AAV-mediated TIMP-1 overexpression in aortic tissue reduces the severity of allograft vasculopathy in mice.

BACKGROUND: Allograft vasculopathy (AV) is the primary limiting factor for long-term graft survival. An increased activity of matrix metalloproteinases (MMPs) contributes to neointima formation in AV and represents a potential therapeutic target. Adeno-associated virus (AAV)-mediated gene therapy comprises a potentially benign vector model for the long-term expression of MMP antagonists. METHODS: Aortic allografts from DBA/2 mice were incubated with control buffer, AAV-enhanced green fluorescence protein (EGFP), or tissue inhibitor of metalloproteinases 1 (TIMP-1)-loaded AAV (AAV-TIMP-1) and transplanted into the infrarenal aorta of C57BL/6 mice. Cyclosporine A (10 mg/kg body weight) was administered daily. Explantation as well as histomorphometric and immunohistochemical evaluation was performed after 30 days. Matrix metalloproteinase (MMP) activity was visualized by gelatin in situ zymography. RESULTS: Intima-to-media area ratio and neointima formation were significantly reduced in the AAV-TIMP-1 treatment group compared with those in the control group (by 40%; p < 0.001) and the AAV-EGFP group (by 38.2%; p < 0.001). TIMP-1 overexpression positively affected several pathomechanisms for the development of AV both in vitro and in vivo as compared to that in the control groups: endothelium integrity was preserved as shown by zona occludens 1 and occludin staining; MMP9 expression and activity were significantly reduced (p = 0.01); and smooth muscle cell migration was significantly reduced as smooth muscle actin positive cells predominantly remained in the aortic media in the treatment group (p = 0.001). Moreover, macrophage infiltration was markedly reduced by 49% in the AAV-TIMP-1 group (p < 0.001). CONCLUSION: Immediate post-harvesting allograft incubation with AAV-TIMP-1 reduces neointima formation and macrophage infiltration, constituting a possible adjunct therapeutic strategy to preserve graft function after transplantation.

AAV serotype 8-mediated liver specific GNMT expression delays progression of hepatocellular carcinoma and prevents carbon tetrachloride-induced liver damage.

Glycine N-methyltransferase (GNMT) is abundantly expressed in normal livers and plays a protective role against tumor formation. GNMT depletion leads to progression of hepatocellular carcinoma (HCC). In this study, we investigated the activity of ectopic GNMT delivered using recombinant adeno-associated virus (AAV) gene therapy in mouse models of liver cirrhosis and HCC. Injection of AAV serotype 8 (AAV8) vector carrying the GNMT gene (AAV8-GNMT) in Gnmt-/- mice increased GNMT expression and downregulated pro-inflammatory responses, resulting in reduced liver damage and incidence of liver tumors. Moreover, AAV8-GNMT resulted in the amelioration of carbon tetrachloride (CCl4)-induced liver fibrosis in BALB/c mice. We showed that AAV8-GNMT protected hepatocytes from CCl4-induced liver damage.  AAV8-GNMT significantly attenuated the levels of pro-fibrotic markers and increased efficiency of hepatocyte proliferation. These results suggest that correction of hepatic GNMT by gene therapy of AAV8-mediated gene enhancement may provide a potential strategy for preventing and delaying development of liver diseases.

Exchange of functional domains between a bacterial conjugative relaxase and the integrase of the human adeno-associated virus.

Endonucleases of the HUH family are specialized in processing single-stranded DNA in a variety of evolutionarily highly conserved biological processes related to mobile genetic elements. They share a structurally defined catalytic domain for site-specific nicking and strand-transfer reactions, which is often linked to the activities of additional functional domains, contributing to their overall versatility. To assess if these HUH domains could be interchanged, we created a chimeric protein from two distantly related HUH endonucleases, containing the N-terminal HUH domain of the bacterial conjugative relaxase TrwC and the C-terminal DNA helicase domain of the human adeno-associated virus (AAV) replicase and site-specific integrase. The purified chimeric protein retained oligomerization properties and DNA helicase activities similar to Rep68, while its DNA binding specificity and cleaving-joining activity at oriT was similar to TrwC. Interestingly, the chimeric protein could catalyse site-specific integration in bacteria with an efficiency comparable to that of TrwC, while the HUH domain of TrwC alone was unable to catalyze this reaction, implying that the Rep68 C-terminal helicase domain is complementing the TrwC HUH domain to achieve site-specific integration into TrwC targets in bacteria. Our results illustrate how HUH domains could have acquired through evolution other domains in order to attain new roles, contributing to the functional flexibility observed in this protein superfamily.

