RESCUE AND AUTONOMOUS REPLICATION OF ADENOASSOCIATED VIRUS TYPE-2 GENOMES CONTAINING REP-BINDING SITE MUTATIONS IN THE VIRAL P5 PROMOTER

The Rep proteins encoded by the adeno-associated virus type 2 (AAV) pl ay a crucial role in the rescue, replication, and integration of the v iral genome. In the absence of a helper virus, little expression of th e AAV Rep proteins occurs, and the AAV genome fails to undergo DNA rep lication. Since previous studies have established that expression of t he Rep78 and Rep68 proteins from the viral p5 promoter is controlled b y the Rep-binding site (RES) and the YY1 factor-binding site (YBS), we constructed a number of recombinant AAV plasmids containing mutations and/or deletions of the RES and the YES in the p5 promoter. These pla smids were transfected in HeLa or 293 cells and analyzed for the poten tial to undergo AAV DNA rescue and replication. Our studies revealed t hat (i) a low-level rescue and autonomous replication of the wild-type AAV genome occurred in 293 but not in HeLa cells; (ii) mutations in t he RES resulted in augmented expression from the p5 promoter, leading to more efficient rescue and/or replication of the AAV genome in 293 b ut not in HeLa cells; (iii) little rescue and/or replication occurred from plasmids containing mutations in the YBS alone in the absence of coinfection with adenovirus; (iv) expression of the adenovirus E1A gen e products was insufficient to mediate rescue and/or replication of th e AAV genome in HeLa cells; (v) autonomously replicated AAV genomes in 293 cells were successfully encapsidated in mature progeny virions th at were biologically active in secondary infection of HeLa cells in th e presence of adenovirus; and (vi) stable transfection of recombinant AAV plasmids containing a gene for resistance to neomycin significantl y affected stable integration only in 293 cells, presumably because re scue and autonomous replication of the AAV genome from these plasmids occurred in 293 cells but not in HeLa or ICE cells. These data suggest that in the absence of adenovirus, the AAV Rep protein-RES interactio n plays a dominant role in down-regulating viral gene expression from the p5 promoter and that perturbation in this interaction is sufficien t to confer autonomous replication competence to AAV in 293 cells.