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-head, and tail-to-tail dimers) is an important hallmark of the productive phase of the adeno-associated virus (AAV) life cycle. These structures are generated by a strand-displacement replication mechanism and believed to be a reservoir for single-stranded DNA genomes. During the course of studies with recombinant versions of AAV (rAAV), we discovered the assembly of circular duplex provirus derivatives in latently infected cell lines under conditions permissive for replication (i.e., helper virus dependent). These novel structures were cloned by bacterial trapping revealing a markedly homogeneous structure that included a single copy of the rAAV genome joined head-to-tail about the inverted terminal repeats (ITR). Restriction and sequence analysis of the point of circularization revealed a so-called "TRT" domain, consisting of a single ITR hairpin palindrome flanked by 5' and 3' D sequence elements. The circular conformation was additionally characterized by Southern blotting and confirmed by purification on an ethidium bromide-CsCl gradient where the buoyant density was consistent with circular supercoiled DNA. These findings suggest that AAV replication is accompanied by the assembly of circular duplex structures.
Musatov
S A
SA
Department of Pathology and Laboratory Medicine, Tulane University Medical Center, New Orleans, Louisiana 70112, USA.
Scully
T A
TA
Dudus
L
L
Fisher
K J
KJ
D015139:Blotting, Southern; D002460:Cell Line; D000229:Dependovirus / Q000502:physiology*; D006801:Humans; D010957:Plasmids / Q000096:biosynthesis*; D016133:Polymerase Chain Reaction; D017735:Virus Latency; D014779:Virus Replication