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

ABSTRACTEpstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus associated with both B cell and epithelial cell malignancies. EBV infection of B cells triggers activation of several signaling pathways that are critical for cell survival, virus latency, and growth transformation. To identify EBV proteins important for regulating cell signaling, we used a proteomic approach to screen viral proteins for AP-1 and NF-κB promoter activity in AP-1– and NF-κB–luciferase reporter assays. We found that EBV BGLF2 activated AP-1 but not NF-κB reporter activity. Expression of EBV BGLF2 in cells activated p38 and c-Jun N-terminal kinase (JNK), both of which are important for mitogen-activated protein kinase (MAPK) signaling. Deletion of the carboxyl-terminal 66 amino acids of BGLF2 reduced the ability of BGLF2 to activate JNK and p38. Expression of BGLF2 enhanced BZLF1 expression in latently EBV-infected lymphoblastoid cell lines, and knockdown of BGLF2 reduced EBV reactivation induced by IgG cross-linking. Expression of BGLF2 induced BZLF1 expression and virus production in EBV-infected gastric carcinoma cells. BGLF2 enhanced BZLF1 expression and EBV production by activating p38; chemical inhibition of p38 and MAPK/ERK kinases 1 and 2 (MEK1/2) reduced expression of BZLF1 and virus production induced by BGLF2. In summary, the EBV tegument protein BGLF2, which is delivered to the cell at the onset of virus infection, activates the AP-1 pathway and enhances EBV reactivation and virus production.IMPORTANCEEpstein-Barr virus (EBV) is associated with both B cell and epithelial cell malignancies, and the virus activates multiple signaling pathways important for its persistence in latently infected cells. We identified a viral tegument protein, BGLF2, which activates members of the mitogen-activated protein kinase signaling pathway. Expression of BGLF2 increased expression of EBV BZLF1, which activates a switch from latent to lytic virus infection, and increased production of EBV. Inhibition of BGFL2 expression or inhibition of p38/MAPK, which is activated by BGLF2, reduced virus reactivation from latency. These results indicate that a viral tegument protein which is delivered to cells upon infection activates signaling pathways to enhance virus production and facilitate virus reactivation from latency.

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Epstein-Barr virus genome packaging factors accumulate in BMRF1-cores within viral replication compartments

PLoS ONE ◽

10.1371/journal.pone.0222519 ◽

2019 ◽

Vol 14 (9) ◽

pp. e0222519 ◽

Cited By ~ 3

Author(s):

Atsuko Sugimoto ◽

Yoriko Yamashita ◽

Teru Kanda ◽

Takayuki Murata ◽

Tatsuya Tsurumi

Keyword(s):

Viral Replication ◽

Epstein Barr Virus ◽

Virus Genome ◽

Genome Packaging ◽

Barr Virus ◽

Epstein Barr

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Interferon Antagonism of Epstein–Barr Virus Tegument Proteins

Proceedings ◽

10.3390/proceedings2020050074 ◽

2020 ◽

Vol 50 (1) ◽

pp. 74

Author(s):

Wai-Yan Lui ◽

Sonia Jangra ◽

Kit-San Yuen ◽

Michael George Botelho ◽

Dong-Yan Jin

Keyword(s):

