Formation of adeno-associated virus circular genomes is differentially regulated by adenovirus E4 ORF6 and E2a gene expression.
A central feature of the adeno-associated virus (AAV) latent life cycle is persistence in the form of both integrated and episomal genomes. However, the molecular processes associated with episomal long-term persistence of AAV genomes are only poorly understood. To investigate these mechanisms, we have utilized a recombinant AAV (rAAV) shuttle vector to identify circular AAV intermediates from transduced HeLa cells and primary fibroblasts. The unique structural features exhibited by these transduction intermediates included circularized monomer and dimer virus genomes in a head-to-tail array, with associated specific base pair alterations in the 5' viral D sequence. In HeLa cells, the abundance and stability of AAV circular intermediates were augmented by adenovirus expressing the E2a gene product. In the absence of E2a, adenovirus expressing the E4 open reading frame 6 gene product decreased the abundance of AAV circular intermediates, favoring instead the linear replication form monomer (Rfm) and dimer (Rfd) structures. In summary, the formation of AAV circular intermediates appears to represent a new pathway for AAV genome conversion, which is consistent with the head-to-tail concatemerization associated with latent-phase persistence of rAAV. A better understanding of this pathway may increase the utility of rAAV vectors for gene therapy.
Duan
D
D
Department of Anatomy and Cell Biology and Department of Internal Medicine at the University of Iowa School of Medicine, Iowa City, USA.
Sharma
P
P
Dudus
L
L
Zhang
Y
Y
Sanlioglu
S
S
Yan
Z
Z
Yue
Y
Y
Ye
Y
Y
Lester
R
R
Yang
J
J
Fisher
K J
KJ
Engelhardt
J F
JF
D017502:Adenovirus E2 Proteins / Q000235:genetics*; D017504:Adenovirus E4 Proteins / Q000235:genetics*; D001483:Base Sequence; D000229:Dependovirus / Q000235:genetics*; D005822:Genetic Vectors; D016679:Genome, Viral; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D011995:Recombination, Genetic
D017502:Adenovirus E2 Proteins; D017504:Adenovirus E4 Proteins