Prolonged siRNA expression in mammalian cells using an Epstein–Barr virus-based plasmid expression system

2020 ◽  
Vol 529 (1) ◽  
pp. 51-56
Author(s):  
Yan Wu ◽  
Tianqiang Song ◽  
Peipei Chen ◽  
Xiaohong Jiang ◽  
Qiang Wang ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Epstein Barr Virus ◽  
Expression System ◽  
Barr Virus ◽  
Sirna Expression ◽  
Epstein Barr ◽  
Expression In Mammalian Cells

scholarly journals Marked Variation in Response of Consensus Binding Elements for the Rta Protein of Epstein-Barr Virus

2005 ◽  
Vol 79 (15) ◽  
pp. 9635-9650 ◽  
Author(s):  
Lee-Wen Chen ◽  
Pey-Jium Chang ◽  
Henri-Jacques Delecluse ◽  
George Miller
Keyword(s):  
Dna Sequences ◽  
Transcriptional Activation ◽  
Mammalian Cells ◽  
Epstein Barr Virus ◽  
Lytic Cycle ◽  
Maximal Response ◽  
Binding Affinities ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT The R transactivator (Rta) protein activates Epstein-Barr virus (EBV) lytic-cycle genes by several distinct mechanisms that include direct binding to viral promoters, synergy with BamHI Z EBV replication activator (ZEBRA), and activation of cellular signaling pathways. In the direct and synergistic mechanisms of action, Rta binds to specific DNA sequences that are present in the promoters of responsive genes. It has been difficult to demonstrate the capacity of Rta expressed in mammalian cells to bind DNA in vitro in order to study the relative affinities of Rta binding elements. We discovered that a short C-terminal region of Rta inhibits the ability of Rta to bind DNA in vitro. C-terminally truncated versions of Rta bind DNA efficiently and thus facilitate a comparison of consensus Rta binding elements (CRBEs) found in promoters of five Rta-responsive genes: BMLF1, BHLF1, BMRF1, BaRF1, and BLRF2. All CRBEs in the promoters of the five genes conform to the proposed recognition sequence GNCCN9GGNG, where N is any nucleotide and N9 represents a sequence of nine nucleotides. Nonetheless, CRBEs varied markedly in their abilities to bind Rta in electrophoretic mobility shift assays. Not all CRBEs bound or responded to Rta. Binding affinities of the CRBEs and the capacity to be activated by Rta in reporter assays were strongly correlated. The CRBEs from the BMLF1 and BHLF1 promoters conferred the greatest response. The response of the BMRF1, BaRF1, and BLRF2 CRBEs was less robust. By creation of chimeras, inversions, and point mutations, differences in binding affinities and transcriptional activation levels could be attributed to N9 sequence variation. The length of N9 was also critical for a maximal response. In Raji and BZLF1-knockout cells, the mRNAs of the five Rta-responsive lytic-cycle genes differed dramatically in kinetics of expression, abundance, and synergistic responses to ZEBRA and Rta. Affinities of Rta response elements for Rta are likely to play an important role in temporal regulation and the level of lytic-cycle EBV gene expression.

scholarly journals Epstein–Barr virus nuclear antigen (EBNA) 3A induces the expression of and interacts with a subset of chaperones and co-chaperones

2008 ◽  
Vol 89 (4) ◽  
pp. 866-877 ◽  
Author(s):  
Paul Young ◽  
Emma Anderton ◽  
Kostas Paschos ◽  
Rob White ◽  
Martin J. Allday
Keyword(s):  
Epstein Barr Virus ◽  
Expression System ◽  
Nuclear Antigen ◽  
Human Diploid Fibroblasts ◽  
Barr Virus ◽  
Cellular Genes ◽  
Infected Cells ◽  
Epstein Barr ◽  
Chaperone Complex

Viral nuclear oncoproteins EBNA3A and EBNA3C are essential for the efficient immortalization of B cells by Epstein–Barr virus (EBV) in vitro and it is assumed that they play an essential role in viral persistence in the human host. In order to identify cellular genes regulated by EBNA3A expression, cDNA encoding EBNA3A was incorporated into a recombinant adenoviral vector. Microarray analysis of human diploid fibroblasts infected with either adenovirus EBNA3A or an empty control adenovirus consistently showed an EBNA3A-specific induction of mRNA corresponding to the chaperones Hsp70 and Hsp70B/B′ and co-chaperones Bag3 and DNAJA1/Hsp40. Analysis of infected fibroblasts by real-time quantitative RT-PCR and Western blotting confirmed that EBNA3A, but not EBNA3C, induced expression of Hsp70, Hsp70B/B′, Bag3 and DNAJA1/Hsp40. This was also confirmed in a stable, inducible expression system. EBNA3A activated transcription from the Hsp70B promoter, but not multimerized heat-shock elements in transient transfection assays, consistent with specific chaperone and co-chaperone upregulation. Co-immunoprecipitation experiments suggest that EBNA3A can form a complex with the chaperone/co-chaperone proteins in both adenovirus-infected cells and EBV-immortalized lymphoblastoid cell lines. Consistent with this, induction of EBNA3A resulted in redistribution of Hsp70 from the cytoplasm to the nucleus. EBNA3A therefore specifically induces (and then interacts with) all of the factors necessary for an active Hsp70 chaperone complex.

