Adeno-associated virus type 2 (AAV) is a non-pathogenic, single-stranded DNA virus that is being developed as a gene therapy vector. AAV requires co-infection with a helper virus, usually an adenovirus or herpesvirus, for efficient productive infection. It is therefore also a good model system for the study of virus-virus interactions. The rep gene of AAV encodes four proteins involved in AAV replication, gene regulation, and preferential integration of the AAV genome into a specific region of human chromosome 19. Rep78 and Rep68 are made from unspliced and spliced transcripts, respectively, from the AAV p5 promoter. Rep52 and Rep40 are encoded by unspliced and spliced transcripts, respectively, from the AAV p19 promoter. The p5 promoter contains a binding site for the host cell transcription factor YY1, at its RNA start site. YY1 is known to mediate gene activation, gene repression, or transcription initiation, depending on context. We mutated this p5 YY1 site in the context of a plasmid containing an infectious AAV genome. The effects of this mutation on Rep protein levels were dependent on the human cell line transfected. Nuclear extracts of adenovirus-infected 293 cells transfected with the YY1 mutant plasmid, had slightly lower levels (less than two-fold difference) of Rep78 and Rep68 than extracts of adenovirus-infected 293 cells transfected with the wild-type AAV genome. However, nuclear extracts of adenovirus-infected HeLa cells transfected with the YY1 mutant plasmid had approximately 4-fold lower levels of Rep78 and Rep68 than extracts of adenovirus-infected HeLa cells transfected with the wild-type AAV genome. Electrophoretic mobility shift analysis, using labeled DNA containing this YY1 binding site and uninfected HeLa or 293 cell nuclear extracts, showed three protein-DNA complexes. Adenovirus infection, which promotes AAV gene expression and replication, appears to diminish one of these complexes and enhance another. We have determined, by competition experiments, that all three complexes are dependent on the YY1 binding site within the DNA. Antibodies against YY1 only supershift one of these complexes. None of the bands were supershifted by antibodies against transcription factors TFIIB or Sp1. We are in the process of purifying the proteins involved in these complexes. Preliminary experiments show that the proteins producing each complex can be separated from each other by column chromatography. We hope that identification of these putative transcription factors will help explain the role of adenovirus co-infection in AAV promoter activation. We have also developed an oligonucleotide-based technique for the selective protection of restriction endonuclease sites. We believe this method will be particularly useful for the cloning of PCR-amplified genomic versions of genes into expression vectors.
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