Overexpression of cyclin A inhibits augmentation of recombinant adeno-associated virus transduction by the adenovirus E4orf6 protein

The 34-kDa product of adenovirus E4 region open reading frame 6 (E4orf6) dr amatically enhances transduction by recombinant adeno-associated virus vect ors (rAAV). This is achieved by promoting the conversion of incoming single -stranded viral genomes into transcriptionally competent duplex molecules. The molecular mechanism for enhancing second-strand synthesis is not fully understood. In this study, we analyzed the cellular consequences of E4orf6 expression and the requirements for efficient rAAV transduction mediated by E4orf6. Expression of E4orf6 in 293 cells led to an inhibition of cell cyc le progression and an accumulation of cells in S phase. This was preceded b y specific degradation of cyclin A and p53, while the levels of other prote ins involved in cell cycle control remained unchanged. In addition, the kin ase activity of cdc2, was inhibited. We further showed that p53 expression is not necessary or inhibitory for augmentation of rAAV transduction by E4o rf6. However, overexpression of cyclin A inhibited E4orf6-mediated enhancem ent of rAAV transduction. A cyclin A mutant incapable of recruiting protein substrates for cdk2 was unable to inhibit E4orf6-mediated augmentation. In addition, we created an E4orf6 mutant that is selectively defective in rAA V augmentation of transduction. Based on these findings,we suggest that cyc lin A degradation represents a viral mechanism to disrupt cell cycle progre ssion, resulting in enhanced viral transduction. Understanding the cellular pathways used during transduction will increase the utility of rAAV vector s in a wide range of gene therapy applications.