Abstract
The four Rep proteins of adeno-associated virus (AAV) orchestrate all aspects of its viral life cycle, including transcription regulation, DNA replication, virus assembly, and site-specific integration of the viral genome into the human chromosome 19. All Rep proteins share a central SF3 superfamily helicase domain. In other SF3 members this domain is sufficient to induce oligomerization. However, the helicase domain in AAV Rep proteins (i.e. Rep40/Rep52) as shown by its monomeric characteristic, is not able to mediate stable oligomerization. This observation led us to hypothesize the existence of an as yet undefined structural determinant that regulates Rep oligomerization. In this document, we described a detailed structural comparison between the helicase domains of AAV-2 Rep proteins and those of the other SF3 members. This analysis shows a major structural difference residing in the small oligomerization sub-domain (OD) of Rep helicase domain. In addition, secondary structure prediction of the linker connecting the helicase domain to the origin-binding domain (OBD) indicates the potential to form α-helices. We demonstrate that mutant Rep40 constructs containing different lengths of the linker are able to form dimers, and in the presence of ATP/ADP, larger oligomers. We further identified an aromatic linker residue (Y224) that is critical for oligomerization, establishing it as a conserved signature motif in SF3 helicases. Mutation of this residue critically affects oligomerization as well as completely abolishes the ability to produce infectious virus. Taken together, our data support a model where the linker residues preceding the helicase domain fold into an α-helix that becomes an integral part of the helicase domain and is critical for the oligomerization and function of Rep68/78 proteins through cooperative interaction with the OBD and helicase domains.
Highlights
The four adeno-associated virus (AAV) Rep proteins are generated from a single open reading frame by the transcriptional use of two different promoters (p5 and p19) and subsequent alternative splicing mechanisms [1,2,3]
We provide evidence that the linker that connects the two domains is an integral feature of the helicase domain and contains a conserved aromatic residue that is critical for oligomerization
We show that in the context of Rep68/78 the required oligomerization is supported by the interdomain linker which is directly involved in oligomerization interface and we provide evidence that the tyrosine residue preceding the start of Rep40/52 (Y224) is critical in the oligomerization and activity of the large AAV Rep proteins
Summary
The four adeno-associated virus (AAV) Rep proteins are generated from a single open reading frame by the transcriptional use of two different promoters (p5 and p19) and subsequent alternative splicing mechanisms [1,2,3]. In terms of domain architecture the AAV Rep proteins resemble other members of the SF3 protein family, the peculiar OBD with its additional nuclease activity and the complex character of their oligomeric properties, set them apart from other SF3 helicases such as simian virus 40 large T antigen (SV40-LTag) and papilloma virus E1 (PV-E1) proteins [10,11,12,13] In both of these proteins, the minimal SF3 helicase domain assembles into a hexameric ring in a process that can be induced by the presence of ATP and/or single-stranded DNA [14,15]. One interface is formed by the interaction of neighbouring N-terminal
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