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Integration into a particular location in human chromosomes is a unique property of the adeno-associated virus (AAV). This reaction requires the viral Rep protein and AAV origin sequences. To understand how Rep recognizes DNA, we have determined the structures of the Rep endonuclease domain separately complexed with two DNA substrates: the Rep binding site within the viral inverted terminal repeat and one of the terminal hairpin arms. At the Rep binding site, five Rep monomers bind five tetranucleotide direct repeats; each repeat is recognized by two Rep monomers from opposing faces of the DNA. Stem-loop binding involves a protein interface on the opposite side of the molecule from the active site where ssDNA is cleaved. Rep therefore has three distinct binding sites within its endonuclease domain for its different DNA substrates. Use of these different interfaces generates the structural asymmetry necessary to regulate later events in viral replication and integration.

Adeno-associated virus (AAV), unique among animal viruses in its ability to integrate into a specific chromosomal location, is a promising vector for human gene therapy. AAV Replication (Rep) protein is essential for viral replication and integration, and its amino terminal domain possesses site-specific DNA binding and endonuclease activities required for replication initiation and integration. This domain displays a novel endonuclease fold and demonstrates an unexpected structural relationship to other viral origin binding proteins such as the papillomavirus E1 protein and the SV40 T antigen. The active site, located at the bottom of a positively charged cleft, is formed by the spatial convergence of a divalent metal ion and two conserved sequence motifs that define the rolling circle replication superfamily.

Adeno-associated virus (AAV) type 2 Rep78 is a multifunctional protein required for AAV DNA replication, integration, and gene regulation. The biochemical activities of Rep78 have been described, but the effects of Rep proteins on the cell have not been characterized. We have analyzed Rep-mediated cytotoxicity. We demonstrated that Rep78 expression is sufficient to induce cell death and disruption of the cell cycle. Cell death was found to be mediated by apoptosis. Rep78 expression resulted in the activation of caspase-3, a terminal caspase directly involved in the execution of cell death. A peptidic inhibitor of caspase-3, Z-Asp-Glu-Val-Asp-fluoromethylketone (Z-DEVD-FMK), abrogated Rep78-induced apoptosis, indicating that Rep78-mediated apoptosis is caspase-3 dependent. Rep78 induced apoptosis in wild-type p53-containing human embryonal carcinoma NT-2 cells and in p53-null promyelocytic human HL-60 cells, indicating that at least one pathway of Rep78-induced apoptosis is p53 independent. Apoptosis was shown to occur during the G(1) and early S phases of the cell cycle. By analyzing the effects of Rep78 mutations on cell viability, the cause of cell death was attributed in part to two biochemical activities of Rep78, DNA binding and ATPase/helicase activity. The endonuclease activity of Rep78 did not contribute to apoptosis induction.

The Rep78 protein of adeno-associated virus (AAV) contains amino acid sequence motifs common to rolling-circle replication (RCR) initiator proteins. In this report, we describe RCR initiator-like activities of Rep78. We demonstrate that a maltose-binding protein (MBP)-Rep78 fusion protein can catalyze the cleavage and ligation of single-stranded DNA substrates derived from the AAV origin of replication. Rep-mediated single-stranded DNA cleavage was strictly dependent on the presence of certain divalent cations (e.g., Mn(2+) or Mg(2+)) but did not require the presence of a nucleoside triphosphate cofactor. Electrophoretic mobility shift assays demonstrated that binding of single-stranded DNA by MBP-Rep78 was influenced by the length of the substrate as well as the presence of potential single-stranded cis-acting sequence elements. Site-directed mutagenesis was used to examine the role of specific tyrosine residues within a conserved RCR motif (motif 3) of Rep78. Replacement of Tyr-156 with phenylalanine abolished the ability of MBP-Rep78 to mediate the cleavage and ligation of single-stranded DNA substrates but not the ability to stably bind single-stranded DNA. The cleaving-joining activity of Rep78 is consistent with the mechanism of replicative intermediate dimer resolution proposed for the autonomous parvoviruses and may have implications for targeted integration of recombinant AAV vectors.

Adeno-associated virus (AAV) replication depends on two viral components for replication: the AAV nonstructural proteins (Rep) in trans, and inverted terminal repeat (ITR) sequences in cis. AAV type 5 (AAV5) is a distinct virus compared to the other cloned AAV serotypes. Whereas the Rep proteins and ITRs of other serotypes are interchangeable and can be used to produce recombinant viral particles of a different serotype, AAV5 Rep proteins cannot cross-complement in the packaging of a genome with an AAV2 ITR. In vitro replication assays indicated that the block occurs at the level of replication instead of at viral assembly. AAV2 and AAV5 Rep binding activities demonstrate similar affinities for either an AAV2 or AAV5 ITR; however, comparison of terminal resolution site (TRS) endonuclease activities showed a difference in specificity for the two DNA sequences. AAV2 Rep78 cleaved only a type 2 ITR DNA sequence, and AAV5 Rep78 cleaved only a type 5 probe efficiently. Mapping of the AAV5 ITR TRS identified a distinct cleavage site (AGTG TGGC) which is absent from the ITRs of other AAV serotypes. Comparison of the TRSs in the AAV2 ITR, the AAV5 ITR, and the AAV chromosome 19 integration locus identified some conserved nucleotides downstream of the cleavage site but little homology upstream.

