Maintenance of Epstein-Barr virus-derived episomal vectors in the murine Sp2/0 myeloma cell line is dependent upon exogenous expression of human EBP2.

Vectors carrying the origin of replication (oriP) and driving expression of the EBNA-1 protein from Epstein-Barr virus (EBV) replicate as extrachromosomal episomes in human cells. Whether these vectors can be maintained as episomes in murine cells is still controversial. Here we demonstrate that EBNA-1 expression alone was unable to maintain episomal expression of an EBV-based vector in the murine Sp2/0 cell line. However, we were able to obtain long-term episome maintenance in Sp2/0 cells after exogenously expressing human EBP2 by genetic engineering. Our results provide further evidence for the fundamental role of human EBP2 in episomal maintenance of EBV-based vectors. Moreover, we demonstrate that EBV-based vectors can be successfully used in cells presumably incompetent for episomal maintenance.     

Live-cell imaging reveals multiple interactions between Epstein-Barr virus nuclear antigen 1 and cellular chromatin during interphase and mitosis

Epstein-Barr virus (EBV) establishes a life-long latent infection in humans. In proliferating latently infected cells, EBV genomes persist as multiple episomes that undergo one DNA replication event per cell cycle and remain attached to the mitotic chromosomes. EBV nuclear antigen 1 (EBNA-1) binding to the episome and cellular genome is essential to ensure proper episome replication and segregation. However, the nature and regulation of EBNA-1 interaction with chromatin has not been clearly elucidated. This activity has been suggested to involve EBNA-1 binding to DNA, duplex RNA, and/or proteins. EBNA-1 binding protein 2 (EBP2), a nucleolar protein, has been proposed to act as a docking protein for EBNA-1 on mitotic chromosomes. However, there is no direct evidence thus far for EBP2 being associated with EBNA-1 during mitosis. By combining video microscopy and Förster resonance energy transfer (FRET) microscopy, we demonstrate here for the first time that EBNA-1 and EBP2 interact in the nucleoplasm, as well as in the nucleoli during interphase. However, in strong contrast to the current proposed model, we were unable to observe any interaction between EBNA-1 and EBP2 on mitotic chromosomes. We also performed a yeast double-hybrid screening, followed by a FRET analysis, that led us to identify HMGB2 (high-mobility group box 2), a well-known chromatin component, as a new partner for EBNA-1 on chromatin during interphase and mitosis. Although the depletion of HMGB2 partly altered EBNA-1 association with chromatin in HeLa cells during interphase and mitosis, it did not significantly impact the maintenance of EBV episomes in Raji cells.     

Long-term transgene expression in proliferating cells mediated by episomally maintained high-capacity adenovirus vectors

High-capacity "gutless" adenovirus vectors (HC-AdV) mediate long-term transgene expression in resting cells in vitro and in vivo because of low toxicity and immunogenicity. However, in proliferating cells, expression is transient since HC-AdV genomes do not possess elements that allow for replication and segregation of the replicated genomes to daughter cells. We developed a binary HC-AdV system that, under certain conditions, allows for significantly prolonged episomal maintenance of HC-AdV genomes in proliferating tissue culture cells, resulting in sustained transgene expression. After transduction of target cells the linear HC-AdV genomes were circularized by the DNA recombinase FLPe, which was expressed from the second HC-AdV. The oriP/EBNA-1 replication system derived from Epstein-Barr virus, as well as the human replication origin from the lamin B2 locus, were used as cis elements to test for replication of the 28-kb circular vector genomes with or without selective pressure. Depending on the system, up to 98% of the circularized genomes were replicated and segregated to daughter cells, as demonstrated by Southern assays and as confirmed by monitoring EGFP transgene expression. Surprisingly, in the absence of FLPe recombinase, a small but significant number of HC-AdV genomes spontaneously circularized after transduction of target cells. These circles, found to contain end-to-end joined adenovirus termini, replicated with increased efficiency compared to vectors circularized by FLPe. After further improvements, this HC-AdV system might be suitable for gene therapy applications requiring long-term transgene expression.     

