Abstract
ABSTRACTThe life cycle of the human parvovirus adeno-associated virus (AAV) is orchestrated by four Rep proteins. The large Rep proteins, Rep78 and Rep68, are remarkably multifunctional and display a range of biochemical activities, including DNA binding, nicking, and unwinding. Functionally, Rep78 and Rep68 are involved in transcriptional regulation, DNA replication, and genomic integration. Structurally, the Rep proteins share an AAA+ domain characteristic of superfamily 3 helicases, with the large Rep proteins additionally containing an N-terminal origin-binding domain (OBD) that specifically binds and nicks DNA. The combination of these domains, coupled with dynamic oligomerization properties, is the basis for the remarkable multifunctionality displayed by Rep68 and Rep78 during the AAV life cycle. In this report, we describe an oligomeric interface formed by Rep68 and demonstrate how disruption of this interface has drastic effects on both the oligomerization and functionality of the Rep proteins. Our results support a role for the four-helix bundle in the helicase domain of Rep68 as a bona fide oligomerization domain (OD). We have identified key residues in the OD that are critical for the stabilization of the Rep68-Rep68 interface; mutation of these key residues disrupts the enzymatic activities of Rep68, including DNA binding and nicking, and compromises viral DNA replication and transcriptional regulation of the viral promoters. Taken together, our data contribute to our understanding of the dynamic and substrate-responsive Rep78/68 oligomerization that is instrumental in the regulation of the DNA transitions that take place during the AAV life cycle. IMPORTANCE The limited genome size of small viruses has driven the evolution of highly multifunctional proteins that integrate different domains and enzymatic activities within a single polypeptide. The Rep68 protein from adeno-associated virus (AAV) combines a DNA binding and endonuclease domain with a helicase-ATPase domain, which together support DNA replication, transcriptional regulation, and site-specific integration. The coordination of the enzymatic activities of Rep68 remains poorly understood; however, Rep68 oligomerization and Rep68-DNA interactions have been suggested to play a crucial role. We investigated the determinants of Rep68 oligomerization and identified a hydrophobic interface necessary for Rep68 activity during the AAV life cycle. Our results provide new insights into the molecular mechanisms underlying the regulation of the versatile Rep proteins. Efficient production of AAV-based gene therapy vectors requires optimal Rep expression levels, and studies such as the one presented here could contribute to further optimization of AAV production schemes.
Highlights
The life cycle of the human parvovirus adeno-associated virus (AAV) is orchestrated by four Rep proteins
Previous studies using Rep68*, a C151S Rep mutant that is functionally equivalent to WT Rep68 but that prevents protein aggregation in solution [25], showed that Rep68 exists as a mixture of oligomers in solution
It has been shown that Rep can form different oligomeric species in vitro both in the absence of DNA and in the presence of different DNA substrates [24, 25], allowing an additional layer of regulation of the Rep activities during the AAV life cycle
Summary
The life cycle of the human parvovirus adeno-associated virus (AAV) is orchestrated by four Rep proteins. The Rep proteins share an AAA؉ domain characteristic of superfamily 3 helicases, with the large Rep proteins containing an N-terminal origin-binding domain (OBD) that binds and nicks DNA The combination of these domains, coupled with dynamic oligomerization properties, is the basis for the remarkable multifunctionality displayed by Rep and Rep during the AAV life cycle. Efficient nicking of the trs at both the viral and cellular origins requires ATP-dependent helicase activity for the generation of an optimal single-stranded substrate [7, 17] Both the OBD and the helicase domain have the ability to mediate the transcriptional regulation of viral and cellular promoters by two independent mechanisms conferring regulatory functions to both small and large Rep proteins [18,19,20]
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