Adeno-associated Virus DNA Replication Research Articles

A Comprehensive Study of the Effects by Sequence Truncation within Inverted Terminal Repeats (ITRs) on the Productivity, Genome Packaging, and Potency of AAV Vectors.

One of the primary challenges in working with adeno-associated virus (AAV) lies in the inherent instability of its inverted terminal repeats (ITRs), which play vital roles in AAV replication, encapsidation, and genome integration. ITRs contain a high GC content and palindromic structure, which occasionally results in truncations and mutations during plasmid amplification in bacterial cells. However, there is no thorough study on how these alterations in ITRs impact the ultimate AAV vector characteristics. To close this gap, we designed ITRs with common variations, including a single B, C, or D region deletion at one end, and dual deletions at both ends of the vector genome. These engineered ITR-carrying plasmids were utilized to generate AAV vectors in HEK293 cells. The crude and purified AAV samples were collected and analyzed for yield, capsid DNA-filled percentage, potency, and ITR integrity. The results show that a single deletion had minor impact on AAV productivity, packaging efficiency, and in vivo potency. However, deletions on both ends, except A, showed significant negative effects on the above characteristics. Our work revealed the role of ITR regions, A, B, C, and D for AAV production and DNA replication, and proposes a new strategy for the quality control of ITR-bearing plasmids and final AAV products.

Adeno-Associated Virus Monoinfection Induces a DNA Damage Response and DNA Repair That Contributes to Viral DNA Replication.

Adeno-associated virus (AAV) belongs to the Dependoparvovirus genus of the Parvoviridae family. AAV replication relies on a helper virus, such as adenovirus (Ad). Co-infection of AAV and Ad induces a DNA damage response (DDR), although its function in AAV DNA replication remains unknown. In this study, monoinfection of AAV2 in HEK293T cells expressing a minimal set of Ad helper genes was used to investigate the role of the DDR solely induced by AAV. We found that AAV2 DNA replication, but not single stranded (ss)DNA genome accumulation and Rep expression only, induced a robust DDR in HEK293T cells. The induced DDR featured the phosphorylation of replication protein A32 (RPA32), histone variant H2AX (H2A histone family member X), and all 3 phosphatidylinositol 3-kinase-related kinases (PIKKs). We also found that the kinase ataxia telangiectasia and Rad3-related protein (ATR) plays a major role in AAV2 DNA replication and that Y family DNA repair DNA polymerases η (Pol η) and Pol κ contribute to AAV2 DNA replication both in vitro and in HEK293T cells. Knockout of Pol η and Pol κ in HEK293T cells significantly decreased wild-type AAV2 replication and recombinant AAV2 production. Thus, our study has proven that AAV2 DNA replication induces a DDR, which in turn initiates a DNA repairing process that partially contributes to the viral genome amplification in HEK293T cells. IMPORTANCE Recombinant AAV (rAAV) has emerged as one of the preferred delivery vectors for clinical gene therapy. rAAV production in HEK293 cells by transfection of a rAAV transgene plasmid, an AAV Rep and Cap expression packaging plasmid, and an Ad helper plasmid remains the popular method. Here, we demonstrated that the high fidelity Y family DNA repair DNA polymerase, Pol η, and Pol κ, plays a significant role in AAV DNA replication and rAAV production in HEK293T cells. Understanding the AAV DNA replication mechanism in HEK293T cells could provide clues to increase rAAV vector yield produced from the transfection method. We also provide evidence that the ATR-mediated DNA repair process through Pol η and Pol κ is one of the mechanisms to amplify AAV genome, which could explain AAV replication and rAAV ssDNA genome conversion in mitotic quiescent cells.

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

My Pathway to Adeno-Associated Virus and Adeno-Associated Virus Gene Therapy: A Personal Perspective.

Impact of the MRN Complex on Adeno-Associated Virus Integration and Replication during Coinfection with Herpes Simplex Virus 1.

