Abstract
Despite increasing utilization of rAAV vectors in gene transfer applications, several aspects of the biology of these vectors remain poorly understood. We have visualized the conversion of rAAV vector genomes from single-stranded to double-stranded DNA in real time. We report that rAAV DNA accumulates into discrete foci inside the nucleus. These rAAV foci are defined in number, increase in size over time after transduction, are relatively immobile, and their presence correlates with the efficiency of cell transduction. These structures overlap with, or lie in close proximity to, the foci in which proteins of the MRN (MRE11-RAD50-NBS1) complex as well as the MDC1 protein accumulate after DNA damage. The downregulation of MRN or MDC1 by RNA interference markedly increases both the formation of rAAV foci and the extent of rAAV transduction. Chromatin immunoprecipitation experiments indicate that the MRE11 protein associates with the incoming rAAV genomes and that this association decreases upon cell treatment with DNA damaging agents. These findings are consistent with a model whereby cellular DNA-damage-response proteins restrict rAAV transduction by negatively regulating rAAV genome processing.
Highlights
The adeno-associated virus (AAV) is a replication defective parvovirus with a single-stranded DNA genome flanked by two short self-complementary palindromic sequences
We report that silencing of NBS1 and mediator of DNA damage checkpoint protein 1 (MDC1) by RNA interference markedly enhances Recombinant AAV (rAAV) transduction, indicating that MRN and MDC1 play an inhibitory role on rAAV genome processing
Visualization of the nuclear sites of dsAAV DNA formation in live cells To address the study of the kinetics of cell transduction with rAAV and, most notably, to visualize the fate of rAAV genomes in living cells, we constructed a rAAV vector (AAVLacO.14), carrying 112 lac repressor (LacR) binding sites (LacO repeats) cloned between the viral ITRs; this vector was used to infect HeLa and MRC5 cell lines that stably express a green fluorescent protein (GFP)-LacR fusion protein with a C-terminal nuclear localization signal (HeLa/GFP-LacR and MRC5/GFP-LacR cells)
Summary
The adeno-associated virus (AAV) is a replication defective parvovirus with a single-stranded (ss) DNA genome flanked by two short self-complementary palindromic sequences (inverted terminal repeats, ITRs). A major effect of these treatments is the improved conversion of the input vector ssDNA genome into double-stranded (ds) DNA (Ferrari et al, 1996; Fisher et al, 1996), which represents an essential requisite for gene expression. These observations have raised the possibility that permissivity to rAAV transduction might be related to the induction of the cellular DNAdamage response (DDR) and, in particular, of the proteins involved in DNA-double-strand break (DSB) repair (Zentilin et al, 2001)
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