Vectors based on the human Adeno-associated virus (AAV) are attractive vehicles for gene delivery. A thorough understanding of the basic biology of this virus is crucial to its successful development and use as a gene transfer vector. Productive AAV infection requires helper functions that can be supplied by co-infection with helper viruses such as adenovirus and herpesvirus. AAV can also replicate when cells are treated with irradiation or genotoxic agents. In the absence of a permissive environment for replication, AAV can integrate into the host genome at a specific locus on human chromosome 19. AAV vectors consist of a transgene flanked by viral inverted terminal repeats (ITRs). The ITR is required for replication, packaging and integration of the virus and is the only viral element that remains in rAAV vectors after integration into the host genome during transduction. Therefore it is important to understand viral and cellular factors that interact with this important structure. The viral Rep protein recognizes a specific motif called the Rep recognition sequence (RRS) within the ITR. This interaction is important for replication and targeted integration. In this proposal we will focus on DNA recognition in vivo by Rep proteins and cellular proteins that are involved in regulating AAV replication and integration. We have identified two cellular proteins that also recognize the RRS and will assess their role in the AAV life-cycle. We have demonstrated that Rep can recruit to the viral origin single-stranded binding proteins from the cell (RPA) or the adenovirus (Ad-DBP) and herpes simplex virus (ICP8) helper viruses. We will characterize how these proteins enhance Rep's functions in replication. In addition we have designed a yeast-based screen to identify proteins that interact with DNA-bound Rep in its native conformation. The role of these Rep-interacting proteins in the viral life-cycle will be examined. These studies will use a combination of biochemical approaches and in vivo assays. Understanding the role of cellular components in regulating AAV replication and integration will enhance our ability both to produce and utilize this promising vector system.
|Chaurushiya, Mira S; Lilley, Caroline E; Aslanian, Aaron et al. (2012) Viral E3 ubiquitin ligase-mediated degradation of a cellular E3: viral mimicry of a cellular phosphorylation mark targets the RNF8 FHA domain. Mol Cell 46:79-90|
|Weitzman, Matthew D; Lilley, Caroline E; Chaurushiya, Mira S (2011) Changing the ubiquitin landscape during viral manipulation of the DNA damage response. FEBS Lett 585:2897-906|
|Weitzman, Matthew D; Linden, R Michael (2011) Adeno-associated virus biology. Methods Mol Biol 807:1-23|
|Lilley, Caroline E; Chaurushiya, Mira S; Weitzman, Matthew D (2010) Chromatin at the intersection of viral infection and DNA damage. Biochim Biophys Acta 1799:319-27|
|Lamarche, Brandon J; Orazio, Nicole I; Weitzman, Matthew D (2010) The MRN complex in double-strand break repair and telomere maintenance. FEBS Lett 584:3682-95|
|Weitzman, Matthew D; Lilley, Caroline E; Chaurushiya, Mira S (2010) Genomes in conflict: maintaining genome integrity during virus infection. Annu Rev Microbiol 64:61-81|
|Chaurushiya, Mira S; Weitzman, Matthew D (2009) Viral manipulation of DNA repair and cell cycle checkpoints. DNA Repair (Amst) 8:1166-76|