CHARACTERIZATION OF THE NONSTRUCTURAL PROTEINS OF ADENO ASSOCIATED VIRUS

腺相关病毒AAV的四种非结构蛋白（Rep）

The four non-structural proteins (Rep) of adeno-associated virus, AAV, are encoded by a single open reading frame, the rep gene, and differ from each other by utilization of an downstream promoter and excision of an intron. The two larger Rep proteins function as replication origin binding and initiation proteins and are required for viral DNA synthesis. Previously, we have demonstrated that the two larger Rep proteins, Rep 68 and Rep 78, are DNA binding proteins that recognize a sequence motif, the prototype is the GAGC repeat within the AAV inverted terminal repeat (ITR). The DNA binding ability of Rep proteins may have explained the regulatory effects of Rep proteins on gene expression that has been observed by several laboratories. However, this effect appears to be independent of the presence of a canonical Rep binding site within the regulatory region of the transcribed gene. Other cellular phenotypes associated with Rep 68 or Rep 78 expression include: loss of viability, inhibition of cell division, and inhibition of transformation by viral oncogenes. These effects may be induced by Rep association with a cellular protein. Domains of Rep proteins that are necessary for Rep oligomerization have been reported previously. However, the residues that are specifically required for these interactions have not been defined. In order to identify which residues are involved with inter-protein interactions, we have made a series of mutations altering a single residue Rep 78. Our results with analytical chromatography demonstrate that Rep 78 or Rep 68 forms a complex with properties consistent with a hexamer similar to other DNA helicase complexes. Defining how a polypeptide interacts with other proteins enables us to generalize and predict what other interactions may occur. Binding of Rep to epitopes displayed on bacteriophage has resulted in selection of clones with a commonly repeated motif. We are in the process of determining whether cellular proteins containing these epitopes interact with Rep 78 and if so, what are the effects on the cellular processes.