Differential contribution of adeno-associated virus type 2 Rep protein expression and nucleic acid elements to inhibition of adenoviral replication in cis and in trans.

UNLABELLED: The helper-dependent adeno-associated virus type 2 (AAV-2) exhibits complex interactions with its helper adenovirus. Whereas AAV-2 is dependent on adenoviral functions for productive replication, it conversely inhibits adenoviral replication, both when its genome is present in trans after coinfection with both viruses and when it is present in cis, as in the production of recombinant adenovirus (rAd)/AAV-2 hybrid vectors. The notion that AAV-mediated inhibition of adenoviral replication is due predominantly to the expression of the AAV-2 Rep proteins was recently challenged by successful Rep78 expression in a rAd5 vector through recoding of the Rep open reading frame (ORF). We closely analyzed the relative contributions of AAV-2 nucleic acid elements and Rep protein expression to the inhibition of adenoviral replication in both of the above scenarios. When present in cis, a sequence element in the 3' part of the rep gene, comprising only the AAV-2 p40 promoter and the AAV-2 intron sequence, which we termed the RIS-Ad, completely blocks adenoviral replication. p5/p19 promoter-driven Rep protein expression, on the other hand, only weakly inhibits rAd/AAV-2 vector propagation, and by inactivation of the RIS-Ad, it is feasible to generate first-generation rAd vectors expressing functional Rep proteins. The RIS-Ad plays no role in the inhibition of adenoviral replication in trans in a model closely mimicking AAV-2-Ad coinfection. In this case, expression of the Rep proteins is required, as well as the presence of an amplifiable inverted terminal repeat (ITR)-containing template. Thus, very different AAV-2 elements and mechanisms are involved in inhibition of adenoviral replication during rAd/AAV-2 vector propagation and after Ad-AAV coinfection. IMPORTANCE: This is the first study to systematically compare the contributions of AAV-2 protein expression and AAV-2 nucleic acid elements to the inhibition of adenoviral replication in rAd/AAV-2 hybrid vector generation and in AAV-2-adenovirus coinfection. This study shows that the two inhibitory processes are very different with regard to AAV-2 functions and the mechanisms involved. Whereas inhibition of rAd/AAV-2 hybrid vector propagation mostly involves a 3' nucleic acid element in the rep gene, inhibition of an adenoviral genome in trans requires the Rep proteins and the AAV ITRs. These findings have important implications both for a basic understanding of the AAV replication cycle and for generation of rAd/AAV-2 hybrid vectors expressing the nonstructural and structural proteins of AAV-2.

Rapid and highly efficient inducible cardiac gene knockout in adult mice using AAV-mediated expression of Cre recombinase.

AIMS: Inducible gene targeting in mice using the Cre/LoxP system has become a valuable tool to analyse the roles of specific genes in the adult heart. However, the commonly used Myh6-MerCreMer system requires time-consuming breeding schedules and is potentially associated with cardiac side effects, which may result in transient cardiac dysfunction. The aim of our study was to establish a rapid and simple system for cardiac gene inactivation in conditional knockout mice by gene transfer of a Cre recombinase gene using adeno-associated viral vectors of serotype 9 (AAV9). METHODS AND RESULTS: AAV9 vectors expressing Cre under the control of a human cardiac troponin T promoter (AAV-TnT-Cre) enabled a highly efficient Cre/LoxP switching in cardiomyocytes 2 weeks after injection into 5- to 6-week-old ROSA26-LacZ reporter mice. Recombination efficiency was at least as high as observed with the Myh6-MerCreMer system. No adverse side effects were detected upon application of AAV-TnT-Cre. As proof of principle, we studied AAV-TnT-Cre in a conditional knockout model (Srf-flex1 mice) to deplete the myocardium of the transcription factor serum response factor (SRF). Four weeks after AAV-TnT-Cre injection, a strong decrease in the cardiac expression of SRF mRNA and protein was observed. Furthermore, mice developed a severe cardiac dysfunction with increased interstitial fibrosis in accordance with the central role of SRF for the expression of contractile and calcium trafficking proteins in the heart. CONCLUSIONS: AAV9-mediated expression of Cre is a promising approach for rapid and efficient conditional cardiac gene knockout in adult mice.

