Relative levels of EBNA1 gene transcripts from the C/W, F and Q promoters in Epstein-Barr virus-transformed lymphoid cells in latent and lytic stages of infection.

Zetterberg, Henrik; Stenglein, Monika; Jansson, Agneta; Ricksten, Anne; Rymo, Lars

doi:10.1099/0022-1317-80-2-457

Cited by 32 publications

(33 citation statements)

References 25 publications

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“…Maintenance of a constant level of the EBNA1 protein could also be a result from IRES-mediated translation. Furthermore, Fp-initiated lytic transcript containing the U exon that is not spliced to the K exon is highly expressed upon induction of the virus lytic cycle (Schaefer et al, 1995a;Zetterberg et al, 1999). Although the intron/exon composition at its 3 0 end is undefined, this transcript indicates a role for the EBNA IRES during productive viral infection.…”

Section: Discussionmentioning

confidence: 99%

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Epstein–Barr virus U leader exon contains an internal ribosome entry site

2003

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Eukaryotic translation can be initiated either by a capdependent mechanism or by internal ribosome entry, a process by which ribosomes are directly recruited to structured regions of mRNA upstream of the initiation codon. Here we report the finding of an internal ribosome entry site (IRES) in the untranslated region of the Epstein-Barr nuclear antigen 1 (EBNA1) gene. EBNA1 is the only nuclear protein expressed in all known states of Epstein-Barr virus (EBV) latency and in the virus lytic cycle, and is required for the maintenance of the EBV episome. Using cDNA reporter constructs and in vitro transfection assays, we found that sequences contained in the 5 0 untranslated region (UTR) of the Fp and Qp initiated EBNA1 mRNA increased the expression level 4-14-fold in different Burkitt lymphoma cell lines. The U leader exon, located within the 5 0 UTR, included in all known EBNA1 transcripts and also contained in the EBNA3, 4 and 6 mRNAs, was demonstrated by bicistronic expression analyses to contain an IRES. The EBNA IRES initiates translation more efficiently than the encephalomyocarditis virus IRES in EBV-positive lymphoma cells. We propose that the EBNA IRES constitute a novel mechanism, whereby EBV regulates latent gene expression.

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Section: Discussionmentioning

confidence: 99%

“…Plasmids for generation of the monocistronic luciferase expression vectors driven by the EBNA1 promoters Fp and Qp (Figure 1) were constructed by cloning PCR-amplified fragments of the pFQUK and the pFQK plasmids previously described (Zetterberg et al, 1999). All PCR-amplified fragments contain 8 nt in the 3 0 end derived from the K exon.…”

Section: Plasmidsmentioning

confidence: 99%

Epstein–Barr virus U leader exon contains an internal ribosome entry site

2003

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“…In type III latency, in addition to the EBERs, EBNA-LP, -2, -3A, -3B, -3C, and -1 are expressed from the C promoter (Cp) (6), whereas LMP-1 and -2B are expressed from the bidirectional LMP1 promoter (46), and a larger splice variant of LMP-2, LMP-2A, is expressed from the TP1 promoter (36). Qp generally is supposed to be silent in type III latency (82,105), although there is also a different view (93). Among the viral proteins expressed in latency type III, EBNA2 plays a central role in switching EBNA transcription from Wp to Cp (W to C switch) (102,104) and in the establishment and maintenance of B-cell transformation (11,28), as EBNA2 transcriptionally activates the expression of the six nuclear antigens from the C promoter (Cp) and the membrane proteins LMP-1 and -2B from the LMP1 promoter (LMP1p), LMP-2A from the TP1 promoter, and a number of cellular proteins associated with the LCL phenotype (1,12,18,39,44,72,76,90,95,98,99,100,101,102,104,110,111).…”

Section: Epstein-barr Virus (Ebv) Infection Is the Cause Of Infectioumentioning

confidence: 99%

Protein-DNA Binding and CpG Methylation at Nucleotide Resolution of Latency-Associated Promoters Qp, Cp, and LMP1p of Epstein-Barr Virus

