Understanding AAV Packaging

Abstract

Adeno-associated virus (AAV) has been utilized as a gene therapy vector, but the biology of the virus is unknown. One of the mysteries of AAV is how exactly the virus packages the genome. That being said, the non-structural Rep proteins appear to be necessary and sufficient for proper AAV packaging. The small Rep proteins serve as the molecular motor to package the DNA into the viral capsid, but the large Rep proteins’ role has not been clarified. Furthermore, there is a consensus all Rep proteins should have the same helicase activity, since they have the same helicase domain, and therefore should serve the same role. That actually isn’t the case since only Rep40 appears to be necessary for this task, so helicase assays were conducted to assess the activity of each of the Rep proteins. We found that Rep40 has a higher helicase activity compared to Rep68 implying its role as the molecular motor as logical. Since Rep68 doesn’t unwind DNA nearly as well as Rep40, we hypothesized another role that is linked to its oligomeric capacity. Using AUC, we have discovered Rep68's ability to not only form higher order molecular complexes on its own, but depending on the DNA substrate, the molecular weight of the DNA/protein complex can change. In the presence of the inverted-terminal repeats (ITR), it forms a structure that is smaller and distinct from the structure formed with ssDNA and Rep68. Currently, we have cryo-EM data on the ssDNA/Rep68 complex and we have negative stain data on the ITR/Rep68 complex. All together, Rep68's flexibility appears to imply a more structural role for Rep68 to help hold the DNA in place as Rep40 pushes the DNA into the capsid.