Epstein Barr Virus ◽

Tyrosine Phosphatase ◽

Host Cells ◽

Tegument Protein ◽

Small Subset ◽

Type I ◽

Type I Ifn ◽

Tegument Proteins ◽

Barr Virus ◽

Epstein Barr

The Epstein–Barr virus (EBV) successfully infects 95% of all adults but causes Burkitt’s lymphoma, Hodgkin’s lymphoma, gastric carcinoma, nasopharyngeal carcinoma or other malignancies in only a small subset of infected individuals. The virus must have developed effective viral countermeasures to evade host innate immunity. In this study, we performed functional screens to identify EBV-encoded interferon (IFN) antagonists. Several tegument proteins were found to be potent suppressors of IFN production and/or signaling. The large tegument protein and deubiquitinase BPLF1 antagonized type I IFN production induced by DNA sensors cGAS and STING or RNA sensors RIG-I and MAVS. BPLF1’s ability to suppress innate immune signaling required its deubiquitinase activity. BPLF1 functioned as a catalytically active deubiquitinase for both K63- and K48-linked ubiquitin chains on STING and TBK1, with no ubiquitin linkage specificity. Induced expression of BPLF1 in EBV-infected cells through CRISPRa led to effective suppression of innate DNA and RNA sensing. Another EBV tegument protein, BGLF2, was found to suppress JAK-STAT signaling. This suppression was ascribed to more pronounced K48-linked polyubiquitination and proteasomal degradation of BGLF2-associated STAT2. In addition, BGLF2 also recruited tyrosine phosphatase SHP1 to inhibit tyrosine phosphorylation of JAK1 and STAT1. A BGLF2-deficient EBV activated type I IFN signaling more robustly. Taken together, we characterized the IFN antagonism of EBV tegument proteins BPLF1 and BGLF2, which modulate ubiquitination of key transducer proteins to counteract type I IFN production and signaling in host cells. Supported by HMRF 17160822, HMRF 18170942, and RGC C7027-16G.

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Epstein-Barr Virus Immediate-Early Protein BRLF1 Induces the Lytic Form of Viral Replication through a Mechanism Involving Phosphatidylinositol-3 Kinase Activation

Journal of Virology ◽

10.1128/jvi.75.13.6135-6142.2001 ◽

2001 ◽

Vol 75 (13) ◽

pp. 6135-6142 ◽

Cited By ~ 69

Author(s):

Catherine Dayle Darr ◽

Amy Mauser ◽

Shannon Kenney

Keyword(s):

Viral Replication ◽

Transcriptional Activation ◽

Epstein Barr Virus ◽

Adenovirus Vector ◽

Pi3 Kinase ◽

Immediate Early ◽

Kinase Activation ◽

Barr Virus ◽

Epstein Barr ◽

Phosphatidylinositol 3

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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Suppression of JAK-STAT signaling by Epstein-Barr virus tegument protein BGLF2 through recruitment of SHP1 phosphatase and promotion of STAT2 degradation

Journal of Virology ◽

10.1128/jvi.01027-21 ◽

2021 ◽

Author(s):

Sonia Jangra ◽

Aradhana Bharti ◽

Wai-Yin Lui ◽

Vidyanath Chaudhary ◽

Michael George Botelho ◽

...

Keyword(s):

Epstein Barr Virus ◽

Suppressive Effect ◽

Host Cells ◽

Interferon Response ◽

Tegument Protein ◽

Type Ii ◽

Barr Virus ◽

Stat Signaling ◽

Epstein Barr ◽

Type Iii Ifn

Some lytic proteins encoded by Epstein-Barr virus (EBV) suppress host interferon (IFN) signaling to facilitate viral replication. In this study we sought to identify and characterize EBV proteins antagonizing IFN signaling. The induction of IFN-stimulated genes (ISGs) by IFN-β was effectively suppressed by EBV. A functional screen was therefore performed to identify IFN-antagonizing proteins encoded by EBV. EBV tegument protein BGLF2 was identified as a potent suppressor of JAK-STAT signaling. This activity was found to be independent of its stimulatory effect on p38 and JNK pathways. Association of BGLF2 with STAT2 resulted in more pronounced K48-linked polyubiquitination and proteasomal degradation of the latter. Mechanistically, BGLF2 promoted the recruitment of SHP1 phosphatase to STAT1 to inhibit its tyrosine phosphorylation. In addition, BGLF2 associated with cullin 1 E3 ubiquitin ligase to facilitate its recruitment to STAT2. Consequently, BGLF2 suppressed ISG induction by IFN-β. Furthermore, BGLF2 also suppressed type II and type III IFN signaling, although the suppressive effect on type II IFN response was milder. When pre-treated with IFN-β, host cells became less susceptible to primary infection of EBV. This phenotype was reversed when expression of BGLF2 was enforced. Finally, genetic disruption of BGLF2 in EBV led to more pronounced induction of ISGs. Taken together, our study unveils the roles of BGLF2 not only in the subversion of innate IFN response but also in lytic infection and reactivation of EBV. Importance Epstein-Barr virus (EBV) is an oncogenic virus associated with the development of lymphoid and epithelial malignancies. EBV has to subvert interferon-mediated host antiviral response to replicate and cause diseases. It is therefore of great interest to identify and characterize interferon-antagonizing proteins produced by EBV. In this study we perform a screen to search for EBV proteins that suppress the action of interferons. We further show that BGLF2 protein of EBV is particularly strong in this suppression. This is achieved by inhibiting two key proteins STAT1 and STAT2 that mediate the antiviral activity of interferons. BGLF2 recruits a host enzyme to remove the phosphate group from STAT1 thereby inactivating its activity. BGLF2 also redirects STAT2 for degradation. A recombinant virus in which BGLF2 gene has been disrupted can activate host interferon response more robustly. Our findings reveal a novel mechanism by which EBV BGLF2 protein suppresses interferon signaling.