Adeno-associated virus (AAV) is a helper-dependent parvovirus that requires coinfection with adenovirus (AdV) or herpes simplex virus 1 (HSV-1) to replicate. In the absence of the helper virus, AAV can persist in an episomal or integrated form. Previous studies have analyzed the DNA damage response (DDR) induced upon AAV replication to understand how it controls AAV replication. In particular, it was shown that the Mre11-Rad50-Nbs1 (MRN) complex, a major player of the DDR induced by double-stranded DNA breaks and stalled replication forks, could negatively regulate AdV and AAV replication during coinfection. In contrast, MRN favors HSV-1 replication and is recruited to AAV replication compartments that are induced in the presence of HSV-1. In this study, we examined the role of MRN during AAV replication induced by HSV-1. Our results indicated that knockdown of MRN significantly reduced AAV DNA replication after coinfection with wild-type (wt) HSV-1 or HSV-1 with the polymerase deleted. This effect was specific to wt AAV, since it did not occur with recombinant AAV vectors. Positive regulation of AAV replication by MRN was dependent on its DNA tethering activity but did not require its nuclease activities. Importantly, knockdown of MRN also negatively regulated AAV integration within the human AAVS1 site, both in the presence and in the absence of HSV-1. Altogether, this work identifies a new function of MRN during integration of the AAV genome and demonstrates that this DNA repair complex positively regulates AAV replication in the presence of HSV-1. Viral DNA genomes trigger a DNA damage response (DDR), which can be either detrimental or beneficial for virus replication. Adeno-associated virus (AAV) is a defective parvovirus that requires the help of an unrelated virus such as adenovirus (AdV) or herpes simplex virus 1 (HSV-1) for productive replication. Previous studies have demonstrated that the cellular Mre11-Rad50-Nbs1 (MRN) complex, a sensor and regulator of the DDR, negatively regulates AAV replication during coinfection with AdV, which counteracts this effect by inactivating the complex. Here, we demonstrate that MRN positively regulates AAV replication during coinfection with HSV-1. Importantly, our study also indicates that MRN also favors integration of AAV genomes within the human AAVS1 site. Altogether, this work indicates that MRN differentially regulates AAV replication depending on the helper virus which is present and identifies a new function of this DNA repair complex during AAV integration.

The Effect of DNA-Dependent Protein Kinase on Adeno-Associated Virus Replication

BackgroundDNA-dependent protein kinase (DNA-PK) is a DNA repair enzyme and plays an important role in determining the molecular fate of the rAAV genome. However, the effect this cellular enzyme on rAAV DNA replication remains elusive.Methodology/Principal FindingsIn the present study, we characterized the roles of DNA-PK on recombinant adeno-associated virus DNA replication. Inhibition of DNA-PK by a DNA-PK inhibitor or siRNA targeting DNA-PKcs significantly decreased replication of AAV in MO59K and 293 cells. Southern blot analysis showed that replicated rAAV DNA formed head-to-head or tail-to-tail junctions. The head-to-tail junction was low or undetectable suggesting AAV-ITR self-priming is the major mechanism for rAAV DNA replication. In an in vitro replication assay, anti-Ku80 antibody strongly inhibited rAAV replication, while anti-Ku70 antibody moderately decreased rAAV replication. Similarly, when Ku heterodimer (Ku70/80) was depleted, less replicated rAAV DNA were detected. Finally, we showed that AAV-ITRs directly interacted with Ku proteins.Conclusion/SignificanceCollectively, our results showed that that DNA-PK enhances rAAV replication through the interaction of Ku proteins and AAV-ITRs.

Creating a Novel Origin of Replication through Modulating DNA-Protein Interfaces

BackgroundWhile the molecular mechanisms of DNA-protein specificity at the origin of replication have been determined in many model organisms, these interactions remain unknown in the majority of higher eukaryotes and numerous vertebrate viruses. Similar to many viral origins of replication, adeno-associated virus (AAV) utilizes a cis-acting origin of replication and a virus specific Replication protein (Rep) to faithfully carry out self-priming replication. The mechanisms of AAV DNA replication are generally well understood. However, the molecular basis of specificity between the Rep protein and the viral origin of replication between different AAV serotypes remains uncharacterized.Methodology/Principal FindingsBy generating a panel of chimeric and mutant origins between two AAV serotypes, we have mapped two independent DNA-Protein interfaces involved in replicative specificity. In vivo replication assays and structural modeling demonstrated that three residues in the AAV2 Rep active site are necessary to cleave its cognate origin. An analogous origin (AAV5) possesses a unique interaction between an extended Rep binding element and a 49 aa region of Rep containing two DNA binding interfaces.Conclusions/SignificanceThe elucidation of these structure-function relationships at the AAV origin led to the creation of a unique recombinant origin and compatible Rep protein with properties independent of either parent serotype. This novel origin may impact the safety and efficacy of AAV as a gene delivery tool. This work may also explain the unique ability of certain AAV serotypes to achieve site-directed integration into the human chromosome. Finally, this result impacts the study of conserved DNA viruses which employ rolling circle mechanisms of replication.

