Adeno-associated Virus (AAV) is a nonpathogenic, defective human parvovirus. AAV requires that a helper virus co-infect the host cell for efficient virus replication. In the absence of helper virus infection, AAV readily integrates its genome into a specific region in the long arm of chromosome 19. AAV's natural defectiveness, nonpathogenic nature and ability to integrate site-specifically have prompted numerous studies on its potential as a human gene therapy vector. Virus replication and site-specific integration is dependent upon two replication proteins, Rep78 and Rep68, that are encoded by the virus. These proteins mediate virus replication and integration by binding to target sites on the viral DNA and the chromosome 19 locus. These proteins also play important roles in regulation of virus and host cell gene expression. Our laboratory has demonstrated that these proteins are phosphorylated on Ser residues. We propose that differential phosphorylation of Rep78 and Rep68 allow for regulation of these proteins. Preliminary results reveal that the level of phosphorylation varies at different times in the virus replication cycle and that phosphatase inhibition results in an increase in phosphorylation. Experiments proposed here will identify the specific residues that are modified in the Rep proteins and reveal the kinases involved in Rep regulation. We will also determine how specific phosphorylation events modulate the known enzymatic activities of the Rep proteins in in vitro as well as in vivo studies. These investigations will provide essential information required for the understanding of the AAV replication cycle and site-specific integration of AAV gene therapy vectors. These studies may also lead to the development of new and improved AAV vectors.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
1R01AI051471-01
Application #
6461747
Study Section
Experimental Virology Study Section (EVR)
Program Officer
Beisel, Christopher E
Project Start
2002-04-01
Project End
2007-03-31
Budget Start
2002-04-01
Budget End
2003-03-31
Support Year
1
Fiscal Year
2002
Total Cost
$329,500
Indirect Cost
Name
University of Toledo
Department
Biochemistry
Type
Schools of Medicine
DUNS #
807418939
City
Toledo
State
OH
Country
United States
Zip Code
43614
Bhrigu, Vipul; Trempe, James P (2009) Adeno-associated virus infection of murine fibroblasts with help provided by mouse adenovirus. Virology 390:22-30
Collaco, Roy F; Bevington, Joyce M; Bhrigu, Vipul et al. (2009) Adeno-associated virus and adenovirus coinfection induces a cellular DNA damage and repair response via redundant phosphatidylinositol 3-like kinase pathways. Virology 392:24-33
Bevington, Joyce M; Needham, Patrick G; Verrill, Kristin C et al. (2007) Adeno-associated virus interactions with B23/Nucleophosmin: identification of sub-nucleolar virion regions. Virology 357:102-13
Dignam, Susan S; Collaco, Roy F; Bieszczad, Jacob et al. (2007) Coupled ATP and DNA binding of adeno-associated virus Rep40 helicase. Biochemistry 46:568-76
Dignam, Susan S; Correia, John J; Nada, Shadia E et al. (2007) Activation of the ATPase activity of adeno-associated virus Rep68 and Rep78. Biochemistry 46:6364-74
Timpe, Jennifer M; Verrill, Kristin C; Black, Bret N et al. (2007) Adeno-associated virus induces apoptosis during coinfection with adenovirus. Virology 358:391-401
Timpe, Jennifer M; Verrill, Kristin C; Trempe, James P (2006) Effects of adeno-associated virus on adenovirus replication and gene expression during coinfection. J Virol 80:7807-15
Needham, Patrick G; Casper, John M; Kalman-Maltese, Vivian et al. (2006) Adeno-associated virus rep protein-mediated inhibition of transcription of the adenovirus major late promoter in vitro. J Virol 80:6207-17
Casper, John M; Timpe, Jennifer M; Dignam, John David et al. (2005) Identification of an adeno-associated virus Rep protein binding site in the adenovirus E2a promoter. J Virol 79:28-38
Collaco, Roy F; Kalman-Maltese, Vivian; Smith, Andrew D et al. (2003) A biochemical characterization of the adeno-associated virus Rep40 helicase. J Biol Chem 278:34011-7

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