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Stimulation of the Replication of ICP0-Null Mutant Herpes Simplex Virus 1 and pp71-Deficient Human Cytomegalovirus by Epstein-Barr Virus Tegument Protein BNRF1

Journal of Virology ◽

10.1128/jvi.01224-16 ◽

2016 ◽

Vol 90 (21) ◽

pp. 9664-9673 ◽

Cited By ~ 6

Author(s):

Yongxu Lu ◽

Anne Orr ◽

Roger D. Everett

Keyword(s):

Epstein Barr Virus ◽

Nuclear Bodies ◽

Regulatory Proteins ◽

Tegument Protein ◽

Null Mutant ◽

Herpes Simplex Virus 1 ◽

Barr Virus ◽

Epstein Barr ◽

Simplex Virus ◽

Hsv 1

ABSTRACT It is now well established that several cellular proteins that are components of promyelocytic leukemia nuclear bodies (PML NBs, also known as ND10) have restrictive effects on herpesvirus infections that are countered by viral proteins that are either present in the virion particle or are expressed during the earliest stages of infection. For example, herpes simplex virus 1 (HSV-1) immediate early (IE) protein ICP0 overcomes the restrictive effects of PML-NB components PML, Sp100, hDaxx, and ATRX while human cytomegalovirus (HCMV) IE protein IE1 targets PML and Sp100, and its tegument protein pp71 targets hDaxx and ATRX. The functions of these viral regulatory proteins are in part interchangeable; thus, both IE1 and pp71 stimulate the replication of ICP0-null mutant HSV-1, while ICP0 increases plaque formation by pp71-deficient HCMV. Here, we extend these studies by examining proteins that are expressed by Epstein-Barr virus (EBV). We report that EBV tegument protein BNRF1, discovered by other investigators to target the hDaxx/ATRX complex, increases the replication of both ICP0-null mutant HSV-1 and pp71-deficient HCMV. In addition, EBV protein EBNA-LP, which targets Sp100, also augments ICP0-null mutant HSV-1 replication. The combination of these two EBV regulatory proteins had a greater effect than each one individually. These findings reinforce the concept that disruption of the functions of PML-NB proteins is important for efficient herpesvirus infections. IMPORTANCE Whether a herpesvirus initiates a lytic infection in a host cell or establishes quiescence or latency is influenced by events that occur soon after the viral genome has entered the host cell nucleus. Certain cellular proteins respond in a restrictive manner to the invading pathogen's DNA, while viral functions are expressed that counteract the cell-mediated repression. One aspect of cellular restriction of herpesvirus infections is mediated by components of nuclear structures known as PML nuclear bodies (PML NBs), or ND10. Members of the alpha-, beta-, and gammaherpesvirus families all express proteins that interact with, degrade, or otherwise counteract the inhibitory effects of various PML NB components. Previous work has shown that there is the potential for a functional interchange between the viral proteins expressed by alpha- and betaherpesviruses, despite a lack of obvious sequence similarity. Here, this concept is extended to include a member of the gammaherpesviruses.

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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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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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