Intrathecal gene therapy corrects CNS pathology in a feline model of mucopolysaccharidosis I.

Enzyme replacement therapy has revolutionized the treatment of the somatic manifestations of lysosomal storage diseases (LSD), although it has been ineffective in treating central nervous system (CNS) manifestations of these disorders. The development of neurotrophic vectors based on novel serotypes of adeno-associated viruses (AAV) such as AAV9 provides a potential platform for stable and efficient delivery of enzymes to the CNS. We evaluated the safety and efficacy of intrathecal delivery of AAV9 expressing α-l-iduronidase (IDUA) in a previously described feline model of mucopolysaccharidosis I (MPS I). A neurological phenotype has not been defined in these animals, so our analysis focused on the biochemical and histological CNS abnormalities characteristic of MPS I. Five MPS I cats were dosed with AAV9 vector at 4-7 months of age and followed for 6 months. Treated animals demonstrated virtually complete correction of biochemical and histological manifestations of the disease throughout the CNS. There was a range of antibody responses against IDUA in this cohort which reduced detectable enzyme without substantially reducing efficacy; there was no evidence of toxicity. This first demonstration of the efficacy of intrathecal gene therapy in a large animal model of a LSD should pave the way for translation into the clinic.

Safety and efficacy of high-dose adeno-associated virus 9 encoding sarcoplasmic reticulum Ca(2+) adenosine triphosphatase delivered by molecular cardiac surgery with recirculating delivery in ovine ischemic cardiomyopathy.

OBJECTIVE: Therapeutic safety and efficacy are the basic prerequisites for clinical gene therapy. We investigated the effect of high-dose molecular cardiac surgery with recirculating delivery (MCARD)-mediated adeno-associated virus 9 (AAV9)/sarcoplasmic reticulum Ca(2+) adenosine triphosphatase (SERCA2a) gene delivery on clinical parameters, oxidative stress, humoral and cellular immune responses, and cardiac remodeling. METHODS: Ischemic cardiomyopathy was generated in a sheep model. The sheep were assigned to 1 of 2 groups: control (n = 10) and study (MCARD, n = 6). The control group underwent no intervention and the study group received 10(14) genome copies of AAV9/SERCA2a 4 weeks after infarction. RESULTS: Our ischemic model produced reliable infarcts leading to heart failure. The baseline ejection fraction in the MCARD group was 57.6% ± 1.6% versus 61.2% ± 1.9% in the control group (P > .05). At 12 weeks after infarction, the MCARD group had superior left ventricular function compared with the control group: stroke volume index, 46.6 ± 1.8 versus 35.8 ± 2.5 mL/m(2) (P < .05); ejection fraction, 46.2% ± 1.9% versus 38.7% ± 2.5% (P < .05); and left ventricular end-systolic and end-diastolic dimensions, 41.3 ± 1.7 versus 48.2 ± 1.4 mm and 51.2 ± 1.5 versus 57.6 ± 1.7 mm, respectively (P < .05). The markers of oxidative stress were significantly reduced in the infarct zone in the MCARD group. No positive T-cell-mediated immune response was seen in the MCARD group at any point. Myocyte hypertrophy was also significantly attenuated in the MCARD group compared with the control group. CONCLUSIONS: Cardiac overexpression of the SERCA2a gene by way of MCARD is a safe therapeutic intervention. It significantly improves left ventricular function, decreases markers of oxidative stress, abrogates myocyte hypertrophy, arrests remodeling, and does not induce a T-cell-mediated immune response.

