R. Michael Linden

The adeno-associated virus (AAV) encodes four regulatory proteins called Rep. The large AAV Rep proteins Rep68 and Rep78 are essential factors required in almost every step of the viral life cycle. Structurally, they share two domains: a modified version of the AAA⁺ domain that characterizes the SF3 family of helicases and an N-terminal domain that binds DNA specifically. The combination of these two domains imparts extraordinary multifunctionality to work as initiators of DNA replication and regulators of transcription, in addition to their essential role during site-specific integration. Although most members of the SF3 family form hexameric rings in vitro, the oligomeric nature of Rep68 is unclear due to its propensity to aggregate in solution. We report here a comprehensive study to determine the oligomeric character of Rep68 using a combination of methods that includes sedimentation velocity ultracentrifugation, electron microscopy, and hydrodynamic modeling. We have determined that residue Cys151 induces Rep68 to aggregate in vitro. We show that Rep68 displays a concentration-dependent dynamic oligomeric behavior characterized by the presence of two populations: one with monomers and dimers in slow equilibrium and a second one consisting of a mixture of multiple-ring structures of seven and eight members. The presence of either ATP or ADP induces formation of larger complexes formed by the stacking of multiple rings. Taken together, our results support the idea of a Rep68 molecule that exhibits the flexible oligomeric behavior needed to perform the wide range of functions occurring during the AAV life cycle.

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. Viruses have to optimize the limited size of their genomes in order to generate the proteins required for infection and replication. Several mechanisms are used to accomplish this including the use of multiple promoters and alternative splicing. These processes generate gene products with diverse functions through the combinatorial assembly of a small number of protein domains. The small genome of the adeno-associated virus has two major open reading frames that generate seven proteins, four non-structural Rep proteins and three capsid proteins. The non-structural Rep proteins share a motor domain that uses hydrolysis of ATP to generate the conformational changes that drive DNA replication, transcriptional regulation, site-specific integration and the packing of viral genome into capsids. These functions depend upon the oligomerization of Rep proteins on specific DNA sites through the cooperation of the N-terminal origin binding domain and the C-terminal helicase domain. 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. This residue emerges to be a signature motif of SF3 helicases and is also present in a subset of bacterial Rep proteins that support rolling circle replication mechanism.

Adeno-associated virus (AAV) was first discovered as a contaminant of adenovirus stocks in the 1960s. The development of recombinant AAV vectors (rAAV) was facilitated by early studies that generated infectious molecular clones, determined the sequence of the genome, and defined the genetic elements of the virus. The refinement of methods and protocols for the production and application of rAAV vectors has come from years of studies that explored the basic biology of this virus and its interaction with host cells. Interest in improving vector performance has in turn driven studies that have provided tremendous insights into the basic biology of the AAV lifecycle. In this chapter, we review the background on AAV biology and its exploitation for vectors and gene delivery.

Adeno-associated viruses (AAV) are widely spread throughout the human population, yet no pathology has been associated with infection. This fact, together with the availability of simple molecular techniques to alter the packaged viral genome, has made AAV a serious contender in the search for an ideal gene therapy delivery vehicle. However, our understanding of the intriguing features of this virus is far from exhausted and it is likely that the mechanisms underlying the viral lifestyle will reveal possible novel strategies that can be employed in future clinical approaches. One such aspect is the unique approach AAV has evolved in order to establish latency. In the absence of a cellular milieu that will support productive viral replication, wild-type AAV can integrate its genome site specifically into a locus on human chromosome 19 (termed AAVS1), where it resides without apparent effects on the host cell until cellular conditions are changed by outside influences, such as adenovirus super-infection, wh...

The nonpathogenic human adeno-associated virus type 2 (AAV-2) has adopted a unique mechanism to site-specifically integrate its genome into the humanMBS85gene, which is embedded inAAVS1on chromosome 19. The fact that AAV has evolved to integrate into this ubiquitously transcribed region and that the chromosomal motifs required for integration are located a few nucleotides upstream of the translation initiation start codon ofMBS85suggests that the transcriptional activity ofMBS85might influence site-specific integration and thus might be involved in the evolution of this mechanism. In order to begin addressing this question, we initiated the characterization of the humanMBS85promoter region and compared its transcriptional activity to that of the AAV-2 p5 promoter. Our results clearly indicate thatAAVS1is defined by a complex transcriptional environment and that theMBS85promoter shares key regulatory elements with the viral p5 promoter. Furthermore, we provide evidence for bidirectionalMBS85promoter activity and demonstrate that the minimal motifs required for AAV site-specific integration are present in the 5′ untranslated region of the gene and play a posttranscriptional role in the regulation ofMBS85expression. These findings should provide a framework to further elucidate the complex interactions between the virus and its cellular host in this unique pathway to latency.

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. Adeno-associated virus (AAV) is a parvovirus with a linear single-stranded DNA genome. Thus far, it is the only eukaryotic virus known to integrate its genome in human cells in a specific region of chromosome 19. Because no pathologies have been associated with AAV, there is great interest in using AAV as a vector for gene therapy. The genetic information of AAV encodes for both the structural Capsid proteins and the Rep proteins. We have studied a protein called Rep68, which is essential for both AAV genome replication and site-specific integration in chromosome 19, and found that it forms distinct structures in the presence of different DNA structures. Of particular interest is the formation of a Rep68 structure composed of two opposite rings, which resemble the structures formed by the large T antigen and E1 viral proteins of the tumor-inducing Simian virus 40 (SV40) and papilloma viruses, respectively. The double-ring structure of these viral proteins is essential for viral DNA replication, which suggests that AAV has evolved a similar mechanism of DNA replication that relies on a double-ring Rep68. Moreover, Rep68 encounters different DNA structures during viral genome replication, and our results show how Rep68 can adapt to these changes.

A variety of viruses establish latency by integrating their genome into the host genome. The integration event generally occurs in a nonspecific manner, precluding the prediction of functional consequences from resulting disruptions of affected host genes. The nonpathogenic adeno-associated virus (AAV) is unique in its ability to stably integrate in a site-specific manner into the human MBS85 gene. To gain a better understanding of the integration mechanism and the consequences of MBS85 disruption, we analyzed the molecular structure of AAV integrants in various latently infected human cell lines. Our study led to the observation that AAV integration causes an extensive but partial duplication of the target gene. Intriguingly, the molecular organization of the integrant leaves the possibility that a functional copy of the disrupted target gene could potentially be preserved despite the resulting rearrangements. A latently infected, Mbs85 -targeted mouse ES cell line was generated to study the functional consequences of the observed duplication-based integration mechanism. AAV-modified ES cell lines continued to self-renew, maintained their multilineage differentiation potential and contributed successfully to mouse development when injected into blastocysts. Thus, our study reveals a viral strategy for targeted genome addition with the apparent absence of functional consequences.