Adeno-associated virus (AAV) is a common, nonpathogenic, helper-dependent human parvovirus. AAV has generated much enthusiasm in recent years because of its potential as a human gene therapy vector. Although vector development is progressing rapidly, there is a lack of understanding of how AAV amplification, gene expression and site-specific integration affect the helper virus and host cell. All three processes are dependent upon de novo expression of, and regulation by, viral replication (Rep) proteins, Rep78 and Rep68. Interestingly, exogenous expression of these proteins has profound effects on the host cell and helper virus. To understand the interactions between AAV and its environment, we have begun a study of the inhibitory effects of AAV on Adenovirus (Ad) replication and gene expression. We present evidence that AAV and its encoded Rep proteins inhibit Ad replication. These inhibitions are due, in part, to inhibition of early gene transcription. The Rep protein-mediated alterations on transcription are due to effects on the Ad early gene promoters as well as on E1A-mediated trans-activation of early gene expression. The following specific aims are designed to elucidate the mechanistic roles played by AAV and its Rep proteins in an Ad co-infection; (1) Characterize early interactions between AAV and Ad in a co-infection, (2) Define the Rep and E1A protein domains and Ad promoter elements involved in Rep regulation of Ad gene expression, (3) Characterize the biochemical mechanisms of Rep-mediated inhibition, and activation, of gene expression. This novel, model system of host-virus interaction benefits from the wealth of knowledge in the literature on Ad biology and the abundance of available Ad reagents for study. Determining how AAV interacts with its most efficient helper virus, we will obtain valuable insights into how AAV interacts with host cells as well. These studies will also have clear ramifications for further AAV vector development, especially with regard to improvement in AAV vector packaging systems.
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