Adeno-associated virus type 2 (AAV) is a non-pathogenic human parvovirus which 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. Helper virus functions are also required for the efficient production of AAV- based gene therapy vectors. RNA initiated from the AAV p5 promoter encodes the Rep68 and Rep78 proteins (Rep68/78), which are involved in AAV replication, gene regulation, and the preferential integration of the AAV genome into a region within the q arm of human chromosome 19 (the only example of site-specific integration of a mammalian virus). There is strong evidence that the helper virus products are not necessarily directly involved in AAV replication, but may alter levels of cellular proteins required for efficient AAV gene expression and replication. We have therefore been looking for cellular transcription factors whose levels are altered by adenovirus infection and attempting to correlate these alterations with changes in protein-DNA complexes on the AAV p5 promoter, as well as changes in AAV gene expression. We have determined that the level of the cellular transcription factor Sp1, which is strongly suspected to be involved in positive regulation of AAV genes by Rep68/78, is increased 4- to 16-fold in human HeLa or 293 cells infected with adenovirus type 5 versus uninfected cells. Adenovirus E1A protein levels actually decrease with adenovirus infection of 293 cells, which contain an integrated E1A gene. We found that the level of cellular transcription factor C/EBP beta is about the same in adenovirus-infected versus uninfected cells. We have also identified an adenovirus-inducible protein-DNA complex at the RNA initiation site of the p5 promoter. We are attempting to purify the protein or proteins which form this complex. Competition and timecourse experiments indicate that this complex does not contain transcription factor Sp1. This line of research may lead to improved AAV gene therapy vector production systems, as well as a better understanding of the basic biology of AAV. - adeno-associated virus, gene regulation, replication, integration
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