Expression Of Rep78 Research Articles

Genetics instability of wtAAV2 genome and AAV promoter activities in the Baculovirus/Sf9 cells system.

The human Adeno-Associated Virus serotype 2 (wtAAV2) is a common non-pathological virus and its recombinant form (rAAV) is widely used as gene therapy vector. Although rAAVs are routinely produced in the Baculovirus/Sf9 cell system, wtAAV2 has never been studied in this context. We tried to produce wtAAV2 in the baculovirus/Sf9 cell system hypothesizing that the wtAAV2 may be considered as a normal recombinant AAV transgene. Through our attempts to produce wtAAV2 in Baculovirus/Sf9, we found that wtAAV2 p5 promoter, which controls the expression of large Rep proteins in mammalian cells, was active in this system. p5 promoter activity in the baculovirus/Sf9 cell system leads to the expression of Rep78 that finally excises wtAAV2 genome from the baculovirus genome during the earliest phases of baculovirus stock production. Via p5 promoter expression kinetics and strand specific RNA-Seq analysis of wtAAV2, rAAV and Rep2/Cap2 cassettes in the baculovirus context we could demonstrate that wtAAV2 native promoters, p5, p19 and p40 are all active in the context of the baculovirus system and lead to the expression of different proteins and peptides. In addition, this study has proven that the baculovirus brings at least some of the helper functions needed in the AAV replication/life cycle.

553. Considering the Wild Type Adeno-Associated Virus of Serotype 2 Genome as a Recombinant AAV in the Baculovirus/Sf9 Cells System

In 2010, a study on the infectivity of wild type Adeno-Associated Virus (WT AAV2) compared to recombinant AAV (rAAV) vectors have been published. The wild type AAV2 had an up to hundred fold higher infectivity compared to rAAVs.With the aim to decipher if this higher infectivity is due to the WT AAV2 production system, or only to the overall nature of the DNA encapsidated in the AAV capsid we have used the baculovirus/Sf9 system to produce rAAV, with the recombinant genome being the Wt AAV2 genome.However, expressing WT AAV2 genome in the baculovirus system has shown an early activity of the p5 promoter leading to the expression of Rep78. The expression of this protein resulted in an excision of the WT AAV2 genome during the first steps of Baculovirus stock formation.The absence of detection of the Rep68 and Rep40 proteins tended point to reduced or impaired splicing of the rep transcripts in the baculovirus/insect cells system. To put this into evidence, we have characterized the activity of the p5 promoter in the baculovirus/Sf9 context and studied the kinetics of expression of the Rep78 protein.

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.

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. 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.

Role and fate of SP100 protein in response to Rep-dependent nonviral integration system

Previously, we studied an AAVS1 site-specific non-viral integration system with a Rep-donor plasmid and a plasmid containing adeno-associated virus integration element. Our earlier study focused on the plasmid vector itself, but the cellular response to the system was still unknown. SP100 is a member of the promyelocytic leukemia nuclear bodies. It is involved in many cellular processes such as transcriptional regulation and the cellular intrinsic immune response against viral infection. In this study, we revealed that SP100 inhibited the Rep-dependent nonviral integration. Conversely, transient expression of Rep78 increased the degradation of SP100. This degradation was inhibited by treatment with MG132, an inhibitor of the ubiquitin proteasome. SP100 and Rep78 are both located in the nucleolus, which provides the spatial possibility for their interaction. Rep78 was coimmunoprecipitated with the enhanced green fluorescent protein (EGFP)-SP100 fusion protein but not EGFP, which verified the interaction between Rep78 and SP100. These results have enriched our knowledge about the cellular protein SP100 and Rep-dependent nonviral integration. It may lead to an improvement in the application of Rep-related transgene integration method and in the selection of target cells.