Salamon

Takács

Ujvári

et al. 2001

J Virol

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Epstein-Barr viral (EBV) latency-associated promotersEpstein-Barr virus (EBV) infection is the cause of infectious mononucleosis and is most closely associated with tumor diseases Burkitt's lymphoma (BL) and nasopharyngeal carcinoma. EBV infection of human B lymphocytes in vitro results in B-cell proliferation and transformation into continuously growing lymphoblastoid cell lines (LCL) (for a review, see reference 42). In latently infected cells, viral genomes are maintained as multiple circular episomal copies which are replicated once per cell cycle (2, 103). Several classes of latency have been described depending on the gene expression pattern (41,77,78). In strict type I latency, represented by BL cells, viral gene expression is restricted to the two RNA polymerase III-transcribed EBER RNA genes and the EBNA1 gene (78) that is transcribed from the Q promoter (Qp) (68). The EBNA1 protein is required for the maintenance of the viral plasmid in dividing cells (45,58). In type III latency, in addition to the EBERs, EBNA-LP, -2, -3A, -3B, -3C, and -1 are expressed from the C promoter (Cp) (6), whereas LMP-1 and -2B are expressed from the bidirectional LMP1 promoter (46), and a larger splice variant of LMP-2, LMP-2A, is expressed from the TP1 promoter (36). Qp generally is supposed to be silent in type III latency (82, 105), although there is also a different view (93). Among the viral proteins expressed in latency type III, EBNA2 plays a central role in switching EBNA transcription from Wp to Cp (W to C switch) (102,104) and in the establishment and maintenance of B-cell transformation (11, 28), as EBNA2 transcriptionally activates the expression of the six nuclear antigens from the C promoter (Cp) and the membrane proteins LMP-1 and -2B from the LMP1 promoter (LMP1p), LMP-2A from the TP1 promoter, and a number of cellular proteins associated with the LCL phenotype (1,12,18,39,44,72,76,90,95,98,99,100,101,102,104,110,111). A crucial mechanism involved in the silencing of Cp and LMP1p in type I latency has been shown to be methylation of CpG dinucleotides (3,15,35,54,60,61,70,73,74,75,84,91,94). In LCL, the EBV genome is mostly free of CpG methylation, whereas in BL cells, EBV genomes are highly methylated. An essential step in understanding the differences between latency types I and III is to elucidate the patterns of methylation and in vivo protein binding of the latency promoters of EBV at nucleotide resolution. Therefore, we decided to examine Qp, Cp, and LMP1p in cells of both latency types.(The contributions of Daniel Salamon to this work were made in partial fulfillment of the requirements for a Ph.D. from Semmelweis University, Budapest, Hungary.) MATERIALS AND METHODSCell lines and tissue culture. LCL 721 is a B95-8-transformed LCL with type III phenotype (40,52,57). Rael (15,43,61) is a group I BL cell line. Mutu BLI-C1216 is a subclone of the BL line Mutu, representative of latency type I (27). Mutu BLIII-C199 is a subclone of the BL line Mutu, representative of latency type III (27). Raji cells expre...

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“…Thus, the ability of EBNA1 to bind to DNA is essential for its activation of replication and transcription through oriP. After induction of the lytic program, EBNA1 transcripts are still synthesized from the F promoter in place of the C promoter (24,25), suggesting an unknown function in lytic infection.…”

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confidence: 99%

In Vivo Dynamics of EBNA1-oriP Interaction during Latent and Lytic Replication of Epstein-Barr Virus

Daikoku

Kudoh

Fujita

et al. 2004

Journal of Biological Chemistry

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The Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA1) is required for maintenance of the viral genome DNA during the latent phase of EBV replication but continues to be synthesized after the induction of viral productive replication. An EBV genome-wide chromatin immunoprecipitation assay revealed that EBNA1 constantly binds to oriP of the EBV genome during not only latent but also lytic infection. Although the total levels of EBNA1 proved constant throughout the latter, the levels of the oriP-bound form were increased as lytic infection proceeded. EBV productive DNA replication occurs at discrete sites in nuclei, called replication compartments, where viral replication proteins are clustered. Confocal laser microscopic analyses revealed that whereas EBNA1 was distributed broadly in nuclei as fine punctate dots during the latent phase of infection, the protein became redistributed to the viral replication compartments and localized as distinct spots within and/or nearby the compartments after the induction of lytic replication. Taking these findings into consideration, oriP regions of the EBV genome might be organized by EBNA1 into replication domains that may set up scaffolding for lytic replication and transcription.The Epstein-Barr virus (EBV) 1 is a human herpes virus that infects 90% of individuals. Primary EBV infection targets resting B lymphocytes, inducing their continuous proliferation. In the B lymphoblastoid cell lines, only limited numbers of viral genes are usually expressed and there is no production of virus particles, this being called latent infection. In the latent state, EBV maintains its 170-kbp genome as complete, multiple copies of plasmids. Latent phase viral replication appears to faithfully mimic cellular replicons; the EBV genomes or small EBNA1-oriP plasmids are synthesized only once in each Sphase by the host cell replication machinery, following the rules of chromosome replication (1).EBV-infected cell lines usually contain a small subpopulation of cells that have switched spontaneously from a latent stage of infection into the lytic cycle. The mechanism of switching is not fully understood, but one of the first detectable changes is expression of the BZLF1 gene product. The BZLF1 protein, together with the protein product of the BRLF1 gene, transactivates viral and certain cellular promoters (2) and leads to an ordered cascade of viral gene expression. The lytic phase of EBV DNA replication is dependent on seven viral replication proteins: BZLF1, an oriLyt-binding protein; BALF5, a DNA polymerase; BMRF1, a polymerase processivity factor; BALF2, a single-stranded DNA-binding protein; and BBLF4, BSLF1, and BBLF2/3, predicted to be helicase-, primase-, and helicase-primase-associated proteins, respectively (3). All of these proteins except for BZLF1 conceivably work together at replication forks to synthesize leading and lagging strands of the concatemeric EBV genome (4). Interestingly, viral lytic replication occurs in discrete sites in nuclei called replication compartments in which...

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Relative levels of EBNA1 gene transcripts from the C/W, F and Q promoters in Epstein-Barr virus-transformed lymphoid cells in latent and lytic stages of infection.

Cited by 32 publications

References 25 publications

Epstein–Barr virus U leader exon contains an internal ribosome entry site

Epstein–Barr virus U leader exon contains an internal ribosome entry site

Protein-DNA Binding and CpG Methylation at Nucleotide Resolution of Latency-Associated Promoters Qp, Cp, and LMP1p of Epstein-Barr Virus

In Vivo Dynamics of EBNA1-oriP Interaction during Latent and Lytic Replication of Epstein-Barr Virus

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