A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-Barr virus

1985 ◽  
Vol 5 (2) ◽  
pp. 410-413
Author(s):  
B Sugden ◽  
K Marsh ◽  
J Yates
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Epstein Barr Virus ◽  
Hygromycin B ◽  
Adherent Cells ◽  
Cis Acting ◽  
E Coli ◽  
Barr Virus ◽  
Ebv Dna ◽  
Epstein Barr

Epstein-Barr virus (EBV) transforms human B-lymphocytes into proliferating blasts which are efficiently established into cell lines. The viral DNA in these cell lines is usually present as complete, unintegrated plasmid molecules. A cis-acting element of EBV, oriP, permits plasmid maintenance in adherent cells that carry EBV DNA. We constructed a vector, pHEBo, that carries oriP and showed that it is also efficiently maintained as a plasmid when introduced into EBV-transformed B-lymphoblasts. The pHEBo vector carries the coding sequences for the hph gene from Escherichia coli such that it can be expressed in mammalian cells and confers resistance to the antibiotic hygromycin B. Hygromycin B kills EBV-transformed lymphoblasts at concentrations of 50 to 300 micrograms/ml. The combination of oriP plus the expressed hph gene makes pHEBo useful for the stable introduction of genes on plasmids into EBV-transformed lymphoblasts. Because pHEBo is derived from the plasmid pBR322 it can be easily isolated from lymphoblasts by reintroduction into E. coli.

scholarly journals Initiation of latent DNA replication in the Epstein-Barr virus genome can occur at sites other than the genetically defined origin.

1995 ◽  
Vol 15 (5) ◽  
pp. 2893-2903 ◽  
Author(s):  
R D Little ◽  
C L Schildkraut
Keyword(s):  
Mammalian Cells ◽  
Epstein Barr Virus ◽  
Virus Genome ◽  
Replication Initiation ◽  
Nuclear Antigen ◽  
Replication Forks ◽  
Small Plasmid ◽  
Barr Virus ◽  
Latently Infected ◽  
Epstein Barr

Our laboratory has previously shown that replication of a small plasmid, p174, containing the genetically defined Epstein-Barr virus (EBV) latent origin of replication, oriP, initiates within oriP at or near a dyad symmetry (DS) element and terminates specifically at a family of repeated sequences (FR), also located within oriP. We describe here an analysis of the replication of intact approximately 170-kb EBV genomes in four latently infected cell lines that uses two-dimensional gel replicon mapping. Initiation was detected at oriP in all EBV genomes examined; however, some replication forks appear to originate from alternative initiation sites. In addition, pausing of replication forks was observed at the two clusters of EBV nuclear antigen 1 binding sites within oriP and at or near two highly expressed viral genes 0.5 to 1 kb upstream of oriP, the EBV-encoded RNA (EBER) genes. In the Raji EBV genome, the relative abundance of these stalled forks and the direction in which they are stalled indicate that most replication forks originate upstream of oriP. We thus searched for additional initiation sites in the Raji EBV and found that the majority of initiation events were distributed over a broad region to the left of oriP. This delocalized pattern of initiation resembles initiation of replication in several well-characterized mammalian chromosomal loci and is the first described for any viral genome. EBV thus provides a unique model system with which to investigate factors influencing the selection of replication initiation and termination sites in mammalian cells.

Expression of the hepatitis B virus surface antigen in mammalian cells using an Epstein-Barr-virus-derived vector

1996 ◽  
Vol 46 (5-6) ◽  
pp. 533-537 ◽  
Author(s):  
S. A. C. Jorge ◽  
C. Hera ◽  
A. M. M. Spina ◽  
R. C. Moreira ◽  
J. R. R. Pinho ◽  
...  
Keyword(s):  
Hepatitis B Virus ◽  
Hepatitis B ◽  
Surface Antigen ◽  
Mammalian Cells ◽  
Epstein Barr Virus ◽  
Virus Surface ◽  
Virus Surface Antigen ◽  
Barr Virus ◽  
B Virus ◽  
Epstein Barr

scholarly journals Development of a Novel Helper-Dependent Adenovirus-Epstein-Barr Virus Hybrid System for the Stable Transformation of Mammalian Cells

2004 ◽  
Vol 78 (12) ◽  
pp. 6556-6566 ◽  
Author(s):  
Oliver Dorigo ◽  
Jose S. Gil ◽  
Sean D. Gallaher ◽  
Brenton T. Tan ◽  
Maria G. Castro ◽  
...  
Keyword(s):  
Hybrid System ◽  
Mammalian Cells ◽  
Transgene Expression ◽  
Epstein Barr Virus ◽  
Nuclear Antigen ◽  
Genetic Defects ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) episomes are stably maintained in permissive proliferating cell lines due to EBV nuclear antigen 1 (EBNA-1) protein-mediated replication and segregation. Previous studies showed the ability of EBV episomes to confer long-term transgene expression and correct genetic defects in deficient cells. To achieve quantitative delivery of EBV episomes in vitro and in vivo, we developed a binary helper-dependent adenovirus (HDA)-EBV hybrid system that consists of one HDA vector for the expression of Cre recombinase and a second HDA vector that contains all of the sequences for the EBV episome flanked by loxP sites. Upon coinfection of cells, Cre expressed from the first vector recombined loxP sites on the second vector. The resulting circular EBV episomes expressed a transgene and contained the EBV-derived family of repeats, an EBNA-1 expression cassette, and 19 kb of human DNA that functions as a replication origin in mammalian cells. This HDA-EBV hybrid system transformed 40% of cultured cells. Transgene expression in proliferating cells was observed for over 20 weeks under conditions that selected for the expression of the transgene. In the absence of selection, EBV episomes were lost at a rate of 8 to 10% per cell division. Successful delivery of EBV episomes in vivo was demonstrated in the liver of transgenic mice expressing Cre from the albumin promoter. This novel gene transfer system has the potential to confer long-term episomal transgene expression and therefore to correct genetic defects with reduced vector-related toxicity and without insertional mutagenesis.

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