Induction of circular episomes during rescue and replication of adeno-associated virus in experimental models of virus latency

The synthesis of linear duplex replicative structures (monomers, head-to-he ad, and tail-to-tail dimers) is an important hallmark of the productive pha se of the adeno-associated virus (AAV) life cycle. These structures are gen erated by a strand-displacement replication mechanism and believed to be a reservoir for single-stranded DNA genomes. During the course of studies wit h recombinant versions of AAV (rAAV), we discovered the assembly of circula r duplex provirus derivatives in latently infected cell lines under conditi ons permissive for replication (i.e., helper virus dependent). These novel structures were cloned by bacterial trapping revealing a markedly homogeneo us structure that included a single copy of the rAAV genome joined head-to- tail about the inverted terminal repeats (ITR). Restriction and sequence an alysis of the point of circularization revealed a so-called "TRT" domain, c onsisting of a single ITR hairpin palindrome flanked by 5' and 3' D sequenc e elements. The circular conformation was additionally characterized by Sou thern blotting and confirmed by purification on an ethidium bromide-CsCl gr adient where the buoyant density was consistent with circular supercoiled D NA These findings suggest that AAV replication is accompanied by the assemb ly of circular duplex structures. (C) 2000 Academic Press.