DNA Structure Modulates the Oligomerization Properties of the AAV Initiator Protein Rep68

Abstract

Rep68 is a multifunctional protein of the adeno-associated virus (AAV), a parvovirus that is mostly known for its promise as a gene therapy vector. In addition to its role as initiator in viral DNA replication, Rep68 is essential for site-specific integration of the AAV genome into human chromosome 19. Rep68 is a member of the superfamily 3 (SF3) helicases, along with the well-studied initiator proteins simian virus 40 large T antigen (SV40-LTag) and bovine papillomavirus (BPV) E1. Structurally, SF3 helicases share two domains, a DNA origin interaction domain (OID) and an AAA+ motor domain. The AAA+ motor domain is also a structural feature of cellular initiators and it functions as a platform for initiator oligomerization. Here, we studied Rep68 oligomerization in vitro in the presence of different DNA substrates using a variety of biophysical techniques and cryo-EM. We found that a dsDNA region of the AAV origin promotes the formation of a complex containing five Rep68 subunits. Interestingly, non-specific ssDNA promotes the formation of a double-ring Rep68, a known structure formed by the LTag and E1 initiator proteins. The Rep68 ring symmetry is 8-fold, thus differing from the hexameric rings formed by the other SF3 helicases. However, similiar to LTag and E1, Rep68 rings are oriented head-to-head, suggesting that DNA unwinding by the complex proceeds bidirectionally. This novel Rep68 quaternary structure requires both the DNA binding and AAA+ domains, indicating cooperativity between these regions during oligomerization in vitro. Our study clearly demonstrates that Rep68 can oligomerize through two distinct oligomerization pathways, which depend on both the DNA structure and cooperativity of Rep68 domains. These findings provide insight into the dynamics and oligomeric adaptability of Rep68 and serve as a step towards understanding the role of this multifunctional protein during AAV DNA replication and site-specific integration.

Structural Insights into the Assembly of the Adeno-associated Virus Type 2 Rep68 Protein on the Integration Site AAVS1

“…The heptameric Rep68-AAVS1 complex is different from other published reports that show a variety of Rep oligomers such as a hexamer of Rep78 on the AAV-2 ITR site (56), a Rep68 octamer bound to a single-stranded poly(dT) DNA (57), and the pentameric complex observed in the AAV5 OBD-RBS structure (26). These large varieties of complexes are the reflections of the versatility of AAV Rep proteins to be modulated depending on the DNA substrate they bind.…”

High-Throughput Sequencing Reveals Principles of Adeno-Associated Virus Serotype 2 Integration

“…The large Rep proteins undergo DNA facilitated oligomerization, where the linker between the DNA binding domain and helicase are critical for complex formation (23)(24)(25). Recent crystal structure and cryo-electron microscopy (cryo-EM) studies have revealed that AAV Rep68/78 can form double octameric or hexameric rings, with the rings facing opposite directions (21,24,26). DNA binding, endonuclease, helicase activity, and Rep oligomerization are required for both viral replication and integration (9,27).…”

Effect of ATP Binding and Hydrolysis on Dynamics of Canine Parvovirus NS1

“…These motifs reside in a stretch of approximately 100 amino acid residues in the middle of NS1 (27, 69). SF3 helicases surround DNA as a ring of six or eight subunits, and the ATP-binding pocket lies between adjacent subunits (30,42). The first subunit provides the A and B motifs, and the arginine residue of the second subunit functions as a trans-acting "arginine finger" sensor for ATP binding and hydrolysis status (56,58).…”