The Epstein-Barr Virus BRRF1 Gene Is Dispensable for Viral Replication in HEK293 cells and Transformation

ABSTRACT Expression of the Epstein-Barr virus (EBV) immediate-early (IE) protein BRLF1 induces the lytic form of viral replication in most EBV-positive cell lines. BRLF1 is a transcriptional activator that binds directly to a GC-rich motif present in some EBV lytic gene promoters. However, BRLF1 activates transcription of the other IE protein, BZLF1, through an indirect mechanism which we previously showed to require activation of the stress mitogen-activated protein kinases. Here we demonstrate that BRLF1 activates phosphatidylinositol-3 (PI3) kinase signaling in host cells. We show that the specific PI3 kinase inhibitor, LY294002, completely abrogates the ability of a BRLF1 adenovirus vector to induce the lytic form of EBV infection, while not affecting lytic infection induced by a BZLF1 adenovirus vector. Furthermore, we demonstrate that the requirement for PI3 kinase activation in BRLF1-induced transcriptional activation is promoter dependent. BRLF1 activation of the SM early promoter (which occurs through a direct binding mechanism) does not require PI3 kinase activation, whereas activation of the IE BZLF1 and early BMRF1 promoters requires PI3 kinase activation. Thus, there are clearly two separate mechanisms by which BRLF1 induces transcriptional activation.

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Functional Interaction between Epstein-Barr Virus Replication Protein Zta and Host DNA Damage Response Protein 53BP1

Journal of Virology ◽

10.1128/jvi.00512-09 ◽

2009 ◽

Vol 83 (21) ◽

pp. 11116-11122 ◽

Cited By ~ 27

Author(s):

Sarah G. Bailey ◽

Elizabeth Verrall ◽

Celine Schelcher ◽

Alex Rhie ◽

Aidan J. Doherty ◽

...

Keyword(s):

Dna Damage ◽

Viral Replication ◽

Epstein Barr Virus ◽

Signal Transduction Pathway ◽

Human Herpesvirus ◽

Lytic Cycle ◽

Barr Virus ◽

Proteomic Approach ◽

Viral Genes ◽

Epstein Barr

ABSTRACT Epstein-Barr virus (EBV; human herpesvirus 4) poses major clinical problems worldwide. Following primary infection, EBV enters a form of long-lived latency in B lymphocytes, expressing few viral genes, and it persists for the lifetime of the host with sporadic bursts of viral replication. The switch between latency and replication is governed by the action of a multifunctional viral protein Zta (also called BZLF1, ZEBRA, and Z). Using a global proteomic approach, we identified a host DNA damage repair protein that specifically interacts with Zta: 53BP1. 53BP1 is intimately connected with the ATM signal transduction pathway, which is activated during EBV replication. The interaction of 53BP1 with Zta requires the C-terminal ends of both proteins. A series of Zta mutants that show a wild-type ability to perform basic functions of Zta, such as dimer formation, interaction with DNA, and the transactivation of viral genes, were shown to have lost the ability to induce the viral lytic cycle. Each of these mutants also is compromised in the C-terminal region for interaction with 53BP1. In addition, the knockdown of 53BP1 expression reduced viral replication, suggesting that the association between Zta and 53BP1 is involved in the viral replication cycle.

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An RS Motif within the Epstein-Barr Virus BLRF2 Tegument Protein Is Phosphorylated by SRPK2 and Is Important for Viral Replication

PLoS ONE ◽

10.1371/journal.pone.0053512 ◽

2013 ◽

Vol 8 (1) ◽

pp. e53512 ◽

Cited By ~ 9

Author(s):

Melissa Duarte ◽

Lili Wang ◽

Michael A. Calderwood ◽

Guillaume Adelmant ◽

Makoto Ohashi ◽

...

Keyword(s):

Viral Replication ◽

Epstein Barr Virus ◽

Tegument Protein ◽

Barr Virus ◽

Epstein Barr

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Epstein-Barr Virus Blocks the Autophagic Flux and Appropriates the Autophagic Machinery To Enhance Viral Replication

Journal of Virology ◽

10.1128/jvi.02199-14 ◽

2014 ◽

Vol 88 (21) ◽

pp. 12715-12726 ◽

Cited By ~ 75

Author(s):

M. Granato ◽

R. Santarelli ◽

A. Farina ◽

R. Gonnella ◽

L. V. Lotti ◽

...

