Binding sites for adeno-associated virus Rep proteins within the human genome.

Abstract

The Rep proteins of adeno-associated virus type 2 (AAV) are known to bind to Rep recognition sequences (RRSs) in the AAV inverted terminal repeats (ITRs), the AAV p5 promoter, and the preferred AAV integration site in human chromosome 19, called AAVS1. Integration of the AAV genome into AAVS1 appears to be mediated by an interaction between the Rep proteins of AAV and Rep binding sites within the viral genome and the integration locus. In an attempt to identify potential alternate integration sites, we looked for recognition sites for AAV Rep proteins in the human genome by performing a BLASTN computerized homology search. We used the 16-mer core sequences of the RRSs in the AAV ITRs and AAVS1 separately as query sequences and identified 18 new RRSs in or flanking the genes coding for the following: tyrosine kinase activator protein 1 (TKA-1); colony stimulating factor-1; insulin-like growth factor binding protein 2 (IGFBP-2); histone H2B.1; basement membrane heparan sulfate proteoglycan, also known as perlecan; the AF-9 gene product, which is involved in the chromosomal translocation t (9:11)(p22:q23); the betaB subunit of the hormone known as inhibin; interleukin-2 enhancer binding factor; an endoplasmic reticulum-Golgi intermediate compartment resident protein called p63; a global transcription activator (hSNF2L); the beta-actin repair domain; a retinoic acid-inducible factor, also known as midkine; a breast tumor autoantigen; a growth-arrest- and DNA-damage-inducible protein called gadd45; the cyclin-dependent kinase inhibitor called KIP2, which inhibits several G1 cyclin-cyclin-dependent kinase complexes; and the hereditary breast and ovarian cancer gene (BRCA1). RRSs were also identified in a newly discovered open reading frame on chromosome 10 and in the ERCC1 locus on human chromosome 19. The ability of a maltose binding protein-Rep68 fusion protein to bind to these sequences was confirmed by electrophoretic mobility shift assays. These sites may serve as alternate integration sites for AAV or play a role in Rep-mediated effects on human cells.