Adeno-associated virus (AAV) is a common, nonpathogenic, helper-dependent human parvovirus. AAV has generated much enthusiasm in recent years because of its potential as a human gene therapy vector. Although vector development is progressing rapidly, there is a lack of understanding of how AAV amplification, gene expression and site-specific integration affect the helper virus and host cell. All three processes are dependent upon de novo expression of, and regulation by, viral replication (Rep) proteins, Rep78 and Rep68. Interestingly, exogenous expression of these proteins has profound effects on the host cell and helper virus. To understand the interactions between AAV and its environment, we have begun a study of the inhibitory effects of AAV on Adenovirus (Ad) replication and gene expression. We present evidence that AAV and its encoded Rep proteins inhibit Ad replication. These inhibitions are due, in part, to inhibition of early gene transcription. The Rep protein-mediated alterations on transcription are due to effects on the Ad early gene promoters as well as on E1A-mediated trans-activation of early gene expression. The following specific aims are designed to elucidate the mechanistic roles played by AAV and its Rep proteins in an Ad co-infection; (1) Characterize early interactions between AAV and Ad in a co-infection, (2) Define the Rep and E1A protein domains and Ad promoter elements involved in Rep regulation of Ad gene expression, (3) Characterize the biochemical mechanisms of Rep-mediated inhibition, and activation, of gene expression. ? ? This novel, model system of host-virus interaction benefits from the wealth of knowledge in the literature on Ad biology and the abundance of available Ad reagents for study. Determining how AAV interacts with its most efficient helper virus, we will obtain valuable insights into how AAV interacts with host cells as well. These studies will also have clear ramifications for further AAV vector development, especially with regard to improvement in AAV vector packaging systems.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM064765-04
Application #
6899689
Study Section
Medical Biochemistry Study Section (MEDB)
Program Officer
Anderson, Richard A
Project Start
2002-07-01
Project End
2008-06-30
Budget Start
2005-07-01
Budget End
2008-06-30
Support Year
4
Fiscal Year
2005
Total Cost
$298,577
Indirect Cost
Name
University of Toledo
Department
Biochemistry
Type
Schools of Medicine
DUNS #
807418939
City
Toledo
State
OH
Country
United States
Zip Code
43614
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
Bhrigu, Vipul; Trempe, James P (2009) Adeno-associated virus infection of murine fibroblasts with help provided by mouse adenovirus. Virology 390:22-30
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
Smith, Andrew D; Collaco, Roy F; Trempe, James P (2003) Enhancement of recombinant adeno-associated virus type 2-mediated transgene expression in a lung epithelial cell line by inhibition of the epidermal growth factor receptor. J Virol 77:6394-404

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