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.     

Differences in human cell lines to support stable replication of Epstein-Barr virus-based vectors

Vectors carrying the origin of replication, ori-P, of the Epstein-Barr virus (EBV) are maintained extrachromosomally in human cells expressing the EBV nuclear antigen 1 (EBNA-1). We have studied the EBV vectors p201 and p292 in which both ori-P and EBNA-1 functions are present using the human cell lines A431 and HeLa. The two lines showed differences in their transfectability by the EBV vectors. Thousands of HeLa transfectants were obtained with either vector and these remained intact as episomes. A431 could only be efficiently transfected with p292 and a high ratio of chromosomal integrations and rearrangements were observed. The vector p292 expressed the EBNA-1 gene more efficiently than p201 and this was found to be associated with a harmful effect on the grown of both HeLa and A431 lines. These results indicate that EBV vectors behave differently, depending on the cell line and that over-expression of EBNA 1 from these vectors may be detrimental to the cells.     

Mapping EBNA-1 domains involved in binding to metaphase chromosomes

The Epstein-Barr virus (EBV) genome can persist in dividing human B cells as multicopy circular episomes. Viral episomes replicate in synchrony with host cell DNA and are maintained at a relatively constant copy number for a long time. Only two viral elements, the replication origin OriP and the EBNA-1 protein, are required for the persistence of viral genomes during latency. EBNA-1 activates OriP during the S phase and may also contribute to the partition and/or retention of viral genomes during mitosis. Indeed, EBNA-1 has been shown to interact with mitotic chromatin. Moreover, viral genomes are noncovalently associated with metaphase chromosomes. This suggests that EBNA-1 may facilitate the anchorage of viral genomes on cellular chromosomes, thus ensuring proper partition and retention. In the present paper, we have investigated the chromosome-binding activity of EBV EBNA-1, herpesvirus papio (HVP) EBNA-1, and various derivatives of EBV EBNA-1, fused to a variant of the green fluorescent protein. The results show that binding to metaphase chromosomes is a common property of EBV and HVP EBNA-1. Further studies indicated that at least three independent domains (CBS-1, -2, and -3) mediate EBNA-1 binding to metaphase chromosomes. In agreement with the anchorage model, two of these domains mapped to a region that has been previously demonstrated to be required for the long-term persistence of OriP-containing plasmids.     

Long-term stability of large insert genomic DNA episomal shuttle vectors in human cells

We have constructed an episomal shuttle vector which can transfer large (>100 kb) human genomic DNA inserts back and forth between bacteria and human cells and which can be tracked in rapidly dividing human cells using a live cell assay. The vector (p5170) is based on the F factor-derived bacterial artificial chromosome cloning vector used in Escherichia coli, with the addition of the family of repeats element from the Epstein-Barr virus (EBV) latent origin of replication. This element provides nuclear retention in cells expressing the EBV protein EBNA-1. We have subcloned a series of genomic DNA inserts into p5170 and transfected the constructs into an EBNA-1(+) human cell line. Episomal mitotic stability was quantitatively analysed using flow cytometry. The episomes were also tracked by time course photography of expanding colonies. A 117 kb episome was retained at approximately 2 copies/cell and could be shuttled unrearranged from the human cells into bacterial cells after 15 months of continuous cell growth. Furthermore, the episome could still be rescued from human cells cultured in the absence of selection for 198 days. Such a trackable E.coli /human cell line shuttle vector system capable of carrying >100 kb of genomic DNA in human cells could prove a valuable tool in gene expression studies.     

