747. Direct Labeling and Tracking of Adeno-Associated Virus Single Stranded DNA in AAV Transduction

Abstract

AAV is a unique gene transfer vector which takes approximately 4–6 week to reach its expression peak. The mechanism for this slow-rise expression profile was proposed to be limited by inefficient second-strand DNA synthesis from single stranded (ss) DNA viral genomes. We generated AAV vectors labeled with BrdU, a nucleotide analog that can be detected by anti-BrdU antibody when the DNA is in ss form, but not in double stranded (ds) form. Vast amounts of ss AAV genome as demonstrated in previous studies should be easily tracked. To our surprise, after AAV transduction, ss AAV genomes were detectable only under denaturing conditions in immunohistochemical analysis or in an ELISA assay. Southern Blot analysis of viral DNA and virion suggested that AAV viral DNA is mostly protected within virions. Extracted cellular fractions demonstrate that viral particles in infected host cells are still infectious. In addition, there is a significant decrease in viral genomes in the first 48 hours post infection. Analysis of the data suggests that free ss DNA seems not abundant during AAV transduction. AAV transduction is limited by the steps that affect AAV uncoating before second strand synthesis can occur. AAV single stranded DNA released from viral uncoating is either converted into double stranded DNA efficiently or degraded by cellular repair mechanisms as damaged DNA. This study may provide a tool to track viral DNA and to investigate fate and mechanisms of viral DNA processing.