Adeno-associated virus encodes four nonstructural proteins, which are known as Rep78, Rep68, Rep52, and Rep40. Expression of these nonstructural proteins affects cell growth and gene expression through processes that have not yet been characterized. Using a yeast two-hybrid screen, we have demonstrated that a stable interaction occurs between the viral proteins Rep78 and Rep52 and the putative protein kinase PrKX, which is encoded on the X chromosome. The stability and specificity of the Rep-PrKX interaction were confirmed by coimmunoprecipitation of complexes assembled in vitro and in vivo. Overexpressed PrKX, which was purified from cos cells, was shown to phosphorylate a synthetic protein kinase A (PKA) substrate. However, this activity was dramatically inhibited by stoichiometric amounts of Rep52 and weakly inhibited with Rep68, which lacks the carboxy-terminal sequence contained in Rep52. Similarly, a stable interaction was observed with Rep78, which also contains the carboxy-terminal sequence of Rep52. A stable interaction and inhibition were also observed between Rep52 and the catalytic subunit of PKA. By using surface plasmon resonance and kinetic studies, Kis of approximately 300 and 167 nM were calculated for Rep52 with PKA and with PrKX, respectively. Thus, Rep52 but not Rep68 can significantly inhibit the trans- and autophosphorylation activities of these kinases. The biological effects of Rep78-specific inhibition of PKA-responsive genes are illustrated by the reduction of steady-state levels of cyclic AMP-responsive-element-binding protein and cyclin A protein.

The rep gene of adeno-associated virus type 2 encodes four overlapping proteins from two separate promoters, termed P5 and P19. The P5-promoted Rep proteins, Rep78 and Rep68, are essential for viral DNA replication, and a wealth of data concerning the biochemical activities of these proteins has been reported. In contrast, data concerning the biochemical functions of the P19-promoted Rep proteins, Rep52 and Rep40, are lacking. Here, we describe enzymatic activities associated with a bacterially expressed maltose-binding protein (MBP)-Rep52 fusion protein. Purified MBP-Rep52 possesses 3'-to-5' DNA helicase activity that is strictly dependent upon the presence of nucleoside triphosphate and divalent cation cofactors. In addition, MBP-Rep52 demonstrates a constitutive ATPase activity that is active in the absence of DNA effector molecules. An MBP-Rep52 chimera bearing a lysine-to-histidine substitution at position 116 (K116H) within a consensus helicase- and ATPase-associated motif (motif I or Walker A site) was deficient for both DNA helicase and ATPase activities. In contrast to a Rep78 A-site mutant protein bearing a corresponding amino acid substitution at position 340 (K340H), the MBP-Rep52 A-site mutant protein failed to exhibit a trans-dominant negative effect when it was mixed with wild-type MBP-Rep52 or MBP-Rep78 in vitro. This lack of trans dominance, coupled with the results of coimmunoprecipitation and gel filtration chromatography experiments reported here, suggests that the ability of Rep52 to engage in multimeric interactions may differ from that of Rep78 or -68.

The Rep78 and Rep68 proteins of adeno-associated virus (AAV) are replication initiator proteins that bind the viral replicative-form origin of replication, nick the origin in a site- and strand-specific fashion, and mediate vectorial unwinding of the DNA duplex via an ATP-dependent helicase activity, thus initiating a strand displacement mechanism of viral DNA replication. Genetic and biochemical studies have identified Rep mutants that demonstrate a trans-dominant negative phenotype in vitro and in vivo, suggesting the possibility that multimerization of Rep is essential for certain replicative functions. In this study, we have investigated the ability of the largest of the Rep proteins, Rep78, to self-associate in vitro and in vivo. Self-association of Rep78 in vivo was demonstrated through the use of a mammalian two-hybrid system. Rep-Rep protein interaction was confirmed in vitro through coimmunoprecipitation experiments with a bacterially expressed maltose-binding protein-Rep78 fusion protein in combination with [35S]methionine-labeled Rep78 synthesized in a coupled in vitro transcription-translation system. Mapping studies with N- and C-terminal truncation mutant forms of Rep indicate that amino acid sequences required for maximal self-association occur between residues 164 and 484. Site-directed mutagenesis identified two essential motifs within this 321-amino-acid region: (i) a putative alpha-helix bearing a 3,4-hydrophobic heptad repeat reminiscent of those found in coiled-coil domains and (ii) a previously recognized nucleoside triphosphate-binding motif. Deletion of either of these regions from the full-length polypeptide resulted in severe impairment of Rep-Rep interaction. In addition, gel filtration chromatography and protein cross-linking experiments indicated that Rep78 forms a hexameric complex in the presence of AAV ori sequences.