Analysis of viral cis elements conferring Kaposi's sarcoma-associated herpesvirus episome partitioning and maintenance

Maintenance of Kaposi's sarcoma-associated herpesvirus (KSHV) episomes in latently infected cells is dependent on the latency-associated nuclear antigen (LANA). LANA binds to the viral terminal repeats (TR), leading to recruitment of cellular origin recognition complex proteins. Additionally, LANA tethers episomes to chromosomes via interactions with histones H2A and H2B (A. J. Barbera et al., Science 311:856-861, 2006). Despite these molecular details, less is known about how episomes are established after de novo infection. To address this, we measured short-term retention rates of green fluorescent protein-expressing replicons in proliferating lymphoid cells. In the absence of antibiotic selection, LANA significantly reduced the loss rate of TR-containing replicons. Additionally, we found that LANA can support long-term stability of KSHV replicons for more than 2 months under nonselective conditions. Analysis of cis elements within TR that confer episome replication and partitioning revealed that these activities can occur independently, and furthermore, both events contribute to episome stability. We found that replication-deficient plasmids containing LANA binding sites (LBS1/2) exhibited measurable retention rates in the presence of LANA. To confirm these observations, we uncoupled KSHV replication and partitioning by constructing hybrid origins containing the Epstein-Barr virus (EBV) dyad symmetry for plasmid replication and KSHV LBS1/2. We demonstrate that multiple LBS1/2 function in a manner analogous to that of the EBV family of repeats by forming an array of LANA binding sites for partitioning of KSHV genomes. Our data suggest that the efficiency with which KSHV establishes latency is dependent on multiple LANA activities, which stabilize viral genomes early after de novo infection.     

Flp recombinase transgenic mice of C57BL/6 strain for conditional gene targeting

We constructed an expression vector of Flp recombinase modified by adding a nuclear localization signal. Injection of the expression vector into fertilized eggs of the C57BL/6 strain yielded transgenic mouse lines expressing the Flp recombinase transgene in the testis. We crossed the transgenic mice to reporter mice carrying the neomycin phosphotransferase gene flanked by target sites of Flp recombinase. Examination of the deletion of the neomycin phosphotransferase gene in the progeny showed that Flp-mediated recombination took place efficiently in vivo in FLP66 transgenic mouse line. These results suggest that the Flp recombinase system is effective in mice and in combination with the Cre recombinase system extends the potentials of gene manipulation in mice. One of the useful applications of FLP66 transgenic mouse line is the removal of marker genes from mice manipulated for the conditional gene targeting with the Cre/loxP system in the pure C57BL/6 genetic background.     

Coupling of mitotic chromosome tethering and replication competence in epstein-barr virus-based plasmids

The Epstein-Barr virus (EBV) replicates once per cell cycle and segregates with high efficiency yet does not encode the enzymes needed for DNA replication or the proteins required to contact mitotic spindles. The virus-encoded EBNA-1 (EBV nuclear antigen 1) and latent replication origin (oriP) are required for both replication and segregation. We developed a sensitive and specific fluorescent labeling strategy to analyze the interactions of both EBNA-1 with viral episomes and viral episomes with host chromosomes. This enabled investigation of the hypothesis that replication and chromosome tethering are linked through the EBNA-1 protein. We show that deleting EBNA-1 or oriP disrupts mitotic chromosome tethering but removing the dyad symmetry element of oriP does not. Microscopic and biochemical approaches demonstrated that an EBNA-1 mutant lacking residues 16 to 372 bound to oriP plasmids but did not support their mitotic chromosome association and that the mutant lost the ability of wild-type EBNA-1 to associate with interphase chromatin. Importantly, the transient-replication abilities of various mutant forms of EBV plasmids, including the mutant form with the EBNA-1 internal deletion, correlated directly with their chromosome-tethering abilities. These data lead us to propose that EBNA-1 recruits oriP-containing plasmids into chromatin subdomains in interphase nuclei to both engage the host replication machinery and enable the plasmids to adhere to host chromosomes to increase their segregation efficiency.     

