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Formation of adeno-associated virus circular genomes is differentially regulated by adenovirus E4 ORF6 and E2a gene
1Department of Anatomy and Cell Biology and Department of Internal Medicine at the University of Iowa School of Medicine, Iowa City, USA.
Abstract:
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.
Insights
Adeno-associated virus (AAV) persistence involves circular DNA forms. Understanding these circular intermediates reveals new pathways for AAV genome conversion, crucial for gene therapy applications.
Area of Science:
- Molecular biology
- Virology
- Gene therapy
Background:
- Adeno-associated virus (AAV) exhibits a latent life cycle characterized by integrated and episomal genomes.
- The molecular mechanisms governing long-term episomal persistence of AAV genomes remain poorly understood.
Purpose of the Study:
- To investigate the molecular processes underlying the episomal long-term persistence of adeno-associated virus (AAV) genomes.
- To identify and characterize circular AAV intermediates formed during the latent life cycle.
Main Methods:
- Utilized a recombinant AAV (rAAV) shuttle vector to identify circular AAV intermediates.
- Analyzed transduced HeLa cells and primary fibroblasts for the presence and structure of viral genomes.
- Investigated the impact of adenovirus gene products (E2a and E4 open reading frame 6) on AAV circular intermediate formation and stability.
Main Results:
- Identified circularized monomer and dimer AAV genomes in a head-to-tail array as key transduction intermediates.
- Observed specific base pair alterations in the 5' viral D sequence of these circular intermediates.
- Adenovirus E2a augmented the abundance and stability of AAV circular intermediates in HeLa cells.
- Adenovirus E4 open reading frame 6 decreased AAV circular intermediates, promoting linear forms (Rfm and Rfd) in the absence of E2a.
Conclusions:
- The formation of AAV circular intermediates represents a novel pathway for AAV genome conversion.
- This pathway is consistent with head-to-tail concatemerization observed during latent-phase persistence of rAAV.
- A deeper understanding of this circularization pathway can enhance the utility of rAAV vectors in gene therapy.