To further define the canonical binding site for the P5-promoted Rep proteins of the adeno-associated virus, a modified random oligonucleotide selection procedure was performed, using purified recombinant Rep protein. These results may explain the effects of Rep on cellular gene expression.

The DNA of human parvovirus adeno-associated virus type 2 (AAV) integrates preferentially into a defined region of human chromosome 19. Southern blots of genomic DNA from latently infected cell lines revealed that the provirus was not simply inserted into the cellular DNA. Both the proviral and adjoining cellular DNA organization indicated that integration occurred by a complex, coordinated process involving limited DNA replication and rearrangements. However, the mechanism for targeted integration has remained obscure. The two larger nonstructural proteins (Rep68 and Rep78) of AAV bind to a sequence element that is present in both the integration locus (P1) and the AAV inverted terminal repeat. This binding may be important for targeted integration. To investigate the mechanism of targeted integration, we tested the cloned integration site subfragment in a cell-free replication assay in the presence or absence of recombinant Rep proteins. Extensive, asymmetric replication of linear or open-circular template DNA was dependent on the presence of P1 sequence and Rep protein. The activities of Rep on the cloned P1 element are analogous to activities on the AAV inverted terminal repeat. Replication apparently initiates from a 3'-OH generated by the sequence-specific nicking activity of Rep. This results in a covalent attachment between Rep and the 5'-thymidine of the nick. The complexity of proviral structures can be explained by the participation of limited DNA replication facilitated by Rep during integration.

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)-Rep68 delta produced in Escherichia coli and wild-type (wt) Rep68 to bind to a linear truncated form of the ITR, delta 57 ITR, with similar affinity as to the wt hairpin ITR. A dissociation constant for MBP-Rep68 delta of approximately 8 x 10(-10) M was determined for the wt ITR and delta 57 ITR probes. Truncation of delta 57 ITR to generate delta 28 ITR, which retains the GCTC repeat motif but not the trs, bound at least 10 times less efficiently than delta 57 ITR. Extension of delta 28 ITR with nonspecific sequence restored the ability of MBP-Rep68 delta to bind to delta 28 ITR. Thus, high-affinity binding would appear to require stabilization by flanking sequence as well as the intact GCTC repeat motif. Cleavage of the delta 57 ITR probe with DdeI, which truncates the flanking sequence and was previously shown to inhibit binding by Rep68, also inhibited the binding of MBP-Rep68 delta. 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-Rep68 delta or wt Rep68 to hairpin ITR or delta 57 ITR was indistinguishable. However, the binding activity of MBP-Rep68 delta to DNA does not appear to correlate with trs endonuclease activity. The nicking and covalent linkage of MBP-Rep68 delta to the nonhairpin delta 57 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-Rep68 delta binding, its presence does affect the trs cleavage activity of the protein.

The large rep gene products (rep68 and rep78) of adeno-associated virus (AAV) are pleiotropic effector proteins which not only play a critical role in AAV DNA replication and in the trans-regulation of AAV promotor elements, but are also known for their onco-suppressive functions. We have previously demonstrated that the large AAV rep protein will strongly inhibit expression from the c-H-ras promoter, but not the murine osteosarcoma virus long terminal repeat (MSV-LTR) promoter. To investigate the possibility that rep may physically bind to these promoter sequences, specifically to GCTC motifs, we conducted electrophoretic mobility shift assays (EMSA) with a maltose binding protein-rep chimeric protein, MBP-rep68 delta, and synthetic double stranded DNA substrates of sequences selected from the c-H-ras and MSV-LTR promoters, as well as with the AAV TR. We find that MPB-rep68 delta bound the AAV TR DNA sequence (three motifs) most strongly, followed by the selected c-H-ras DNA sequence (two noninterfering motifs), and most poorly to the MSV-LTR DNA (one motif). These data are consistent with our previous study and suggest a direct mechanism of action for AAV rep inhibition of the c-H-ras promoter. Furthermore, the results suggest that the number of GCTC motifs, when closely associated, affect the affinity of rep binding. Finally, we find that MBP-rep68 delta also binds to the c-H-ras oligomer substrates which have secondary hairpin structures.

AAV is unique among eukaryotic viruses in the ability of its DNA to integrate preferentially into a specific region of the human genome. Understanding AAV integration may aid in developing gene therapy systems with predictable integration sites. Using a gel mobility-shift assay, we have identified a DNA sequence within the AAV integration locus on human chromosome 19 which is specifically bound by the AAV Rep78 and Rep68 proteins. This Rep recognition sequence is a GCTC repeating motif very similar to sequences within the inverted terminal repeats of the AAV genome which are also bound by Rep78 and Rep68. Cloned oligonucleotides containing the recognition sequence can direct specific binding by Rep proteins. Binding assays with mutant Rep proteins show that the amino-terminal portion of Rep78 and Rep68 can direct binding to either the AAV terminal repeat hairpin DNA or chromosome 19. This human genomic DNA can be complexed with AAV DNA by Rep proteins as demonstrated by a dual-label (32P/biotin) assay. These results suggest a role for Rep in targeting viral integration.

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.