Adeno-Associated Virus Type 2 p5 Promoter: a Rep-Regulated DNA Switch Element Functioning in Transcription, Replication, and Site-Specific Integration

Abstract

The large Rep proteins, p68 and p78, function as master controllers of the adeno-associated virus type 2 (AAV2) life cycle, involved in transcriptional control, in latency, in rescue, and in viral DNA replication. The p5 promoter may be the nucleic acid complement to the large Rep proteins. It drives expression of the large Rep proteins, it undergoes autoregulation by Rep, it undergoes induction by helper virus, it is a target substrate for Rep-mediated site-specific integration (RMSSI), and it can function as a replicative origin. To better understand the relationship between each of the p5 functions, we have determined the effects of p5 promoter mutations (p5 integration efficiency element, or p5IEE) on transcription, integration, and replication using RMSSI transfection protocols in HeLa cells. The data demonstrate that the organization of the p5 promoter provides a unique platform for regulated AAV2 template transcription and subsequent repression by Rep through direct and indirect mechanisms. The elements of the p5IEE that define its function as a promoter also define its function as a highly optimized substrate for Rep-mediated site-specific integration and replication. The p5 Rep binding element (RBE) is essential in RMSSI and Rep-dependent replication; however, replacement of the p5 RBE with either the AAV2 inverted terminal repeat or the AAVS1 RBE sequence elements neither enhances nor severely compromises RMSSI activity of p5IEE. The RBE by itself or in combination with the YY1؉1 initiator/terminal resolution sequence element does not mediate efficient site-specific integration. We found that replication and integration were highly sensitive to sequence manipulations of the p5 TATA/RBE/YY1؉1 core structure in a manner that reflects the function of these elements in transcription. The data presented support a model where, depending on the state of the cell (Rep expression and helper virus influences), the p5IEE operates as a transcription/integration switch sequence element.Adeno-associated virus type 2 (AAV2) is a nonpathogenic single-stranded DNA parvovirus that requires coinfection with a helper virus (adenovirus [Ad] or herpes virus) to stimulate progression through the viral replicative cycle. The Ad helper virus expresses the immediate early E1A gene products which have been shown to transactivate the AAV2 p5 promoter (1, 2), resulting in expression of two alternatively spliced p5 transcripts that yield the p68 and p78 (p68/78) Rep polypeptides (8,33). The large Rep polypeptides possess endonuclease, helicase, and ATPase enzymatic activities (reviewed in reference 21). In addition to enzymatic properties, the N-terminal domain of Rep p68/78 has sequence-specific DNA binding capabilities that recognize a GAGC sequence motif (3,18,24,28). This motif is imperfectly repeated four times in the AAV2 origin element located in the hairpin inverted terminal repeat (ITR) present at both ends of the viral genome (18,24). Both the enzymatic and DNA binding functions of p68/78 are essential for full-length re...

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

“…5E). In addition to the ITR, the p5 promoter has been shown to contain a functional RBS/TRS, and in plasmid systems, the p5 promoter is able to independently enhance AAVS1 integration efficiency (13,38,39,62). The combination of a p5 transcriptional complex localized near the leftend ITR may present a unique structural domain to specifically interact with the integration complex forming on a genomic site.…”

Basic Biology of Adeno-Associated Virus (AAV) Vectors Used in Gene Therapy

“…This primarily drives the expression of Rep proteins as well as cellular factors such as YY1, a human GLI-Kruppel-related protein [21,133]. Both P5 and P19 promoters act as a switch element involved in transcription, replication, and site-specific integration [134,135]. The activation of these promoters leads to the generation of Rep78, Rep68, Rep 52 and Rep 40.…”

Relative Influence of the Adeno-Associated Virus (AAV) Type 2 p5 Element for Recombinant AAV Vector Site-Specific Integration

“…These studies also indicated that the TATA box flanking the Rep binding site was absolutely required for in vivo replication of the p5 element, likely through TATA binding protein-mediated enhancement of Rep-dependent binding and nicking. Similarly, Murphy et al recently confirmed that mutating the TATA box severely compromised replication of the p5 region and also found that RMSSI of p5-containing plasmids was sensitive to the same mutations as those affecting replication of this element (7). However, these studies, like the previous ones, were performed by analyzing stable cell clones derived from actively dividing cells, such as HeLa or 293 cells, transfected with p5-containing plasmids.…”

“…A first study was conducted to determine if the minimal 55-bp p5 element, identified as an efficient replication origin (3), was also able to increase RMSSI of rAAV plasmid as previously documented for the entire p5 region. To answer this question, we used the procedure that was previously employed by other investigators (7,10,11), consisting of the analysis of stable cell clones isolated from HeLa cells transfected with the rAAV plasmid containing different versions of the p5 element inserted between the ITRs (Fig. 1).…”