Keyword(s):

Viral Replication ◽

Epstein Barr Virus ◽

Autophagic Flux ◽

Barr Virus ◽

Epstein Barr

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Efficient Replication of Epstein-Barr Virus-Derived Plasmids Requires Tethering by EBNA1 to Host Chromosomes

Journal of Virology ◽

10.1128/jvi.01606-13 ◽

2013 ◽

Vol 87 (23) ◽

pp. 13020-13028 ◽

Cited By ~ 13

Author(s):

Theresa L. Hodin ◽

Tanbir Najrana ◽

John L. Yates

Keyword(s):

Dna Binding ◽

Epstein Barr Virus ◽

Binding Domain ◽

Hek293 Cells ◽

Plasmid Replication ◽

Nuclear Fraction ◽

Dna Binding Domain ◽

Host Chromosome ◽

Barr Virus ◽

Epstein Barr

The EBNA1 protein of Epstein-Barr virus enables plasmids carryingoriPboth to duplicate and to segregate efficiently in proliferating cells. EBNA1 recruits the origin recognition complex (ORC) to establish a replication origin at one element oforiP, DS (dyadsymmetry); at another element, FR (family ofrepeats), EBNA1 binds to an array of sites from which it tethers plasmids to host chromosomes for mitotic stability. We report experiments leading to the conclusion that tethering by EBNA1 to host chromosomes is also needed within interphase nuclei in order for plasmids to be replicated efficiently fromoriP. The DNA-binding domain of EBNA1, which lacks chromosome-binding ability, was found to support weak, DS-specific replication in HEK293 cells after transient transfection, being 17% as active as wild-type EBNA1. The low efficiency of replication was not due to the failure of the DNA-binding domain to retain plasmids within nuclei, because plasmids were recovered in similar amounts and entirely from the nuclear fraction of these transiently transfected cells. A derivative of EBNA1 with its chromosome-tethering domains replaced by a 22-amino-acid nucleosome-binding domain was fully active in supportingoriPfunctions. The implication is that EBNA1's DNA-binding domain is able to recruit ORC to DS, but either this step or subsequent replication is only efficient if the plasmid is tethered to a host chromosome. Finally, with some cell lines, DS can hardly support even transient plasmid replication without FR. A loss of plasmids lacking FR from nuclei cannot account for this requirement, suggesting that the stronger tethering to chromosomes by FR is needed for plasmid replication within the nuclei of such cells.

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Cellular transcription factors recruit viral replication proteins to activate the Epstein–Barr virus origin of lytic DNA replication, oriLyt

The EMBO Journal ◽

10.1038/sj.emboj.7592140b ◽

2000 ◽

Vol 19 (2) ◽

pp. 315-315

Author(s):

M. Baumann ◽

R. Feederle ◽

E. Kremmer ◽

W. Hammerschmidt

Keyword(s):

Transcription Factors ◽

Dna Replication ◽

Viral Replication ◽

Epstein Barr Virus ◽

Replication Proteins ◽

Barr Virus ◽

Virus Origin ◽

Epstein Barr ◽

Lytic Dna Replication ◽

Cellular Transcription

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Epstein-Barr Virus BZLF1-Mediated Downregulation of Proinflammatory Factors Is Essential for Optimal Lytic Viral Replication

Journal of Virology ◽

10.1128/jvi.01921-15 ◽

2015 ◽

Vol 90 (2) ◽

pp. 887-903 ◽

Cited By ~ 6

Author(s):

Yuqing Li ◽

Xubing Long ◽

Lu Huang ◽

Mengtian Yang ◽

Yan Yuan ◽

...

