No abstract

The Rep78 and Rep68 proteins of adeno-associated virus (AAV) bind to the AAV terminal repeat hairpin DNA and are required for viral replication. We have expressed a series of mutant rep genes from the human immunodeficiency virus type 1 long terminal repeat promoter in human 293 cells and in an in vitro transcription-translation system. Mutant proteins were analyzed for AAV hairpin DNA binding and AAV terminal resolution functions. Deletion of amino acid residues 523 through 621 of Rep78 had no effect on these functions. Amber mutant Rep proteins truncated at either amino acid 237 or amino acid 243 showed no detectable hairpin DNA binding or terminal resolution activity. A frameshift mutant Rep protein which contained Rep78 amino acids 1 through 241 lacked terminal resolution functions but bound specifically to the AAV hairpin DNA. The carboxyl-terminal missense sequence in this mutant appeared to have complemented an AAV-specific DNA-binding domain within the amino terminus of the Rep protein. A mutant Rep protein in which methionine 225 of Rep78 was deleted (M225dl) was reduced threefold in AAV hairpin binding and had no terminal resolution functions. A mutant Rep protein in which a glycine was substituted at position 225 (M225G) was fully functional in these assays. When M225dl extract was mixed with wild-type Rep78 extract, AAV terminal resolution by Rep78 was inhibited. These results suggest that the amino-terminal portion of Rep78 and Rep68 contains a domain which can direct binding to AAV terminal hairpin DNA and that elements within the central region of the protein stabilize binding.

Four Rep proteins are encoded by the human parvovirus adeno-associated virus type 2 (AAV). The two largest proteins, Rep68 and Rep78, have been shown in vitro to perform several activities related to AAV DNA replication. The Rep78 and Rep68 proteins are likely to be involved in the targeted integration of the AAV DNA into human chromosome 19, and the full characterization of these proteins is important for exploiting this phenomenon for the use of AAV as a vector for gene therapy. To obtain sufficient quantities for facilitating the characterization of the biochemical properties of the Rep proteins, the AAV rep open reading frame was cloned and expressed in Escherichia coli as a fusion protein with maltose-binding protein (MBP). Recombinant MBP-Rep68 and MBP-Rep78 proteins displayed the following activities reported for wild-type Rep proteins when assayed in vitro: (i) binding to the AAV inverted terminal repeat (ITR), (ii) helicase activity, (iii) site-specific (terminal resolution site) endonuclease activity, (iv) binding to a sequence within the integration locus for AAV DNA on human chromosome 19, and (v) stimulation of radiolabeling of DNA containing the AAV ITR in a cell extract. These five activities have been described for wild-type Rep produced from mammalian cell extracts. Furthermore, we recharacterized the sequence requirements for Rep binding to the ITR and found that only the A and A' regions are necessary, not the hairpin form of the ITR. The left open reading frame (ORF) of adeno-associated virus type 2 (AAV) encodes the so-called Rep proteins. Two promoters located at map positions 5 and 19 (p5 and p19, respectively) control expression of the four proteins derived from this ORF (6, 24, 31). Processing of a common intron results in two gene products which are derived from transcripts that are initiated from either promoter (and are designated by the apparent mass of the protein in kilodaltons); Rep78 and Rep68 are produced from p5-initiated transcripts, and Rep52 and Rep4O are produced from pl9-initiated transcripts (22). Plasmids containing cloned AAV yielded wild-type infectious AAV when transfected into adenovirus-infected cells (23, 28). However, mutations within the Rep gene blocked DNA replication (9). The Rep proteins have pleiotropic effects on infected or transfected cells. Properties of the p5-initiated Rep proteins determined in vivo include the abilities to regulate AAV transcription (2, 21), activate replication of AAV (19), inhibit transcription of heterologous promoters (8, 20), and inhibit cellular transformation by bovine papilloma virus (7) or adenovirus Ela plus an activated ras oncogene (15). By in vitro analysis, p5-derived Rep proteins have been shown to bind to the AAV inverted terminal repeat (ITR) (11, 12, 26), have sequence-and strand-specific endonuclease activity and helicase activity (12), and bind to a defined region of human chromosome 19 at the integration locus for AAV provirus (35). These in vitro activities are thought to be essential for the viral life cycle via...

Replication of the palindromic inverted terminal repeats (ITRs) of adeno-associated virus type 2 requires several functions of the viral nonstructural Rep proteins. These include binding to the ITR, nicking of the double-stranded replication intermediate at the terminal resolution site (trs), and then strand displacement and synthesis from the nick. This report demonstrates the ability of both recombinant fusion maltose-binding protein (MBP)-Rep68A produced in Escherichia coli and wild-type (wt) Rep68 to bind to a linear truncated form of the ITR, A57 ITR, with similar affinity as to the wt hairpin ITR. A dissociation constant for MBP-Rep68A of approximately 8 x 10-10 M was determined for the wt ITR and 457 ITR probes. Truncation of 457 ITR to generate 428 ITR, which retains the GCTC repeat motif but not the trs, bound at least 10 times less efficiently than 457 ITR. Extension of A28 ITR with nonspecific sequence restored the ability of MBP-Rep684 to bind to A28 ITR. Thus, high-affinity binding would appear to require stabilization by flanking sequence as well as the intact GCTC repeat motif. Cleavage of the A57 ITR probe with DdeI, which truncates the flanking sequence and was previously shown to inhibit binding by Rep68, also inhibited the binding of MBP-Rep684. The requirements for stable binding were further defined with a series of oligonucleotide probes which spanned the region protected by MBP-Rep78 in DNase I footprinting. The binding activity of either MBP-Rep684 or wt Rep68 to hairpin ITR or A57 ITR was indistinguishable. However, the binding activity of MBP-Rep684 to DNA does not appear to correlate with trs endonuclease activity. The nicking and covalent linkage of MBP-Rep68A to the nonhairpin 457 ITR was approximately 100-fold less efficient than its linkage to a hairpin-containing ITR. Therefore, although the hairpin portion of the ITR does not appear to play a role in recognition and stabilization of MBP-Rep684 binding, its presence does affect the trs cleavage activity of the protein.

Lung diseases such as cystic fibrosis (CF) might be treated by gene therapy using viral vectors delivered to the airway. One potential vector is the defective human parvovirus, adeno-associated virus (AAV). We examined the AAV p5 transcription promoter for gene expression in immortalized cell lines derived from the airway (IB3-1) or pancreas (CFPAC-1) of CF patients. AAV vectors expressing the prokaryotic genes cat (pAAVp5cat) or neo (pAAVp5neo) from the p5 promoter were evaluated after introduction into IB3-1 or CFPAC-1 cells by lipofection. In transient assays in both cell lines, the cat gene was expressed 5- to 10-fold more efficiently from the p5 promoter than from a simian virus 40 early gene promoter (pSVcat). IB3-1 cells were transformed stably to geneticin resistance by pAAVp5neo at a 5-fold higher efficiency than by an SVneo vector. The AAV inverted terminal repeat (ITR) region immediately upstream of the p5 promoter appears to have an enhancer effect and the promoter also contains a CREB site which confers a response to forskolin. In IB3-1 cells, expression of the cat gene from a p5 promoter was decreased about 5-fold by deletion of both the upstream ITR and the CREB site. The AAVp5neo vector was also packaged into AAV particles and used to infect IB3-1 cells as a transducing virus. Under these conditions, 60 to 70% of the cells could be stably transformed to geneticin resistance. Thus, AAV transducing vectors appear to be a highly efficient delivery system for stable integration and expression of genes in cultured airway epithelial cells.