Adeno-associated virus is a dependent parvovirus requiring coinfection with another virus in order to undergo a productive infection in cultured cells. In the absence of coinfection with helper virus, the AAV genome integrates via its ends into the host genome and can later be """"""""rescued"""""""" upon infection of the latent cell with adenovirus. The overall objective of the proposed work is to utilize the infectious AAV recombinant clone to understand the mechanism(s) of integration and rescue in human cells. Three goals are proposed: (1) To determine whether AAV-encoded activities are required for specific integration a AAV DNA (in a way that permits subsequent rescue). Preliminary experiments suggest that this may be the case. Two approaches will be taken to address this problem. First, the latently infected Detroit 6 cell line will be infected with a selectable AAV virus, AAV/NEO. The resulting neo cell lines will then be assayed for rescue upon superinfection with adenovirus. Second, cell lines expressing specific AAV genes will be generated and assayed as described for the detroit 6 line. (2) To develop an efficient system for retrieving and cloning integrated AAV sequences, and to then determine the sequences at the viral-host junctions. The lambda repressor-binding sequence will be introduced into an AAV recombinant clone, and the feasibility of retrieving a single copy of this DNA from genomic DNA by binding to a filter containing the repressor will be tested. If successful, the procedure will be utilized to clone a number of integration sites and they will be analyzed. And (3) To utilize bacterial cells for the recovery of products of the rescue process from HeLa cells transfected with the infectios clone. The products will then be analyzed to obtain clues about possible rescue mechanisms. If the products of the rescue process can be routinely isolated and identified the assay will be utilized to study the requirements for AAV rescue.
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