Cre levels limit packaging signal excision efficiency in the Cre/loxP helper-dependent adenoviral vector system

Helper-dependent (HD) adenovirus vectors devoid of all viral coding sequences have a large cloning capacity and provide long-term transgene expression in vivo with negligible toxicity, making them attractive vectors for gene therapy. Currently, the most efficient means of producing HD vectors involves coinfecting 293 cells expressing Cre with the HD vector and a helper virus bearing a packaging signal flanked by loxP sites. Cre-mediated packaging signal excision renders the helper virus genome unpackageable but still able to replicate and provide helper functions for HD vector propagation. Typically, helper virus contamination is < or =1% pre- and < or =0.1% postpurification by CsCl banding. While these contamination levels are low, further reduction is desirable. However, this objective has not been realized since the Cre/loxP system was first developed. This lack of progress is due, at least in part, to our lack of understanding of the origins of the contaminating helper virus, thus rendering its reduction or elimination difficult to achieve. This study was designed to investigate the possible sources of contaminating helper virus persisting during HD vector amplification. The results revealed that Cre is limiting in helper virus-infected Cre-expressing 293 cells, thereby permitting helper viruses to escape packaging signal excision and propagate. The results of this study should provide a foundation for developing rational strategies to further reduce or possibly eliminate the contaminating helper virus.     

Reduction of Cre recombinase toxicity in proliferating Drosophila cells by estrogen-dependent activity regulation

The Cre/loxP site-specific recombination system has been used successfully for genome manipulation in a wide range of species. However, in Drosophila melanogaster, a major model organism for genetic analyses, the alternative FLP/FRT system, which is less efficient at least in mammalian cells, has been established, primarily for the generation of genetic mosaics for clonal analyses. To extend genetic methodology in D. melanogaster, we have created transgenic lines allowing tissue-specific expression of Cre recombinase with the UAS/GAL4 system. Surprisingly, chronic expression of Cre recombinase from these transgenes (UAST-cre) was found to be toxic for proliferating cells. Therefore, we also generated transgenic lines allowing the expression of Cre recombinase fused to the ligand-binding domain of the human estrogen receptor (UASP-cre-EBD). We demonstrate that recombination can be efficiently dissociated from toxicity by estrogen-dependent regulation of recombinase activity of the UASP-cre-EBD transgene products.     

An OriP/EBNA-1-based baculovirus vector with prolonged and enhanced transgene expression

BACKGROUND: The baculovirus Autographa californica multiple nucleopolyhedrovirus (AcMNPV) has been explored as a gene delivery vehicle for a variety of mammalian cell lines. However, the transient expression nature due to its incapability to replicate in mammalian cells and insufficient transduction efficiency limit its application. METHODS: Recombinant baculovirus vectors containing genetic elements from Epstein-Barr virus (EBV), OriP and EBNA-1, which are essential for the episomal maintenance of the EBV genome in latently infected cells, were constructed and tested for their ability to sustain and express transgene (enhanced green fluorescence protein (egfp)) in mammalian cells. RESULTS: The recombinant baculovirus containing OriP and EBNA-1 genes driven by the cytomegalovirus (CMV) promoter was capable of persisting in a significant proportion of infected mammalian cells, HEK293, Vero, Cos-7, and Hone-1, without any selective pressure. In HEK293, the expression of EGFP lasted for 60 days with markedly enhanced expression level. The persistence of baculovirus genome correlated with the expression of EBNA-1. CONCLUSIONS: The improved baculovirus vector could mediate prolonged and enhanced foreign gene expression in some mammalian cells. Furthermore, an adequate level of the EBNA-1 protein was essential for the maintenance of the OriP-containing baculovirus genome. The new vector has potential for use in gene therapy.     

