RESCUE AND REPLICATION OF ADENOASSOCIATED VIRUS TYPE-2 AS WELL AS VECTOR DNA-SEQUENCES FROM RECOMBINANT PLASMIDS CONTAINING DELETIONS IN THE VIRAL INVERTED TERMINAL REPEATS - SELECTIVE ENCAPSIDATION OF VIRAL GENOMES IN PROGENY VIRIONS

The adeno-associated virus type 2 (AAV) genome can be successfully res cued from recombinant plasmids following transfection in adenovirus-in fected human cells, However, following rescue, the AAV genome undergoe s preferential replication and encapsidation, whereas little replicati on and packaging of the vector DNA sequences occur, In view of the cru cial role in the rescue, replication, and packaging of the proviral ge nome played by the AAV inverted terminal repeats (ITRs), which consist of a palindromic hairpin (HP) structure and a 20-nucleotide stretch, designated the D-sequence, that is not involved in the WP formation, w e evaluated the involvement of the individual ITRs as well as their co mponents in the selective viral DNA replication and encapsidation, A n umber of recombinant AAV plasmids that contained deletions-substitutio ns in different regions of the individual ITRs were constructed and ex amined for their potential to allow rescue, replication, and/or packag ing in adenovirus-infected human cells in vivo, The results reported h ere document that (i) two HP structures and one D-sequence are suffici ent for efficient rescue and preferential replication of the AAV DNA, (ii) two HP structures alone allow a low-level rescue and replication of the AAV DNA, but rescue and replication of the vector DNA sequences also occur in the absence of the D-sequences, (iii) one HP structure and two D-sequences, but not one HP structure and one D-sequence, also allow rescue and replication of the AAV as well as the vector DNA seq uences, (iv) one HP structure alone or two D-sequences, but not one D- sequence alone, allow replication of the full-length plasmid DNA, but no rescue of the AAV genome occurs, (v) no rescue-replication occurs i n the absence of the HP structures and the D-sequences, (vi) in the ab sence of the D-sequences, the HP structures are insufficient for succe ssful encapsidation of the AAV genomes, and (vii) the AAV genomes cont aining only one ITR structure can be packaged into biologically active virions, Thus, the D-sequence plays a crucial role in the efficient r escue and selective replication and encapsidation of the AAV genome, F urthermore, the D-sequence specifically interacts with a hitherto unkn own host-cell protein that we have designated the D-sequence-binding p rotein (D-BP), These studies illustrate that the D-sequcnce-D-BP inter action constitutes an important step in the AAV life cycle.