A Helper-Dependent Capsid-Modified Adenovirus Vector Expressing Adeno-Associated Virus Rep78 Mediates Site-Specific Integration of a 27-Kilobase Transgene Cassette

Random integration of viral gene therapy vectors and subsequent activation or disruption of cellular genes poses safety risks. Major efforts in the field are aimed toward targeting vector integration to specific sites in the host genome. The adeno-associated virus (AAV) Rep78 protein is able to target AAV integration to a specific site on human chromosome 19, called AAVS1. We studied whether this ability could be harnessed to achieve site-specific integration of a 27-kb transgene cassette into a model cell line for human hematopoietic cells (Mo7e). To deliver rep78 and the transgene to Mo7e cells, we used helper-dependent adenovirus (Ad) vectors containing Ad serotype 35 fiber knob domains (HD-Ad). An HD-Ad vector containing the rep78 gene under the control of the globin locus control region (LCR) (Ad.LCR-rep78) conferred Rep78 expression on Mo7e cells. Upon coinfection of Ad.LCR-rep78 with an HD-Ad vector containing a 27-kb globin-LCR-green fluorescent protein (GFP) transgene cassette flanked by AAV inverted terminal repeats (ITRs) (Ad.AAV-LCR-GFP), transduced cells were cloned and expanded (without selection pressure), and vector integration was analyzed in clones with more than 30% GFP-positive cells. Vector integration into the AAVS1 region was seen in 30% of analyzed integration sites, and GFP expression from these integrants was stable over time. Of the remaining integration sites, 25% were within the genomic globin LCR. In almost 90% of sites, transgene integration occurred via the Ad ITR. This indicates that rescue of the AAV ITR-flanked transgene cassette from Ad.AAV-LCR-GFP is not required for Rep78-mediated integration into AAVS1 and that free ends within the vector genome can be created by breaks within the Ad ITRs, whose structure is apparently recognized by cellular "nicking" enzymes. The finding that 55% of all analyzed integration sites were either within the AAVS1 or globin LCR region demonstrates that a high frequency of targeted integration of a large transgene cassette can be achieved in human hematopoietic stem cell lines.

880. AAV-Rep 78 Mediated Rescue of a 27kb Transgene Cassette from a Helper-Dependent Ad Vector Genome

AAV Rep78 is thought to increase site-specific integration of AAV vectors into the AAVS1 site within human chromosome 19. We have constructed helper-dependent (HD) adenovirus vectors containing a 27kb globin LCR-GFP expression cassette flanked by AAV ITRs (HD-Ad.AAV-GFP). Our goal was to test whether transient Rep78 expression would rescue this large expression cassette from the HD-Ad genome and increase its site-specific integration. We planned to use HD-Ad5/35 vectors to deliver the rep78 gene to our target cells, MO7e cells, which are a human erythroleukemic cell line. Construction of Rep78-expressing Ad vectors is problematic, however, because trace amounts of Rep78 inhibit Ad replication. We therefore placed the rep78 gene under the control of the â-globin promoter, which was found (in preliminary studies) to have low activity in 293 (producer) cells but high activity in target MO7e cells. We produced this vector (HD-Ad.LCR-rep78) at titers of 4.1x1012 genomes per ml. Western blot analysis with Rep-specific antibodies confirmed rep78 expression from HD-Ad.LCR-rep78. We then performed rescue assays in MO7e cells by infection with HD-Ad.LCR-rep78 and HD-Ad.AAV-GFP. While no rescue of the AAV-LCR-GFP cassette was detectable by Southern blot analysis upon infection with the two HD-vectors, addition of a first-generation Ad vector caused rescue. This effect was nullified by addition of hydroxyurea, an inhibitor of adenoviral DNA replication. We drew the conclusions that a large (27kb) transgene cassette can be excised from HD-Ad genomes by Rep78, and E1-deleted first-generation vectors are able to replicate their DNA and the DNA of co-infected HD-Ad vectors in MO7e cells, and Rep78 mediated rescue in MO7e cells requires Ad proteins and/or viral replication of HD-Ad.AAV genomes. We are currently attempting to dissect these two possibilities. We also performed transduction studies with HD-Ad.LCR-rep78 and HD-Ad.AAV-GFP vector. Co-infection with HD-Ad.LCR-rep78 increased the overall stable transduction frequency of HD.Ad.AAV-LCR in MO7e cells about two-fold. We are currently analyzing the integration frequency into AAV-S1 in the presence of HD-Ad.LCR-rep78 and the results will be presented.