DNA Structure Modulates the Oligomerization Properties of the AAV Initiator Protein Rep68

Rep68 is a multifunctional protein of the adeno-associated virus (AAV), a parvovirus that is mostly known for its promise as a gene therapy vector. In addition to its role as initiator in viral DNA replication, Rep68 is essential for site-specific integration of the AAV genome into human chromosome 19. Rep68 is a member of the superfamily 3 (SF3) helicases, along with the well-studied initiator proteins simian virus 40 large T antigen (SV40-LTag) and bovine papillomavirus (BPV) E1. Structurally, SF3 helicases share two domains, a DNA origin interaction domain (OID) and an AAA+ motor domain. The AAA+ motor domain is also a structural feature of cellular initiators and it functions as a platform for initiator oligomerization. Here, we studied Rep68 oligomerization in vitro in the presence of different DNA substrates using a variety of biophysical techniques and cryo-EM. We found that a dsDNA region of the AAV origin promotes the formation of a complex containing five Rep68 subunits. Interestingly, non-specific ssDNA promotes the formation of a double-ring Rep68, a known structure formed by the LTag and E1 initiator proteins. The Rep68 ring symmetry is 8-fold, thus differing from the hexameric rings formed by the other SF3 helicases. However, similiar to LTag and E1, Rep68 rings are oriented head-to-head, suggesting that DNA unwinding by the complex proceeds bidirectionally. This novel Rep68 quaternary structure requires both the DNA binding and AAA+ domains, indicating cooperativity between these regions during oligomerization in vitro. Our study clearly demonstrates that Rep68 can oligomerize through two distinct oligomerization pathways, which depend on both the DNA structure and cooperativity of Rep68 domains. These findings provide insight into the dynamics and oligomeric adaptability of Rep68 and serve as a step towards understanding the role of this multifunctional protein during AAV DNA replication and site-specific integration.

Definition of Herpes Simplex Virus Type 1 Helper Activities for Adeno-Associated Virus Early Replication Events

The human parvovirus Adeno-Associated Virus (AAV) type 2 can only replicate in cells co-infected with a helper virus, such as Adenovirus or Herpes Simplex Virus type 1 (HSV-1); whereas, in the absence of a helper virus, it establishes a latent infection. Previous studies demonstrated that the ternary HSV-1 helicase/primase (HP) complex (UL5/8/52) and the single-stranded DNA-Binding Protein (ICP8) were sufficient to induce AAV-2 replication in transfected cells. We independently showed that, in the context of a latent AAV-2 infection, the HSV-1 ICP0 protein was able to activate rep gene expression. The present study was conducted to integrate these observations and to further explore the requirement of other HSV-1 proteins during early AAV replication steps, i.e. rep gene expression and AAV DNA replication. Using a cellular model that mimics AAV latency and composite constructs coding for various sets of HSV-1 genes, we first confirmed the role of ICP0 for rep gene expression and demonstrated a synergistic effect of ICP4 and, to a lesser extent, ICP22. Conversely, ICP27 displayed an inhibitory effect. Second, our analyses showed that the effect of ICP0, ICP4, and ICP22 on rep gene expression was essential for the onset of AAV DNA replication in conjunction with the HP complex and ICP8. Third, and most importantly, we demonstrated that the HSV-1 DNA polymerase complex (UL30/UL42) was critical to enhance AAV DNA replication to a significant level in transfected cells and that its catalytic activity was involved in this process. Altogether, this work represents the first comprehensive study recapitulating the series of early events taking place during HSV-1–induced AAV replication.

