Adeno-associated virus (AAV) is a non-pathogenic human DNA virus which contains a linear, single-stranded, DNA genome, approximately 5kb long. One of its open reading frames, the rep gene, codes for a family of 4 viral nonstructural proteins and is absolutely required for DNA replication. In addition to being essential for DNA replication, the two larger Rep proteins (Rep68 and 78) also are required for transactivation of AAV transcription, repression of AAV and heterologous gene expression, and the repression of oncogenic transformation by papilloma virus. In our previous work we purified the AAV Rep68 and 78 proteins and we found that they contain three biochemical activities: 1) an ATP dependent site-specific and strand-specific endonuclease activity which specifically cuts the AAV origin at the terminal resolution site (trs), 2) the ability to covalently attach to the 5' end of the cut site, and 3) a DNA helicase activity. These results provide a plausible role for the AAV Rep proteins in DNA replication and we will continue this work by characterizing in detail the biochemical activities of Rep68 and 78. In addition to our work with purified Rep proteins we have also developed an in vitro terminal resolution assay using crude AAV infected extracts. In this reaction AAV terminal repeats which are in the hairpined, covalently closed conformation can be converted to a normal extended duplex form. The reaction relies on the Rep protein and cellular enzymes and it should be useful for identifying which cellular DNA polymerase and accessory proteins are required for AAV DNA replication. Finally, in the absence of a helper virus AAV integrates into human chromosomes and persists as a provirus. The frequency of integration is extraordinarily high and the reason for this appears to be that AAV genomes preferentially integrate into a sequence present on human chromosome 19. In our previous work we generated a large number of proviral cell lines and we now have an opportunity to determine the sequence of the AAV proviruses and deduce a mechanism for AAV integration.
Our aims are: 1) to characterize in detail the biochemical activities of the purified Rep proteins, 2) to identify the features of the AAV terminal repeat that are recognized by the Rep proteins, 3) to identify the cellular DNA replication enzymes which are required for AAV DNA replication, and 4) to continue our studies of the mechanism of AAV integration.
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