Positive and negative effects of adeno-associated virus Rep on AAVS1-targeted integration.

Adeno-associated virus type 2 integrates preferentially into the AAVS1 locus on chromosome 19 of the human genome. It was reported previously that transfection with two plasmids, one for Rep and the other carrying a transgene flanked by inverted terminal repeats (ITRs), enables preferential integration of the latter into AAVS1. Aiming at increasing the frequency of AAVS1-specific integration, the Rep- to transgene-plasmid ratio necessary to achieve a higher frequency of site-specific integration was examined. 293 cells were co-transfected with the Rep78 plasmid and an ITR-flanked Neo gene at different ratios. G418-resistant clones were selected randomly. Extensive Southern blot analysis showed an optimum range of Rep78 expression. In that range, approximately 20 % of clones harboured the Neo gene at AAVS1. Excess Rep expression, however, resulted in 'abortive' integration of the Neo gene, a rearrangement of AAVS1 without transgene integration. Rep78 appeared to cause abortive integration more extensively than Rep68. Deleterious effects of the Rep protein on the AAVS1 locus should be considered to develop an improved AAVS1-targeted system.

Site-specific integration of an adeno-associated virus vector plasmid mediated by regulated expression of rep based on Cre-loxP recombination.

Recombinant adeno-associated virus (AAV) type 2 has attracted attention because it appears to have the potential to serve as a vector for human gene therapy. An interesting feature of wild-type AAV is its site-specific integration into AAVS1, a defined locus on chromosome 19. This reaction requires the presence of two viral elements: inverted terminal repeats and Rep78/68. Accordingly, current AAV vectors lacking the rep gene lack the capacity for site-specific integration. In this report, we describe the use of Cre-loxP recombination in a novel system for the regulated, transient expression of Rep78, which is potentially cytotoxic when synthesized constitutively. We constructed a plasmid in which the p5 promoter was situated downstream of the rep coding sequence; in this configuration, rep expression is silent. However, Cre circularizes the rep expression unit, directly joining the p5 promoter to the 5' end of the rep78 coding sequence, resulting in expression of Rep78. Such structural and functional changes were confirmed by detailed molecular analysis. A key feature of this system is that Rep expression was terminated when the circular molecule was linearized and integrated into the chromosome. Using this regulated expression system, we attempted site-specific integration of AAV vector plasmids. A PCR-based assay and analysis of fluorescence in situ hybridization showed that the AAV vector sequence was integrated into chromosome 19. Sequence analysis also confirmed that transient expression of Rep78 was sufficient for site-specific integration at the AAVS1 locus, as is observed with integration of wild-type AAV.

Adeno-associated virus type 2 Rep78 induces apoptosis through caspase activation independently of p53.

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.

Adeno-associated virus Rep78 protein and terminal repeats enhance integration of DNA sequences into the cellular genome.

Two adeno-associated virus (AAV) elements are necessary for the integration of the AAV genome: Rep78/68 proteins and inverted terminal repeats (ITRs). To study the contribution of the Rep proteins and the ITRs in the process of integration, we have compared the integration efficiencies of three different plasmids containing a green fluorescent protein (GFP) expression cassette. In one plasmid, no viral sequences were present; a second plasmid contained AAV ITRs flanking the reporter gene (integration cassette), and a third plasmid consisted of an integration cassette plus a Rep78 expression cassette. One day after transfection of 293 cells, fluorescent cells were sorted by flow cytometry and plated at 1 cell per well. Two weeks after sorting, colonies were monitored for stable expression of GFP. Transfection with the GFP plasmid containing no viral sequences resulted in no stable fluorescent colonies. Transfection with the plasmid containing the integration cassette alone (GFP flanked by ITRs) produced stable fluorescent colonies at a frequency of 5.3% +/- 1.0% whereas transfection with the plasmid containing both the integration cassette and Rep78 expression cassette produced stable fluorescent colonies at a frequency of 47% +/- 7.5%. Southern blot analysis indicated that in the presence of Rep78, integration is targeted to the AAVSI site in more than 50% of the clones analyzed. Some clones also showed tandem arrays of the integrated GFP cassette. Both head-to-head and head-to-tail orientations were detected. These findings indicate that the presence of AAV ITRs and the Rep78 protein enhance the integration of DNA sequences into the cellular genome and that the integration cassette is targeted to AAVS1 in the presence of Rep78.