Adeno-associated Virus of a Single-polarity DNA Genome Is Capable of Transduction In Vivo

The adeno-associated virus (AAV) is a promising vector for gene therapy. Further improvement of the virus for clinical application depends on better understanding of the molecular structure and fate of the vector genome. AAV vectors with wild-type inverted terminal repeats package either the plus- or the minus-strand DNA genomes with equal frequency. By creating a series of deletions within the, we have developed a genetic approach that can generate an AAV vector that packages its single-stranded DNA genome predominantly in a single polarity (99.4%). This novel reagent efficiently transduced muscle, brain and liver in whole animals. The transduction efficiencies were similar to those of the control mixed-polarity vectors. Our results showed that reannealing of plus- and minus-strand DNA was not required for AAV-mediated transduction in vivo, supporting the hypothesis that second-strand DNA synthesis is a primary pathway in converting the single-stranded AAV genome into double-stranded forms. The availability of the single-polarity AAV vector would aid further studies on the mechanism of AAV transduction as well as the application of AAV vector for gene replacement therapy.

AAV-2 Rep78 and HPV-16 E1 Interact in Vitro, Modulating Their ATPase Activity

Adeno-associated virus (AAV) is a nonpathogenic single-stranded human parvovirus which usually requires the presence of a "helper" virus for strong DNA replication. In addition to adeno- and herpes viruses, human papillomavirus (HPV) can serve as an AAV helper. We recently published that HPV type 16 (HPV-16) E1 protein contributes significantly as an individual helper gene for AAV-2 DNA replication and transcription. As Rep78 and E1 are the corresponding DNA helicase/replication proteins of AAV and HPV, respectively, and Rep78 and E1 have a degree of homology, we assayed whether these two proteins interact physically. The full length proteins were purified from bacteria as GST-E1 and MBP-Rep78 and used in five assays to observe Rep78-E1 interactions. All five assays (pull-down, coimmunoprecipitation, enzyme-linked immunosorbent assay (ELISA), chemical cross-linking, and ATPase activity) provided evidence consistent with Rep78-E1 interaction. Most intriguing, an overall decrease in ATPase activity was observed when both proteins were present together. These data strongly suggest that E1 and Rep78 interact and that this interaction modulates at least some of their individual biochemical functions. This study adds to our understanding of AAV-HPV interaction biology, E1's modulation of Rep78 biochemistry, Rep78's modulation of E1 biochemistry and provides initial clues which may lead to the underlying mechanism of HPV E1 helper function for AAV DNA replication.

Cis Effects in Adeno-Associated Virus Type 2 Replication

We have utilized deletion mutants of adeno-associated virus (AAV) to investigate which elements of the AAV genome are required in cis for high yields of the wild-type virus in a plasmid transfection assay and in addition whether these elements affect primarily AAV DNA replication or encapsidation. All tested deletions from within the Rep region demonstrated a modest, approximately threefold, decrease in viral production. Deletions within the cap region resulted in markedly less virus. Previous observations suggested that in cells in which recombinant AAV (rAAV) was produced, as in our assay with the helper plasmid pDG, there is a substantial excess of empty capsids. Co-transfections of high- and low-yielding constructs demonstrated that under conditions where Cap is abundant, the constructs with cap deletions did not package efficiently. These observation suggest that the lower yields of rAAV cannot be entirely due to lack of capsids but that elements within the cap region of the wild-type genome are important for efficient encapsidation. The production of virus by the mutants we tested was, however, not consistent with the disruption of a cis-acting packaging signal. Apparently, when Cap is provided "in trans," encapsidation is inefficient. A second observation is that there were equivalent amounts of replicated but unencapsidated viral DNA in cells transfected with each of our constructs. We propose that, in accord with the previously proposed link between DNA replication and encapsidation, the total amount of AAV DNA replication can be limited by the efficiency of encapsidation.

Purification of Host Cell Enzymes Involved in Adeno-Associated Virus DNA Replication

