Identification and characterization of an adeno-associated virus integration site in CV-1 cells from the African green monkey.
Adeno-associated virus (AAV) is a classification given to a group of nonpathogenic, single-stranded DNA viruses known to reside latently in primates. During latency in humans, AAV type 2 (AAV2) preferentially integrates at a site on chromosome 19q13.3ter by targeting a sequence composed of an AAV Rep binding element (RBE), a spacer, and a nicking site. Here, we report the DNA sequence of an African green monkey AAV integration site isolated from CV-1 cells. Overall, it has 98% homology to the analogous human site, including identical spacer and nicking sequences. However, the simian RBE is expanded, having five perfect directly repeated GAGC tetramers. We carried out a number of in vitro and in vivo assays to determine the effect of this expanded RBE sequence on the Rep-RBE interaction and AAV targeted integration. Using electromobility shift assays it was demonstrated that AAV4 Rep68 bound the expanded RBE with a sixfold-greater affinity than the human RBE. To determine the basis for the affinity increase, DNase I protection and methylation interference (MI) assays were performed. Comparison of footprints on both the human and simian RBEs revealed nearly identical protection; however, MI analysis suggested greater interaction with the guanine nucleotides of the expanded RBE, thus providing a biochemical basis for the increased binding activity. In vivo, integration targeted to the simian RBE was demonstrated by PCR analysis of latently infected Cos-7 cells. Interestingly, the frequency of site-specific integration was twofold greater in Cos-7 cells than in HeLa cells. Overall, these experiments establish that the simian RBE, identified in CV-1 cells, functions analogously to the human RBE and provide further evidence for a developing model that proposes individual roles for the RBE and the spacer and nicking site elements.
Amiss
Terry J
TJ
Department of Pharmacology. Gene Therapy Center. Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
McCarty
Doug M
DM
Skulimowski
Anna
A
Samulski
R Jude
RJ
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002522:Chlorocebus aethiops; D019175:DNA Methylation; D004279:DNA, Viral / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000378:metabolism; D000229:Dependovirus / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D014764:Viral Proteins / Q000378:metabolism; D016662:Virus Integration
D004279:DNA, Viral; D004268:DNA-Binding Proteins; D014764:Viral Proteins; C068734:rep proteins, Adeno-associated virus 2