Cardiac I-1c overexpression with reengineered AAV improves cardiac function in swine ischemic heart failure.

Cardiac gene therapy has emerged as a promising option to treat advanced heart failure (HF). Advances in molecular biology and gene targeting approaches are offering further novel options for genetic manipulation of the cardiovascular system. The aim of this study was to improve cardiac function in chronic HF by overexpressing constitutively active inhibitor-1 (I-1c) using a novel cardiotropic vector generated by capsid reengineering of adeno-associated virus (BNP116). One month after a large anterior myocardial infarction, 20 Yorkshire pigs randomly received intracoronary injection of either high-dose BNP116.I-1c (1.0 × 10(13) vector genomes (vg), n = 7), low-dose BNP116.I-1c (3.0 × 10(12) vg, n = 7), or saline (n = 6). Compared to baseline, mean left ventricular ejection fraction increased by 5.7% in the high-dose group, and by 5.2% in the low-dose group, whereas it decreased by 7% in the saline group. Additionally, preload-recruitable stroke work obtained from pressure-volume analysis demonstrated significantly higher cardiac performance in the high-dose group. Likewise, other hemodynamic parameters, including stroke volume and contractility index indicated improved cardiac function after the I-1c gene transfer. Furthermore, BNP116 showed a favorable gene expression pattern for targeting the heart. In summary, I-1c overexpression using BNP116 improves cardiac function in a clinically relevant model of ischemic HF.

Breaking and joining single-stranded DNA: the HUH endonuclease superfamily.

HUH endonucleases are numerous and widespread in all three domains of life. The major function of these enzymes is processing a range of mobile genetic elements by catalysing cleavage and rejoining of single-stranded DNA using an active-site Tyr residue to make a transient 5'-phosphotyrosine bond with the DNA substrate. These enzymes have a key role in rolling-circle replication of plasmids and bacteriophages, in plasmid transfer, in the replication of several eukaryotic viruses and in various types of transposition. They have also been appropriated for cellular processes such as intron homing and the processing of bacterial repeated extragenic palindromes. Here, we provide an overview of these fascinating enzymes and their functions, using well-characterized examples of Rep proteins, relaxases and transposases, and we explore the molecular mechanisms used in their diverse activities.

Cardiac-selective expression of extracellular superoxide dismutase after systemic injection of adeno-associated virus 9 protects the heart against post-myocardial infarction left ventricular remodeling.

BACKGROUND: Cardiac magnetic resonance imaging has not been used previously to document the attenuation of left ventricular (LV) remodeling after systemic gene delivery. We hypothesized that targeted expression of extracellular superoxide dismutase (EcSOD) via the cardiac troponin-T promoter would protect the mouse heart against both myocardial infarction (MI) and subsequent LV remodeling. METHODS AND RESULTS: Using reporter genes, we first compared the specificity, time course, magnitude, and distribution of gene expression from adeno-associated virus (AAV) 1, 2, 6, 8, and 9 after intravenous injection. The troponin-T promoter restricted gene expression largely to the heart for all AAV serotypes tested. AAV1, 6, 8, and 9 provided early-onset gene expression that approached steady-state levels within 2 weeks. Gene expression was highest with AAV9, which required only 3.15×10(11) viral genomes per mouse to achieve an 84% transduction rate. AAV9-mediated, cardiac-selective gene expression elevated EcSOD enzyme activity in heart by 5.6-fold (P=0.015), which helped protect the heart against both acute MI and subsequent LV remodeling. In acute MI, infarct size in EcSOD-treated mice was reduced by 40% compared with controls (P=0.035). In addition, we found that cardiac-selective expression of EcSOD increased myocardial capillary fractional area and decreased neutrophil infiltration after MI. In a separate study of LV remodeling, after a 60-minute coronary occlusion, cardiac magnetic resonance imaging revealed that LV volumes at days 7 and 28 post-MI were significantly lower in the EcSOD group compared with controls. CONCLUSIONS: Cardiac-selective expression of EcSOD from the cardiac troponin-T promoter after systemic administration of AAV9 provides significant protection against both acute MI and LV remodeling.

