CHARACTERIZATION OF THE NONSTRUCTURAL PROTEINS OF ADENO ASSOCIATED VIRUS

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I. Self-association of Rep 78. The replication initiator proteins of AAV-2, Rep78 and Rep68, are essential viral gene products that bind the viral origin of replication (ori), nick the ori DNA in a site- and strand-specific fashion (thus providing a 3' -OH group with which to prime viral DNA synthesis), and subsequently unwind the viral genomic duplex ahead of the advancing polymerase complex. We have investigated the ability of Rep78 to self-associate in vitro and in vivo. Site-directed Rep78 mutants were used to identify two motifs essential to oligomerization. These are i) a putative helix consisting of a 3,4-hydrophobic heptad repeat (or coiled-coil domain) within aa residues 160-185, and ii) a previously recognized nucleoside triphosphate (NTP)-binding motif occurring within aa residues 332-346. The assembly state of Rep78 oligomers was ascertained by gel filtration chromatography in the presence or absence of ori substrate and by chemical cross-linking analyses. These experiments demonstrated that Rep78 exists predominantly as a monomeric species in the absence of DNA substrate; however, in the presence of AAV ori sequences, Rep78 forms a hexameric protein complex with a Stokes radius of approximately 6.3nm. These results demonstrate that Rep78 is a hexameric helicase. II. Heterologous intermolecular interactions. Transient expression of Rep has a dramatic phenotypic affect ranging from repression of cellular gene expression to cytostasis and cell death. The Rep induced phenotype may be attributable in part to the DNA binding activities of Rep, however, since promoters which lack a Rep binding sequence are also repressed, an alternative explanation is required. It is likely then, that these effects are due to interactions between Rep and cellular proteins. Using a yeast two hybrid screening system, several cDNAs have been isolated which encode proteins that interact with Rep. One of these candidates is a novel cellular kinase that binds to Rep 52. Rep 52 and Rep 78 are readily co-immunoprecipitated with the kinase whereas Rep 68 is only weakly co-immunoprecipitated. Deletional analysis confirms that the carboxy terminus of Rep 52 and Rep 78 are necessary for the interaction. Trans- and auto- phosphorylation activity of this kinase is inhibited by Rep 52. Thus, we have identified a cellular kinase that differentially interacts with the unspliced and spliced forms of Rep, i.e. Rep 78, Rep 52 and Rep 68, Rep 40 respectively.