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.

FIG. 1.

The p5IEE functions as a target for Rep-mediated site-specific integration. (A) Diagrammatic representation of the p5CAT plasmid used as a target substrate for replication and integration assays. The p5 element or p5IEE indicated in black is functioning as the promoter for the CAT reporter gene. The CAT coding region used for Southern analysis is indicated by shading, and the MboI restriction sites at 691 and 1561 are shown. (B) Sequence of the p5IEE flanked by NotI and SrfI restriction sites. (C) Restriction digestion map illustrating genomic sequence in the area of AAVS1. Map depicts genomic fragments for EcoRV and BamHI digests used to characterize AAVS1 disruptions and integrants and the AAVS1 genomic region used as a probe in Southern blot analysis. Representative Southern blots of genomic DNA from individual cell lines transfected with p5CAT and T7Rep78 6 weeks posttransfection, hybridized with AAVS1 probe (D) or CAT probe (E) are shown. Arrowheads at the bottom of panel E indicate lanes where restriction fragment bands revealed by both AAVS1 and CAT probes were found to comigrate.

FIG. 2.

Replication of p5CAT in HeLa cells. Replication assay of the ITR-containing plasmid pTRUF or p5CAT, cotransfected with a control plasmid pCMVGFP, pT7Rep, or CMVRep, in the presence of wt Ad2 or mock (medium) helper virus infection. DNA harvested from duplicate transfections was digested with MboI or DpnI to identify replication products (MboI) or transfected plasmid (DpnI). (A) pTRUF2 replication products detected by Southern analysis when probed for pTRUF2 are indicated by arrows (542 and 621 bp). Boxed areas highlight the locations of expected Rep-dependent replication products. (B) Ethidium bromide staining of gel in panel A before transfer. (C) p5CAT replication products detected by Southern analysis when probed with CAT. The expected 861-bp replication product should be present within the boxed area. (D) Ethidium bromide staining of gel in panel C before transfer.

FIG. 3.

Characterization of p5IEE origin elements associated with the RBE and the TRS site (YY1). Plasmids containing only the p5 RBEYY1+1 sequence region of p5 or only the p5 RBE (A) were analyzed for CAT expression following transient transfection in the absence (−Rep) or presence of Rep (+Rep) (B). Constructs were characterized for site-specific integration efficiency following transfection of each construct in the presence of T7Rep (C) and by a DNA replication assay (D) as described in the text.

FIG. 4.

Characterization of p5IEE plasmids containing mutations in the RBE region. (A) Diagram of p5CAT constructs. The RBE was mutated to a non-Rep binding sequence (p5RBEmut), or a wt RBE was reinserted flanked by the restriction sites PacI and ClaI (p5RBEfix) or with PacI only (p5PacRBE) or ClaI only (p5ClaRBE). Constructs were characterized for CAT expression (+Rep or −Rep) (B) site-specific integration efficiency (C), or p5 replication function (D) as previously described.

FIG. 5.

Characterization of RBE replacement domains from AAVS1 and AAVITR in p5IEE. The p5 RBE was replaced with the RBE sequence present in the AAV2 ITR origin domain or the human chromosome 19 AAVS1 integration site RBE (A) and characterized for CAT activity in the presence (+Rep) or absence (−Rep) of T7Rep (B), site-specific integration efficiency with T7Rep (C), or DNA replication with CMVRep (D), as previously described.

FIG. 6.

The role of p5IEE promoter elements in transcription, replication, and integration. The p5 promoter includes sequence elements associated with specific transcription factor binding sites as previously indicated. Constructs containing the indicated transcription factor binding site mutations (A) were constructed and characterized for promoter activity (B), for site-specific integration (C), or for Rep-dependent replication (D) carried out as previously described.