High-level expression of adeno-associated virus (AAV) Rep78 or Rep68 protein is sufficient for infectious-particle formation by a rep-negative AAV mutant.

Adeno-associated virus (AAV) codes for four closely related nonstructural proteins (Rep) required for AAV DNA replication and gene regulation. In vitro studies have revealed that either Rep78 or Rep68 alone is sufficient for AAV DNA replication. Rep52 and Rep40 are not required for DNA replication but have been reported to enhance the efficiency of accumulation of single-stranded progeny DNA. Previous studies on rep-expressing cell lines had indicated that only a subset of the four Rep proteins are required for the production of infectious AAV. We therefore set out to determine the minimal set of Rep proteins sufficient for the generation of infectious AAV. Transient cotransfections in HeLa cells of constructs for high-level expression of individual Rep proteins with a rep-negative AAV genome revealed that either Rep78 or Rep68 alone could complement for a full replication cycle yielding infectious virus. This result was confirmed by transfection studies in the cell line HeM2, which selectively expresses Rep78 at rather low levels under the control of the glucocorticoid-responsive mouse mammary tumor virus long terminal repeat (C. Hölscher, M. Hörer, J. A. Kleinschmidt, H. Zentgraf, A. Bürkle, and R. Heilbronn, J. Virol. 68:7169-7177, 1994). Increasing the level of Rep78 expression by transfection of a glucocorticoid receptor expression construct resulted in a higher level of DNA replication of a cotransfected rep-negative AAV genome and in the production of infectious rep-negative AAV particles. We further report on the generation of a new rep-expressing cell line, HeCM1, which was obtained by stable supertransfection of a construct for constitutive Rep40 expression into HeM1 cells (Hölscher et al., J. Virol. 68:7169-7177). Transfection of rather large amounts of rep-negative AAV DNA led to detectable virus production in HeCM1 cells even in the absence of the cotransfected glucocorticoid receptor expression construct, but higher yields were obtained after increasing the Rep78 level by coexpression of the glucocorticoid receptor. These data demonstrate that all Rep functions required for the productive replication of AAV in HeLa cells are contained within both Rep78 and Rep68.

Cell lines inducibly expressing the adeno-associated virus (AAV) rep gene: requirements for productive replication of rep-negative AAV mutants.

The adeno-associated virus (AAV) rep gene codes for a family of nonstructural proteins which are required for AAV gene regulation and DNA replication. In addition, rep has been implicated in a variety of activities outside the AAV life cycle which have been difficult to study, since attempts to achieve separate and constitutive expression of rep in stable cell lines have failed so far. Here we report the generation of two cell lines which inducibly express Rep78 under the control of the glucocorticoid-responsive mouse mammary tumor virus promoter. In addition, one of the cell lines constitutively expresses relatively high levels of Rep52. Both cell lines showed similar plating efficiencies with and without induction of Rep78 expression, which rules out cytotoxic effects of Rep78. The cell lines efficiently support DNA replication of a rep-negative AAV genome and initiate the formation of AAV particles. However, despite the correct sizes and stoichiometry of the three capsid proteins, the AAV particles were noninfectious. This was found to be due to a defect in the accumulation of single-stranded AAV DNA. Transient transfection of single expression constructs for constitutive, high-level expression of individual Rep proteins (either Rep78, Rep68, Rep52, or Rep40) complemented this defect. Infectious rep-negative AAV progeny was produced at varying efficiencies depending on the rep expression construct used. These data show that functional expression of full-length Rep in recombinant cell lines is possible and that the state of Rep expression is critical for the infectivity of AAV progeny produced.