Adeno-associated virus (AAV) replicates its DNA by a modified rolling-circle mechanism that exclusively uses leading strand displacement synthesis. To identify the enzymes directly involved in AAV DNA replication, we fractionated adenovirus-infected crude extracts and tested them in an in vitro replication system that required the presence of the AAV-encoded Rep protein and the AAV origins of DNA replication, thus faithfully reproducing in vivo viral DNA replication. Fractions that contained replication factor C (RFC) and proliferating cell nuclear antigen (PCNA) were found to be essential for reconstituting AAV DNA replication. These could be replaced by purified PCNA and RFC to retain full activity. We also found that fractions containing polymerase delta, but not polymerase epsilon or alpha, were capable of replicating AAV DNA in vitro. This was confirmed when highly purified polymerase delta complex purified from baculovirus expression clones was used. Curiously, as the components of the DNA replication system were purified, neither the cellular single-stranded DNA binding protein (RPA) nor the adenovirus-encoded DNA binding protein was found to be essential for DNA replication; both only modestly stimulated DNA synthesis on an AAV template. Also, in addition to polymerase delta, RFC, and PCNA, an as yet unidentified factor(s) is required for AAV DNA replication, which appeared to be enriched in adenovirus-infected cells. Finally, the absence of any apparent cellular DNA helicase requirement led us to develop an artificial AAV replication system in which polymerase delta, RFC, and PCNA were replaced with T4 DNA polymerase and gp32 protein. This system was capable of supporting AAV DNA replication, demonstrating that under some conditions the Rep helicase activity can function to unwind duplex DNA during strand displacement synthesis.

Live Covisualization of Competing Adeno-Associated Virus and Herpes Simplex Virus Type 1 DNA Replication: Molecular Mechanisms of Interaction

We performed live cell visualization assays to directly assess the interaction between competing adeno-associated virus (AAV) and herpes simplex virus type 1 (HSV-1) DNA replication. Our studies reveal the formation of separate AAV and HSV-1 replication compartments and the inhibition of HSV-1 replication compartment formation in the presence of AAV. AAV Rep is recruited into AAV replication compartments but not into those of HSV-1, while the single-stranded DNA-binding protein HSV-1 ICP8 is recruited into both AAV and HSV-1 replication compartments, although with differential staining patterns. Slot blot analysis of coinfected cells revealed a dose-dependent inhibition of HSV-1 DNA replication by wild-type AAV but not by rep-negative recombinant AAV. Consistent with this, Western blot analysis indicated that wild-type AAV affects the levels of the HSV-1 immediate-early protein ICP4 and the early protein ICP8 only modestly but strongly inhibits the accumulation of the late proteins VP16 and gC. Furthermore, we demonstrate that the presence of Rep in the absence of AAV DNA replication is sufficient for the inhibition of HSV-1. In particular, Rep68/78 proteins severely inhibit the formation of mature HSV-1 replication compartments and lead to the accumulation of ICP8 at sites of cellular DNA synthesis, a phenomenon previously observed in the presence of viral polymerase inhibitors. Taken together, our results suggest that AAV and HSV-1 replicate in separate compartments and that AAV Rep inhibits HSV-1 at the level of DNA replication.

Regulation of Adeno-Associated Virus DNA Replication by the Cellular TAF-I/Set Complex

The Rep proteins of the adeno-associated virus (AAV) are required for viral replication in the presence of adenovirus helper functions and as yet poorly characterized cellular factors. In an attempt to identify such factors, we purified Flag-Rep68-interacting proteins from human cell lysates. Several polypeptides were identified by mass spectrometry, among which was ANP32B, a member of the acidic nuclear protein 32 family which takes part in the formation of the template-activating factor I/Set oncoprotein (TAF-I/Set) complex. The N terminus of Rep was found to specifically bind the acidic domain of ANP32B; through this interaction, Rep was also able to recruit other members of the TAF-I/Set complex, including the ANP32A protein and the histone chaperone TAF-I/Set. Further experiments revealed that silencing of ANP32A and ANP32B inhibited AAV replication, while overexpression of all of the components of the TAF-I/Set complex increased de novo AAV DNA synthesis in permissive cells. Besides being the first indication that the TAF-I/Set complex participates in wild-type AAV replication, these findings have important implications for the generation of recombinant AAV vectors since overexpression of the TAF-I/Set components was found to markedly increase viral vector production.