The adeno-associated virus type 5 small rep proteins expressed via internal translation initiation are functional.

Although precluded from using splicing to produce multiple small Rep proteins, adeno-associated virus type 5 (AAV5) generates a Rep40-like protein by alternative translation initiation at an internal AUG. A defined region upstream of the internal AUG was both required and sufficient to program internal initiation within AAV5 and may act similarly in heterologous contexts. The internally initiated AAV5 Rep40-like protein was functional and had helicase activity similar to that of AAV2 Rep40. Surprisingly, both the AAV5 Rep40-like protein and Rep52 were able to be translated from the AAV5 upstream P7-generated RNAs; however, the relative level of small to large Rep proteins was reduced compared to that of the wild type. A P19 mutant AAV5 infectious clone generated near-wild-type levels of the double-stranded monomer replicative form (mRF) replicative intermediate but reduced levels of virus, consistent with the previously defined role of Rep40-like proteins in genome encapsidation. Levels of mutant virus were dramatically reduced upon amplification.

Evidence for pH-dependent protease activity in the adeno-associated virus capsid.

Incubation of highly purified adeno-associated virus (AAV) capsids in vitro at pH 5.5 induced significant autocleavage of capsid proteins at several amino acid positions. No autocleavage was seen at pH 7.5. Examination of other AAV serotypes showed at least two different pH-induced cleavage patterns, suggesting that different serotypes have evolved alternative protease cleavage sites. In contrast, incubation of AAV serotypes with an external protease substrate showed that purified AAV capsid preparations have robust protease activity at neutral pH but not at pH 5.5, opposite to what is seen with capsid protein autocleavage. Several lines of evidence suggested that protease activity is inherent in AAV capsids and is not due to contaminating proteins. Control virus preparations showed no protease activity on external substrates, and filtrates of AAV virus preparations also showed no protease activity contaminating the capsids. Further, N-terminal Edman sequencing identified unique autocleavage sites in AAV1 and AAV9, and mutagenesis of amino acids adjacent to these sites eliminated cleavage. Finally, mutation of an amino acid in AAV2 (E563A) that is in a conserved pH-sensitive structural region eliminated protease activity on an external substrate but did not seem to affect autocleavage. Taken together, our data suggested that AAV capsids have one or more protease active sites that are sensitive to pH induction. Further, it appears that acidic pHs comparable to those seen in late endosomes induce a structural change in the capsid that induces autolytic protease activity. The pH-dependent protease activity may have a role in viral infection.

DNA-binding activity of adeno-associated virus Rep is required for inverted terminal repeat-dependent complex formation with herpes simplex virus ICP8.

Herpes simplex virus (HSV) helper functions for (AAV) replication comprise HSV ICP8 and helicase-primase UL5/UL52/UL8. Here we show that N-terminal amino acids of AAV Rep78 that contact the Rep-binding site within the AAV inverted terminal repeat (ITR) are required for ternary-complex formation with infected-cell protein 8 (ICP8) on AAV single-strand DNA (ssDNA) in vitro and for colocalization in nuclear replication domains in vivo. Our data suggest that HSV-dependent AAV replication is initiated by Rep contacting the AAV ITR and by cooperative binding of ICP8 on AAV ssDNA.

The amino acid linker between the endonuclease and helicase domains of adeno-associated virus type 5 Rep plays a critical role in DNA-dependent oligomerization.