Keyword(s):

Viral Replication ◽

Epstein Barr Virus ◽

Lytic Cycle ◽

Inflammatory Factors ◽

Barr Virus ◽

Ebv Infection ◽

Tnf Α ◽

Ifn Γ ◽

Epstein Barr ◽

Proinflammatory Factors

ABSTRACTElevated secretion of inflammatory factors is associated with latent Epstein-Barr virus (EBV) infection and the pathology of EBV-associated diseases; however, knowledge of the inflammatory response and its biological significance during the lytic EBV cycle remains elusive. Here, we demonstrate that the immediate early transcriptional activator BZLF1 suppresses the proinflammatory factor tumor necrosis factor alpha (TNF-α) by binding to the promoter of TNF-α and preventing NF-κB activation. A BZLF1Δ207-210 mutant with a deletion of 4 amino acids (aa) in the protein-protein binding domain was not able to inhibit the proinflammatory factors TNF-α and gamma interferon (IFN-γ) and reduced viral DNA replication with complete transcriptional activity during EBV lytic gene expression. TNF-α depletion restored the viral replication mediated by BZLF1Δ207-210. Furthermore, a combination of TNF-α- and IFN-γ-neutralizing antibodies recovered BZLF1Δ207-210-mediated viral replication, indicating that BZLF1 attenuates the antiviral response to aid optimal lytic replication primarily through the inhibition of TNF-α and IFN-γ secretion during the lytic cycle. These results suggest that EBV BZLF1 attenuates the proinflammatory responses to facilitate viral replication.IMPORTANCEThe proinflammatory response is an antiviral and anticancer strategy following the complex inflammatory phenotype. Latent Epstein-Barr virus (EBV) infection strongly correlates with an elevated secretion of inflammatory factors in a variety of severe diseases, while the inflammatory responses during the lytic EBV cycle have not been established. Here, we demonstrate that BZLF1 acts as a transcriptional suppressor of the inflammatory factors TNF-α and IFN-γ and confirm that BZLF1-facilitated escape from the TNF-α and IFN-γ response during the EBV lytic life cycle is required for optimal viral replication. This finding implies that the EBV lytic cycle employs a distinct strategy to evade the antiviral inflammatory response.

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Architecture of Replication Compartments Formed during Epstein-Barr Virus Lytic Replication

Journal of Virology ◽

10.1128/jvi.79.6.3409-3418.2005 ◽

2005 ◽

Vol 79 (6) ◽

pp. 3409-3418 ◽

Cited By ~ 61

Author(s):

Tohru Daikoku ◽

Ayumi Kudoh ◽

Masatoshi Fujita ◽

Yutaka Sugaya ◽

Hiroki Isomura ◽

...

Keyword(s):

Dna Replication ◽

Viral Replication ◽

Epstein Barr Virus ◽

Protein A ◽

Lytic Replication ◽

Viral Dna ◽

Barr Virus ◽

Epstein Barr ◽

Cellular Replication ◽

Cellular Dna

ABSTRACT Epstein-Barr virus (EBV) productive DNA replication occurs at discrete sites, called replication compartments, in nuclei. In this study we performed comprehensive analyses of the architecture of the replication compartments. The BZLF1 oriLyt binding proteins showed a fine, diffuse pattern of distribution throughout the nuclei at immediate-early stages of induction and then became associated with the replicating EBV genome in the replication compartments during lytic infection. The BMRF1 polymerase (Pol) processivity factor showed a homogenous, not dot-like, distribution in the replication compartments, which completely coincided with the newly synthesized viral DNA. Inhibition of viral DNA replication with phosphonoacetic acid, a viral DNA Pol inhibitor, eliminated the DNA-bound form of the BMRF1 protein, although the protein was sufficiently expressed in the cells. These observations together with the findings that almost all abundantly expressed BMRF1 proteins existed in the DNA-bound form suggest that the BMRF1 proteins not only act at viral replication forks as Pol processive factors but also widely distribute on newly replicated EBV genomic DNA. In contrast, the BALF5 Pol catalytic protein, the BALF2 single-stranded-DNA binding protein, and the BBLF2/3 protein, a component of the helicase-primase complex, were colocalized as distinct dots distributed within replication compartments, representing viral replication factories. Whereas cellular replication factories are constructed based on nonchromatin nuclear structures and nuclear matrix, viral replication factories were easily solubilized by DNase I treatment. Thus, compared with cellular DNA replication, EBV lytic DNA replication factories would be simpler so that construction of the replication domain would be more relaxed.

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