Function Of Dna Virus Genomes In Animal Cells

Diabetes, Digestive, Kidney Diseases

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Abstract

Adeno-associated virus type 2 (AAV2) is a non-pathogenic, human parvovirus that is being developed as a gene therapy vector. AAV2 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 AAV2 encodes four Rep proteins involved in AAV2 replication and gene regulation. Rep78 and Rep68 are made from unspliced and spliced transcripts, respectively, from the AAV2 p5 promoter. The p5 promoter contains a binding site for the host cell transcription factor YY1, at its RNA start site. Conservation of this YY1 binding site amongst related parvoviruses suggests its importance in the AAV2 life cycle. YY1 is known to mediate gene activation, gene repression, or transcription initiation, depending on context. We had previously demonstrated, using electrophoretic mobility shift assays, that the amount of YY1-containing protein-DNA complexes at the AAV2 p5 promoter's RNA start site is higher when nuclear extracts of permissive cells infected with adenovirus type 5 (Ad5) are used, as opposed to nuclear extracts of uninfected cells. We have now shown that there is a corresponding increase in YY1 protein levels in these extracts in the presence of Ad5 infection, as detected by western blot analysis. In the absence of helper virus, AAV2 DNA integrates into the host genome with a strong preference (70%-90% of integration events) for a 4 kb region of human chromosome 19, designated AAVS1 (the only example of site-specific integration in a mammalian virus system). This ability to specifically integrate also contributes to AAV2's attractiveness as a vector for gene therapy, since this could potentially limit the dangers associated with insertional mutagenesis. During AAV2 replication, the Rep68 and Rep78 proteins (Rep68/78) of AAV2 make a site- and strand-specific nick at the terminal resolution site (trs) within the stem of the hairpin structure formed by the inverted terminal repeats (ITRs) of AAV2 DNA. The nicking activity of Rep68/78 has also been found to be essential to the preferential integration process and a nicking site has been identified within AAVS1. In all six AAV serotypes, for which DNA sequence data is available, the nicking site is flanked by a sequence that can theoretically form a stem-loop with standard Watson-Crick base pairing (i.e. A-T and G-C). The region flanking the nicking site in AAVS1 cannot form a similar stem-loop structure. Therefore, we searched for a non Watson-Crick secondary structure. In comparing the migration of radiolabeled DNA containing sequences from the AAVS1 and AAV2-ITR nicking sites we have found evidence that is consistent with the hypothesis that there is a secondary structure within AAVS1. We have so far identified two G residues that appear to be important in forming this putative secondary structure near the AAVS1 nicking site. These results may further help to explain the specificity of AAV2 integration.