Role of the Herpes Simplex Virus Helicase-Primase Complex during Adeno-Associated Virus DNA Replication

A subset of DNA replication proteins of herpes simplex virus (HSV) comprising the single-strand DNA-binding protein, ICP8 (UL29), and the helicase-primase complex (UL5, UL8, and UL52 proteins) has previously been shown to be sufficient for the replication of adeno-associated virus (AAV). We recently demonstrated complex formation between ICP8, AAV Rep78, and the single-stranded DNA AAV genome, both in vitro and in the nuclear HSV replication domains of coinfected cells. In this study the functional role(s) of HSV helicase and primase during AAV DNA replication were analyzed. To differentiate between their necessity as structural components of the HSV replication complex or as active enzymes, point mutations within the helicase and primase catalytic domains were analyzed. In two complementary approaches the remaining HSV helper functions were either provided by infection with HSV mutants or by plasmid transfection. We show here that upon cotransfection of the minimal four HSV proteins (i.e., the four proteins constituting the minimal requirements for basal AAV replication), UL52 primase catalytic activity was not required for AAV DNA replication. In contrast, UL5 helicase activity was necessary for fully efficient replication. Confocal microscopy confirmed that all mutants retained the ability to support formation of ICP8-positive nuclear replication foci, to which AAV Rep78 colocalized in a manner strictly dependent on the presence of AAV single-stranded DNA (ssDNA). The data indicate that recruitment of AAV Rep78 and ssDNA to nuclear replication sites by the four HSV helper proteins is maintained in the absence of catalytic primase or helicase activities and suggest an involvement of the HSV UL5 helicase activity during AAV DNA replication.

762. Human Papillomavirus Type 16 Helper Functions for Adeno-Associated Virus Type 2 Replication

Top of pageAbstract Human papillomavirus (HPV) is known to “help” adeno-associated virus (AAV) to replicate to high levels during skin co-infection. However, unlike AAV's other helper viruses adenovirus and herpes simplex, very little is known about the HPV genes which help AAV. In this study we surveyed four HPV-16 early genes, E1, E2, E6, and E7, for their ability to increase the basal level of AAV replication in a stratifying squamous epithelium. It was found that the HPV-16 E1, E2, and E6 genes were able to stimulate higher AAV replication in keratinocytes cultures upon co-transfection during wild type AAV-2 infection. E1 had the strongest enhancing effect upon AAV DNA replication (Southern blot), RNA expression (RT-PCR), protein expression (Western blot) and AAV virion production (2 plate-Southern blot). Further study of partial E1 sequence mutants showed that the carboxy-half of E1, the helicase/ATPase domain, was the main contributor to HPV E1 helper activity. These latter data suggest that helicase activity may be E1's mechanism of action for helping AAV replication. These data are important for understanding the HPV-AAV interaction and its effect on cervical cancer risk. These data are also important as HPV helper genes may be potentially useful in generating recombinant AAV virions for gene therapy purposes as the adenovirus and herpes virus genes are now used.

799. Initial Characterization of a Super-Permissive Cell Line for Adeno-Associated Virus (AAV): Microarray-Intimated CK2 Helps AAV Replication

Adeno-associated virus (AAV) infection is negatively associated with cervical cancer and is being used as a human gene therapy vector. However, AAV is very selective as to the specific conditions under which it replicates. The identification of cellular genes involved in AAV replication will allow for a better understanding of AAV's molecular biology and AAV's oncoprotective phenotype, as well as allow for the easier generation of recombinant (r)AAV for gene therapy. Here we identify a primary cervical cancer cell line, PT3, which allowed for 10 fold higher AAV DNA replication levels than other isolates and normal primary keratinocytes. Microarray gene expression comparative analysis of PT3 cells to others identified many differences in gene expression. Casein kinase 2 (CK2) was found to be the most highly expressed protein kinase in PT3 cells. Following this lead CK2 stimulated AAV DNA replication in normal keratinocyte-based organotypic epithelial rafts (skin) both as an expressed cDNA ( subunit) or as transfected protein. CK2 also phosphorylated the AAV encoded Rep78, major replication/regulatory protein in vitro. These data suggest that CK2 may be a cellular helper gene for AAV, possibly through phosphorylating Rep78. However, the number of genes identified by microarray comparative analysis suggested that, in addition to UP-regulated genes such as CK2, the genes which are DOWN-regulated might also be significant for AAV replication as four fold more such down-regulated genes were identified.

Spatial and temporal organization of adeno-associated virus DNA replication in live cells.

