rep68 and rep78) of adeno-associated virus (AAV) are pleiotropic effector proteins which not only play a critical role in AAV DNA replication and in the trans-regulation of AAV promotor elements, but are also known for their onco-suppressive functions. We have previously demonstrated that the large AAV rep protein will strongly inhibit expression from the c-H-ras promoter, but not the murine osteosarcoma virus long terminal repeat (MSV-LTR) promoter. To investigate the possibility that rep may physically bind to these promoter sequences, specifically to GCTC motifs, we conducted electrophoretic mobility shift assays (EMSA) with a maltose binding protein-rep chimeric protein, MBP-rep68 delta, and synthetic double stranded DNA substrates of sequences selected from the c-H-ras and MSV-LTR promoters, as well as with the AAV TR. We find that MPB-rep68 delta bound the AAV TR DNA sequence (three motifs) most strongly, followed by the selected c-H-ras DNA sequence (two noninterfering motifs), and most poorly to the MSV-LTR DNA (one motif). These data are consistent with our previous study and suggest a direct mechanism of action for AAV rep inhibition of the c-H-ras promoter. Furthermore, the results suggest that the number of GCTC motifs, when closely associated, affect the affinity of rep binding. Finally, we find that MBP-rep68 delta also binds to the c-H-ras oligomer substrates which have secondary hairpin structures.