RECOMBINANT JUNCTIONS FORMED BY SITE-SPECIFIC INTEGRATION OF ADENOASSOCIATED VIRUS INTO AN EPISOME

A model system using an episomal Epstein-Barr virus shuttle vector was recently developed to study the adeno-associated virus (AAV) site-spe cific integration event in chromosome 19q13.3-qter (C, Giraud, E, Wino cour, and K, I, Berns, Proc, Natl, Acad, Sci, USA 91:10039-10043, 1994 ), In this study, we analyze the recombinant junctions generated after integration of the AAV genome into an Epstein-Barr virus shuttle vect or carrying 8.2, 1.6, or 0.51 kb of the chromosome 19 preintegration s equence (AAVS1 locus), In most of the recombinants, one end of the vir al genome was joined to a portion of the AAVS1 DNA previously shown to be a minimum target for AAV integration, Within this AAVS1 segment, t he AAV insertion points were strikingly clustered around a binding sit e for the AAV regulatory protein, In all cases, the second junction wi th AAV occurred with vector DNA outside of the AAVS1 segment, With res pect to the viral genome, one junction with the shuttle vector DNA occ urred either within the AAV inverted terminal repeat (itr), or near th e P5 promoter, approximately 100 nucleotides distal to a modified itr, The modified itr in 5 of 11 recombinants involved a head-to-tail orga nization, In one such instance, the AAV insert contained slightly more than one genome equivalent arranged in a head-to-tail manner with a j unction close to the P5 promoter; the AAV insert in this recombinant e pisome could be rescued by adenovirus infection and replicated to viru s particles, The significance of the head-to tail organization is disc ussed in terms of the possible circularization of AAV DNA before or du ring integration.