Transgene Coplacement and High Efficiency Site-Specific Recombination with the Cre/Loxp System in Drosophila

Studies of gene function and regulation in transgenic Drosophila are often compromised by the possibility of genomic position effects on gene expression. We have developed a method, called transgene coplacement, in which any two sequences can be positioned at exactly the same site and orientation in the genome. Transgene coplacement makes use of the bacteriophage P1 system of Cre/loxP site-specific recombination, which we have introduced into Drosophila. In the presence of a cre transgene driven by a dual hsp70-Mos1 promoter, a white reporter gene flanked by loxP sites is excised with virtually 100% efficiency both in somatic cells and in germ cells. A strong maternal effect, resulting from Cre recombinase present in the oocyte, is observed as white or mosaic eye color in F(1) progeny. Excision in germ cells of the F(1) yields a strong grand-maternal effect, observed as a highly skewed ratio of eye-color phenotypes in the F(2) generation. The excision reactions of Cre/loxP and the related FLP/FRT system are used to create Drosophila lines in which transgenes are at exactly allelic sites in homologous chromosomes.     

Epstein-Barr virus (EBV) nuclear antigen 1 colocalizes with cellular replication foci in the absence of EBV plasmids

Epstein-Barr virus (EBV) EBNA-1 is the only EBV-encoded protein that is essential for the once-per-cell-cycle replication and maintenance of EBV plasmids in latently infected cells. EBNA-1 binds to the oriP region of latent EBV plasmids and cellular metaphase chromosomes. In the absence of oriP-containing plasmids, EBNA-1 was highly colocalized with cellular DNA replication foci that were identified by immunostaining S-phase cells for proliferating cell nuclear antigen and replication protein A (RP-A) in combination with DNA short pulse-labeling. For the association of EBNA-1 with the cellular replication focus areas, the EBNA-1 regions of amino acids (aa) 8 to 94 and/or aa 315 to 410, but not the RP-A-interacting carboxy-terminal region, were necessary. These results suggest a new aspect of latent virus-cell interactions.     

LoxP-directed cloning: use of Cre recombinase as a universal restriction enzyme.

We have developed a novel way to use the Cre/loxP system for in vitro manipulation of DNA and a technique to clone DNA into circular episomes. The method is fast, reliable, and allowsflexible cloning of DNA fragments into episomes containing a loxP site. We show that a loxP site can serve as a universal target site to clone a DNA fragment digested with any restriction enzyme(s). This technique abolishes the need for compatible restriction sites in cloning vectors and targets by generating custom-designed 5' 3', or blunt ends in the desired orientation and reading frame in the vector Therefore, this method eliminates the limitations encountered when DNA fragments are cloned into vectors with a confined number of cloning sites. The 34-bp loxP sequence assures uniqueness, even when large episomes are manipulated. We present three examples, including the manipulation of a bacterial artificial chromosome. Because DNA manipulation takes place at a loxP site, we refer to this technique as loxP-directed cloning.     

Sequences adjacent to oriP improve the persistence of Epstein-Barr virus-based episomes in B cells

Epstein-Barr virus (EBV) oriP and the EBV nuclear antigen 1 (EBNA-1) protein allow persistence of EBV-based episomes. A nuclear matrix attachment region (MAR) spans oriP and the adjacent region of the EBV genome containing the EBV-expressed RNAs. Here, we show that episomes with the MAR are retained significantly more efficiently in EBV-positive B cells than episomes containing oriP alone.     

Efficient long-term gene transfer into muscle tissue of immunocompetent mice by adeno-associated virus vector.

Muscle-directed gene transfer is being considered for the treatment of several metabolic diseases, including hemophilia and Duchene's muscular dystrophy. Previous efforts to target this tissue for somatic delivery with various vector systems have resulted in transient expression due to silencing of the transgene or to an immune response against the vector-transduced cells. We introduced recombinant adeno-associated virus vector (rAAV) carrying a lacZ reporter into muscle tissue of immunocompetent mice. The lacZ reporter gene was efficiently transduced and expressed with no evidence of a cellular immune response. Moreover, gene expression persisted for more than 1.5 years. Molecular characterization of rAAV vector DNA suggests a mechanism for persistence, since vector episomes convert to high-molecular-weight genomic DNA. These data provide the first report for establishing long-term gene transduction into mammalian muscle cells in vivo without the need for immune modulation of the organism.