Adeno-associated virus type 2 (AAV) is a non-pathogenic human parvovirus which is being developed as a gene therapy vector. The Rep78 and Rep68 proteins encoded by AAV are DNA-binding proteins involved in AAV replication, AAV gene regulation, and the preferential integration of the AAV genome into a region within the q arm of human chromosome 19. These proteins also inhibit cell proliferation and the replication of human immunodeficiency virus type 1 (HIV). The preferential integration of AAV DNA into a region of human chromosome 19( the only example of site-specific integration in a mammalian virus) involves both the DNA binding and site-specific endonuclease activity of Rep78 and Rep68. We have demonstrated previously that DNA binding activity is mediated by an interaction between the amino terminal portion of the Rep proteins and an imperfect 4x (GAGC) motif. The endonuclease activity occurs at a site 9-14 bases away, within a second motif whose consensus sequence is GTTGG. We have identified and examined over 20 Rep binding sites within the human genome. Of these sites , only the site at the chromosome 19 preferred integration locus has a nearby consensus endonuclease site. A tyrosine is believed to be part of the endonuclease active site. After using truncation analysis to identify the minimal functional Rep endonuclease, we systematically mutated tyrosines in this region to phenylalanines, within the context of a nearly full-length protein expressed as maltose binding protein fusions in Escherichia coli. Mutation of tyrosine 152 specifically knocked out the endonuclease activity without affecting DNA binding or DNA helicase activity. Tyrosine 152 is therefore the prime candidate for the active-site tyrosine. This also provided the first evidence for separate active sites for the helicase and endonuclease activities. We have also begun a collaborative project to develop AAV-based gene therapy vectors for lysosomal storage diseases.
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