The adeno-associated virus (AAV) genome encodes four Rep proteins, all of which contain an SF3 helicase domain. The larger Rep proteins, Rep78 and Rep68, are required for viral replication, whereas Rep40 and Rep52 are needed to package AAV genomes into preformed capsids; these smaller proteins are missing the site-specific DNA-binding and endonuclease domain found in Rep68/78. Other viral SF3 helicases, such as the simian virus 40 large T antigen and the papillomavirus E1 protein, are active as hexameric assemblies. However, Rep40 and Rep52 have not been observed to form stable oligomers on their own or with DNA, suggesting that important determinants of helicase multimerization lie outside the helicase domain. Here, we report that when the 23-residue linker that connects the endonuclease and helicase domains is appended to the adeno-associated virus type 5 (AAV5) helicase domain, the resulting protein forms discrete complexes on DNA consistent with single or double hexamers. The formation of these complexes does not require the Rep binding site sequence, nor is it nucleotide dependent. These complexes have stimulated ATPase and helicase activities relative to the helicase domain alone, indicating that they are catalytically relevant, a result supported by negative-stain electron microscopy images of hexameric rings. Similarly, the addition of the linker region to the AAV5 Rep endonuclease domain also confers on it the ability to bind and multimerize on nonspecific double-stranded DNA. We conclude that the linker is likely a key contributor to Rep68/78 DNA-dependent oligomerization and may play an important role in mediating Rep68/78's conversion from site-specific DNA binding to nonspecific DNA unwinding.

Mosaic analysis of gene function in postnatal mouse brain development by using virus-based Cre recombination.

Normal brain function relies not only on embryonic development when major neuronal pathways are established, but also on postnatal development when neural circuits are matured and refined. Misregulation at this stage may lead to neurological and psychiatric disorders such as autism and schizophrenia. Many genes have been studied in the prenatal brain and found crucial to many developmental processes. However, their function in the postnatal brain is largely unknown, partly because their deletion in mice often leads to lethality during neonatal development, and partly because their requirement in early development hampers the postnatal analysis. To overcome these obstacles, floxed alleles of these genes are currently being generated in mice. When combined with transgenic alleles that express Cre recombinase in specific cell types, conditional deletion can be achieved to study gene function in the postnatal brain. However, this method requires additional alleles and extra time (3-6 months) to generate the mice with appropriate genotypes, thereby limiting the expansion of the genetic analysis to a large scale in the mouse brain. Here we demonstrate a complementary approach that uses virally-expressed Cre to study these floxed alleles rapidly and systematically in postnatal brain development. By injecting recombinant adeno-associated viruses (rAAVs) encoding Cre into the neonatal brain, we are able to delete the gene of interest in different regions of the brain. By controlling the viral titer and coexpressing a fluorescent protein marker, we can simultaneously achieve mosaic gene inactivation and sparse neuronal labeling. This method bypasses the requirement of many genes in early development, and allows us to study their cell autonomous function in many critical processes in postnatal brain development, including axonal and dendritic growth, branching, and tiling, as well as synapse formation and refinement. This method has been used successfully in our own lab (unpublished results) and others, and can be extended to other viruses, such as lentivirus, as well as to the expression of shRNA or dominant active proteins. Furthermore, by combining this technique with electrophysiology as well as recently-developed optical imaging tools, this method provides a new strategy to study how genetic pathways influence neural circuit development and function in mice and rats.

Intrinsic phospholipase A2 activity of adeno-associated virus is involved in endosomal escape of incoming particles.

The unique region of the VP1 capsid protein of adeno-associated viruses (AAV) in common with autonomously replicating parvoviruses comprises a secreted phospholipase A2 (sPLA2) homology domain. While the sPLA2 domain of Minute Virus of Mice has recently been shown to mediate endosomal escape by lipolytic pore formation, experimental evidence for a similar function in AAV infection is still lacking. Here, we explored the function of the sPLA2 domain of AAV by making use of the serotype 2 mutant (76)HD/AN. The sPLA2 defect in (76)HD/AN, which severely impairs AAV's infectivity, could be complemented in trans by co-infection with wild-type AAV2. Furthermore, co-infection with endosomolytically active, but not with inactive adenoviral variants partially rescued (76)HD/AN, providing the first evidence for a function of this domain in endosomal escape of incoming AAV particles.

NLX-P101, an adeno-associated virus gene therapy encoding glutamic acid decarboxylase, for the potential treatment of Parkinson's disease.

