Organization of adeno-associated virus DNA in latently infected Detroit 6 cells.
The DNA of a human dependovirus, adeno-associated virus (AAV), integrates into the cellular genome under conditions nonpermissive for viral DNA replication. The AAV DNA can be rescued from the cellular genome by superinfection with a helper virus, e.g., adenovirus. To characterize the organization of the proviral DNA in greater detail, we isolated three proviral subfragments from a latently infected human cell line. Our findings, based on sequence analysis, restriction enzyme mapping, and genomic blots, demonstrate that the viral terminal repeats (trs) are present at or near the cellular/viral DNA junctions, but significant deletions of the tr sequences have occurred. One of the proviral clones has extensive rearrangements which apparently occurred by nonhomologous recombination. The tail-to-tail arrangement of one of the proviral clones is consistent with limited DNA replication prior to integration by a mechanism similar to that seen in a productive infection. Although there are BamH1 fragments in the cell line we have characterized, which are the right size to have come from a head-to-tail tandem repeat, critical double digests show no evidence for the presence of a head-to-tail tandem repeat of wild-type proviral DNA. Use of probes derived from the flanking cellular DNA enabled us to determine that in this cell line the viral DNA had integrated into a site composed of nonrepetitive sequences.
Kotin
R M
RM
Department of Microbiology, Cornell University Medical College, New York, New York 10021.
Berns
K I
KI
D001483:Base Sequence; D015139:Blotting, Southern; D002460:Cell Line; D003001:Cloning, Molecular; D015342:DNA Probes; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D000229:Dependovirus / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D015183:Restriction Mapping
D015342:DNA Probes; D004279:DNA, Viral