Abstract
BackgroundWhile the molecular mechanisms of DNA-protein specificity at the origin of replication have been determined in many model organisms, these interactions remain unknown in the majority of higher eukaryotes and numerous vertebrate viruses. Similar to many viral origins of replication, adeno-associated virus (AAV) utilizes a cis-acting origin of replication and a virus specific Replication protein (Rep) to faithfully carry out self-priming replication. The mechanisms of AAV DNA replication are generally well understood. However, the molecular basis of specificity between the Rep protein and the viral origin of replication between different AAV serotypes remains uncharacterized.Methodology/Principal FindingsBy generating a panel of chimeric and mutant origins between two AAV serotypes, we have mapped two independent DNA-Protein interfaces involved in replicative specificity. In vivo replication assays and structural modeling demonstrated that three residues in the AAV2 Rep active site are necessary to cleave its cognate origin. An analogous origin (AAV5) possesses a unique interaction between an extended Rep binding element and a 49 aa region of Rep containing two DNA binding interfaces.Conclusions/SignificanceThe elucidation of these structure-function relationships at the AAV origin led to the creation of a unique recombinant origin and compatible Rep protein with properties independent of either parent serotype. This novel origin may impact the safety and efficacy of AAV as a gene delivery tool. This work may also explain the unique ability of certain AAV serotypes to achieve site-directed integration into the human chromosome. Finally, this result impacts the study of conserved DNA viruses which employ rolling circle mechanisms of replication.
Highlights
General understanding of the mechanisms required for function at origins of replication has grown immensely since the first prokaryotic origins were characterized
While the DNA-protein interactions necessary for replication in prokaryotes, lower eukaryotes, and bacteriophages are generally well understood, mechanisms employed in the majority of higher eukaryotes and vertebrate viruses, such as adeno-associated virus (AAV), are still being determined
The inverted terminal repeats (ITRs) of AAV and other Parvoviruses act as the origin of replication
Summary
General understanding of the mechanisms required for function at origins of replication has grown immensely since the first prokaryotic origins were characterized. While the DNA-protein interactions necessary for replication in prokaryotes, lower eukaryotes, and bacteriophages are generally well understood, mechanisms employed in the majority of higher eukaryotes and vertebrate viruses, such as adeno-associated virus (AAV), are still being determined. Discovering the mechanisms which drive Rep-ITR specificity promises to advance our understanding of DNA-protein interactions at viral origins of replication. These findings promise to shed light on how eukaryotic and prokaryotic proteins achieve selectivity to DNA substrates. While the molecular mechanisms of DNA-protein specificity at the origin of replication have been determined in many model organisms, these interactions remain unknown in the majority of higher eukaryotes and numerous vertebrate viruses. The molecular basis of specificity between the Rep protein and the viral origin of replication between different AAV serotypes remains uncharacterized
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