Parkinson's disease (PD) is a neurodegenerative disease affecting nigrostriatal dopaminergic neurons. Dopamine depletion in the striatum leads to functional changes in several deep brain nuclei, including the subthalamic nucleus (STN), which becomes disinhibited and perturbs the control of body movement. Although there is no cure for PD, some pharmacological and surgical treatments can significantly improve the functional ability of patients, particularly in the early stages of the disease. Among neurodegenerative diseases, PD is a particularly suitable target for gene therapy because the neuropathology is largely confined to a relatively small region of the brain. Neurologix Inc is developing NLX-P101 (AAV2-GAD), an adeno-associated viral vector encoding glutamic acid decarboxylase (GAD), for the potential therapy of PD. As GAD potentiates inhibitory neurotransmission from the STN, sustained expression of GAD in the STN by direct delivery of NLX-P101 decreases STN overactivation. This procedure was demonstrated to be a safe and efficient method of reducing motor deficits in animal models of PD. A phase I clinical trial has demonstrated that NLX-P101 was safe and indicated the efficacy of this approach in patients with PD. Results from an ongoing phase II clinical trial of NLX-P101 are awaited to establish the clinical efficacy of this gene therapy.

Activation of the ATPase activity of adeno-associated virus Rep68 and Rep78.

Rep68 and Rep78 DNA helicases, encoded by adeno-associated virus 2 (AAV2), are required for replication of AAV viral DNA in infected cells. They bind to imperfect palindromic elements in the inverted terminal repeat structures at the 3'- and 5'-ends of virion DNA. The ATPase activity of Rep68 and Rep78 is stimulated up to 10-fold by DNA containing the target sequence derived from the inverted terminal repeat; nontarget DNA stimulates ATPase activity at 50-fold higher concentrations. Activation of ATPase activity of Rep68 by DNA is cooperative with a Hill coefficient of 1.8 +/- 0.2. When examined by gel filtration at 0.5 M NaCl in the absence of DNA, Rep68 self-associates in a concentration-dependent manner. In the presence of DNA containing the binding element, Rep68 (and Rep78) forms protein-DNA complexes that exhibit concentration-dependent self-association in gel filtration analysis. The ATPase activity of the isolated Rep68-DNA and Rep78-DNA complexes is not activated by additional target DNA. Results of sedimentation velocity experiments in the presence of saturating target DNA are consistent with Rep68 forming a hexamer of the protein with two copies of the DNA element. Activation of the ATPase activity of Rep68 is associated with the formation of a protein-DNA oligomer.

Coupled ATP and DNA binding of adeno-associated virus Rep40 helicase.

Adeno-associated virus 2 Rep40 helicase is involved in packaging single-stranded genomic DNA into virions. ATPase activity was stimulated 5-10-fold by DNA, depending upon assay conditions. The concentration dependence of Rep40 ATPase activity in the absence and presence of DNA indicates that the monomer is inactive and that the active enzyme is at least a dimer. Binding to oligonucleotides, examined by fluorescence anisotropy, was positively cooperative and required ATP or ATPgammaS; ADP and AMPPCP did not promote binding. The cooperativity and the nucleotide requirement were also demonstrated by surface plasmon resonance. Although the Rep40 behaves as a monomer in solution, it binds to DNA as an oligomer. The requirement of a nucleotide for DNA binding and the stimulation of ATPase activity by DNA indicate that the two processes are linked. Glutaraldehyde cross-linking generated a species that migrates as a trimer on sodium dodecyl sulfate (SDS) gel electrophoresis; ATPS promoted the formation of this species and higher order oligomers. The predominant cross-linked species was a trimer in the absence of ATPgammaS, regardless of whether duplex or single-stranded DNA was present. In the presence of duplex or single-stranded DNA and ATPgammaS, glutaraldehyde cross-linking generated a species that behaved as a dimer on SDS gel elctrophoresis. Sucrose-gradient velocity sedimentation of Rep40 gave an S20,w of 3 in the absence of ligands or in the presence of a 26 bp duplex DNA. The S20,w was 3.5 in the presence of ATPgammaS and 7 and 7.6 in the presence of DNA and ATPgammaS.