Upon cell entry, the genomes of herpes simplex virus type 1 (HSV-1) and adenovirus (Ad) associate with distinct nuclear structures termed ND10 or promyelocytic leukemia (PML) nuclear bodies (NBs). PML NB morphology is altered or disrupted by specific viral proteins as replication proceeds. We examined whether adeno-associated virus (AAV) replication compartments also associate with PML NBs, and whether modification or disruption of these by HSV-1 or Ad, both of which are helper viruses for AAV, is necessary at all. Furthermore, to add a fourth dimension to our present view of AAV replication, we established an assay that allows visualization of AAV replication in live cells. A recombinant AAV containing 40 lac repressor binding sites between the AAV inverted terminal repeats was constructed. AAV Rep protein and helper virus-mediated replication of this recombinant AAV genome was visualized by binding of enhanced yellow fluorescent protein-lac repressor fusion protein to double-stranded AAV replication intermediates. We demonstrate in live cells that AAV DNA replication occurs in compartments which colocalize with AAV Rep. Early after infection, the replication compartments were small and varied in numbers from 2 to more than 40 per cell nucleus. Within 4 to 8 h, individual small replication compartments expanded and fused to larger structures which filled out much of the cell nucleus. We also show that AAV replication compartments can associate with modified PML NBs in Ad-infected cells. In wild-type HSV-1-infected cells, AAV replication compartments and PML NBs did not coexist, presumably because PML was completely disrupted by the HSV-1 ICP0 protein. However, alteration or disruption of PML appears not to be a prerequisite for AAV replication, as the formation of replication compartments was normal when the ICP0 mutants HSV-1 dl1403 and HSV-1 FXE, which do not affect PML NBs, were used as the helper viruses; under these conditions, AAV replication compartments did not associate with PML NBs.

Efficient generation and amplification of high-capacity adeno-associated virus/adenovirus hybrid vectors.

Effective gene therapy is dependent on safe gene delivery vehicles that can achieve efficient transduction and sustained transgene expression. We are developing a hybrid viral vector system that combines in a single particle the large cloning capacity and efficient cell cycle-independent nuclear gene delivery of adenovirus (Ad) vectors with the long-term transgene expression and lack of viral genes of adeno-associated virus (AAV) vectors. The strategy being pursued relies on coupling the AAV DNA replication mechanism to the Ad encapsidation process through packaging of AAV-dependent replicative intermediates provided with Ad packaging elements into Ad capsids. The generation of these high-capacity AAV/Ad hybrid vectors takes place in Ad early region 1 (E1)-expressing cells and requires an Ad vector with E1 deleted to complement in trans both AAV helper functions and Ad structural proteins. The dependence on a replicating helper Ad vector leads to the contamination of AAV/Ad hybrid vector preparations with a large excess of helper Ad particles. This renders the further propagation and ultimate use of these gene delivery vehicles very difficult. Here, we show that Cre/loxP-mediated genetic selection against the packaging of helper Ad DNA can reduce helper Ad vector contamination by 99.98% without compromising hybrid vector rescue. This allowed amplification of high-capacity AAV/Ad hybrid vectors to high titers in a single round of propagation.

Adeno-associated virus (AAV) site-specific recombination does not require a Rep-dependent origin of replication within the AAV terminal repeat.

Adeno-associated virus (AAV) is the only known eukaryotic virus capable of targeted integration in human cells. An AAV Rep binding element (RBE) and terminal resolution site (trs) identical to the viral terminal repeats required for AAV DNA replication are located on chromosome (ch) 19. Both ch-19 RBE and trs elements have been shown to be essential for viral targeting to this locus. To characterize the role of the AAV inverted terminal repeat (ITR) cis-acting sequences in targeted integration an AAV trs mutant incapable of supporting viral replication was tested. Wild-type and mutant substrates were assayed for targeted integration after cotransfection in the presence or absence of Rep. Our results demonstrated that, in the presence of Rep78, both ITR substrates targeted to ch-19 with similar frequency. Molecular characterization of the mutant ITR integrants confirmed the presence of the trs mutation in the majority of samples tested. Complementation analysis confirmed that the mutant targeted viral genomes were unable to rescue and replicate. In addition, Rep78 induced extensive rearrangement and amplification of ch-19 sequences independent of wild-type or mutant targeting substrate. These studies demonstrate that Rep-dependent nicking of the viral cis-acting trs sequence is not a prerequisite for site-specific recombination and suggests AAV targeting is mediated by Rep78/68-dependent replication from the ch-19 origin of replication (ori). These studies have significant impact toward the understanding of AAV site-specific recombination and the development of targeting vectors.