Adeno-Associated Virus type 2 (AAV-2) is unique among animal viruses in its ability to establish a latent infection by integrating site-specifically into a locus of chromosome 19. Moreover, because of its apparent non- pathogenicity, broad cell and tissue tropism and ability to infect in a cell-cycle independent fashion, AAV has emerged as one of the most promising vectors for gene therapy. A number of studies indicate that AAV- mediated site-specific integration requires only three players: a) the AAV Rep78 or Rep68 non-structural proteins, b) an AAV DNA element containing a Rep Binding Site (RBS) and c) the cellular AAVS1 integration site. During a decisive event, these players come together to form a protein-DNA complex that serves as the starting point for an integration process that is considered to be parallel to the AAV replication initiation process. This pre-integration complex is dependent on the ability of Rep68/78 proteins to bind the integration site AAVS1, promote its melting and catalyze a strand-specific nicking reaction that leaves a Rep68/78 protein covalently bound to the integration site. Although a general view of this mechanism has been achieved through the study of AAV DNA replication, little is known regarding the architecture of the Rep-AAVS1 complex and the steps that lead to its formation. The long-term goal of this proposal is to establish a structural, biophysical and biochemical framework required to understand the mechanism of AAV-mediated site-specific integration. Our approach is to couple structural studies using X-ray crystallography, cryo-EM and small angle X-ray scattering (SAXS) with biophysical, kinetic and functional studies to get a complete understanding of the events that lead to site-specific integration. We expect that the results obtained in this research proposal will serve both as a starting point to generate accurate models to understand this unique mechanism and also will give us the knowledge and tools to potentially design Rep proteins with novel specificities that could be used to develop new technologies in the gene therapy field.

Public Health Relevance

This study will contribute significantly toward understanding the mechanism of AAV mediated site-specific integration. In addition, it will contribute to our understanding of origin DNA replication initiation. Knowledge gained from this proposal will serve as the basis for designing novel gene therapy vectors that could be targeted to a specific region in the chromosome.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM092854-03
Application #
8324224
Study Section
Virology - A Study Section (VIRA)
Program Officer
Janes, Daniel E
Project Start
2010-09-30
Project End
2014-08-31
Budget Start
2012-09-01
Budget End
2013-08-31
Support Year
3
Fiscal Year
2012
Total Cost
$288,610
Indirect Cost
$95,560
Name
Virginia Commonwealth University
Department
Physiology
Type
Schools of Medicine
DUNS #
105300446
City
Richmond
State
VA
Country
United States
Zip Code
23298
Zarate-Perez, Francisco; Santosh, Vishaka; Bardelli, Martino et al. (2017) Determination of Adeno-associated Virus Rep DNA Binding Using Fluorescence Anisotropy. Bio Protoc 7:
Jaiswal, Rahul; Choudhury, Malay; Zaman, Shamsu et al. (2016) Functional architecture of the Reb1-Ter complex of Schizosaccharomyces pombe. Proc Natl Acad Sci U S A 113:E2267-76
Bardelli, Martino; Zárate-Pérez, Francisco; Agúndez, Leticia et al. (2016) Identification of a Functionally Relevant Adeno-Associated Virus Rep68 Oligomeric Interface. J Virol 90:6612-6624
Musayev, Faik N; Zarate-Perez, Francisco; Bishop, Clayton et al. (2015) Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1. J Biol Chem 290:27487-99
Remesh, Soumya G; Santosh, Vishaka; Escalante, Carlos R (2015) Structural Studies of IRF4 Reveal a Flexible Autoinhibitory Region and a Compact Linker Domain. J Biol Chem 290:27779-90
Musayev, Faik N; Zarate-Perez, Francisco; Bardelli, Martino et al. (2015) Structural Studies of AAV2 Rep68 Reveal a Partially Structured Linker and Compact Domain Conformation. Biochemistry 54:5907-19
Jaiswal, Rahul; Singh, Samarendra K; Bastia, Deepak et al. (2015) Crystallization and preliminary X-ray characterization of the eukaryotic replication terminator Reb1-Ter DNA complex. Acta Crystallogr F Struct Biol Commun 71:414-8
Dutheil, Nathalie; Smith, Sarah C; Agúndez, Leticia et al. (2014) Adeno-associated virus Rep represses the human integration site promoter by two pathways that are similar to those required for the regulation of the viral p5 promoter. J Virol 88:8227-41
Zarate-Perez, Francisco; Mansilla-Soto, Jorge; Bardelli, Martino et al. (2013) Oligomeric properties of adeno-associated virus Rep68 reflect its multifunctionality. J Virol 87:1232-41
Zarate-Perez, Francisco; Bardelli, Martino; Burgner 2nd, John W et al. (2012) The interdomain linker of AAV-2 Rep68 is an integral part of its oligomerization domain: role of a conserved SF3 helicase residue in oligomerization. PLoS Pathog 8:e1002764

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