Infectious molecular clones of adeno-associated virus isolated directly from human tissues.
Adeno-associated virus (AAV) replication and biology have been extensively studied using cell culture systems, but there is precious little known about AAV biology in natural hosts. As part of our ongoing interest in the in vivo biology of AAV, we previously described the existence of extrachromosomal proviral AAV genomes in human tissues. In the current work, we describe the molecular structure of infectious DNA clones derived directly from these tissues. Sequence-specific linear rolling-circle amplification was utilized to isolate clones of native circular AAV DNA. Several molecular clones containing unit-length viral genomes directed the production of infectious wild-type AAV upon DNA transfection in the presence of adenovirus help. DNA sequence analysis of the molecular clones revealed the ubiquitous presence of a double-D inverted terminal repeat (ITR) structure, which implied a mechanism by which the virus is able to maintain ITR sequence continuity and persist in the absence of host chromosome integration. These data suggest that the natural life cycle of AAV, unlike that of retroviruses, might not have genome integration as an obligatory component.
Schnepp
Bruce C
BC
The Research Institute, Nationwide Children's Hospital, Columbus, Ohio, USA.
Jensen
Ryan L
RL
Clark
K Reed
KR
Johnson
Philip R
PR
D000293:Adolescent; D001483:Base Sequence; D015153:Blotting, Western; D002648:Child; D002675:Child, Preschool; D003001:Cloning, Molecular; D017931:DNA Primers; D000229:Dependovirus / Q000235:genetics / Q000302:isolation & purification / Q000472:pathogenicity*; D006367:HeLa Cells; D006801:Humans; D016133:Polymerase Chain Reaction; D014774:Virulence
D017931:DNA Primers