Epstein-Barr Virus MicroRNA miR-BART20-5p Suppresses Lytic Induction by InhibitingBAD-Mediatedcaspase-3-Dependent Apoptosis

ABSTRACTEpstein-Barr virus (EBV) is a human gammaherpesvirus associated with a variety of tumor types. EBV can establish latency or undergo lytic replication in host cells. In general, EBV remains latent in tumors and expresses a limited repertoire of latent proteins to avoid host immune surveillance. When the lytic cycle is triggered by some as-yet-unknown form of stimulation, lytic gene expression and progeny virus production commence. Thus far, the exact mechanism of EBV latency maintenance and thein vivotriggering signal for lytic induction have yet to be elucidated. Previously, we have shown that the EBV microRNA miR-BART20-5p directly targets the immediate early genesBRLF1andBZLF1as well asBcl-2-associated death promoter (BAD) in EBV-associated gastric carcinoma. In this study, we found that both mRNA and protein levels ofBRLF1andBZLF1were suppressed in cells followingBADknockdown and increased afterBADoverexpression. Progeny virus production was also downregulated by specific knockdown ofBAD. Our results demonstrated thatcaspase-3-dependent apoptosis is a prerequisite forBAD-mediated EBV lytic cycle induction. Therefore, our data suggest that miR-BART20-5p plays an important role in latency maintenance and tumor persistence of EBV-associated gastric carcinoma by inhibitingBAD-mediatedcaspase-3-dependent apoptosis, which would trigger immediate early gene expression.IMPORTANCEEBV has an ability to remain latent in host cells, including EBV-associated tumor cells hiding from immune surveillance. However, the exact molecular mechanisms of EBV latency maintenance remain poorly understood. Here, we demonstrated that miR-BART20-5p inhibited the expression of EBV immediate early genes indirectly, by suppressingBAD-inducedcaspase-3-dependent apoptosis, in addition to directly, as we previously reported. Our study suggests that EBV-associated tumor cells might endure apoptotic stress to some extent and remain latent with the aid of miR-BART20-5p. Blocking the expression or function of BART20-5p may expedite EBV-associated tumor cell death via immune attack and apoptosis.

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Induction of Epstein-Barr Virus Latent Membrane Protein 1 by a Lytic Transactivator Rta

Journal of Virology ◽

10.1128/jvi.78.23.13028-13036.2004 ◽

2004 ◽

Vol 78 (23) ◽

pp. 13028-13036 ◽

Cited By ~ 30

Author(s):

Yao Chang ◽

Heng-Huan Lee ◽

Shih-Shin Chang ◽

Tsuey-Ying Hsu ◽

Pei-Wen Wang ◽

...

Keyword(s):

Membrane Protein ◽

Epstein Barr Virus ◽

Latent Membrane Protein ◽

Lytic Cycle ◽

Immediate Early ◽

Latent Membrane Protein 1 ◽

Virus Reactivation ◽

Barr Virus ◽

Lmp1 Expression ◽

Epstein Barr

ABSTRACT Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is a transforming protein that affects multiple cell signaling pathways and contributes to EBV-associated oncogenesis. LMP1 can be expressed in some states of EBV latency, and significant induction of full-length LMP1 is also observed frequently during virus reactivation into the lytic cycle. It is still unknown how LMP1 expression is regulated during the lytic stage and whether any EBV lytic protein is involved in the induction of LMP1. In this study, we first identified that LMP1 expression is associated with the spontaneous virus reactivation in EBV-infected 293 cells and that its expression is a downstream event of the lytic cycle. We further found that LMP1 can be induced by ectopic expression of Rta, an EBV immediate-early lytic protein. The Rta-mediated LMP1 induction is independent of another immediate-early protein, Zta. Northern blotting and reverse transcription-PCR analysis revealed that Rta upregulates LMP1 at the RNA level. Reporter gene assays further demonstrated that Rta activates both the proximal and distal promoters of the LMP1 gene in EBV-negative cells. Both the amino and carboxyl termini of the Rta protein are required for the induction of LMP1. In addition, Rta transactivates LMP1 not only in epithelial cells but also in B-lymphoid cells. This study reveals a new mechanism to upregulate LMP1 expression, expanding the knowledge of LMP1 regulation in the EBV life cycle. Considering an equivalent case of Kaposi's sarcoma-associated herpesvirus, induction of a transforming membrane protein by a key lytic transactivator during virus reactivation is likely to be a conserved event for gammaherpesviruses.

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Retrograde Regulation by the Viral Protein Kinase Epigenetically Sustains the Epstein-Barr Virus Latency-to-Lytic Switch To Augment Virus Production

Journal of Virology ◽

10.1128/jvi.00572-19 ◽

2019 ◽

Vol 93 (17) ◽

Cited By ~ 4

Author(s):

Xiaofan Li ◽

Sergei V. Kozlov ◽

Ayman El-Guindy ◽

Sumita Bhaduri-McIntosh

Keyword(s):

Gene Expression ◽

Protein Kinase ◽

Epstein Barr Virus ◽

Infectious Virus ◽

Virus Production ◽

Lytic Cycle ◽

Barr Virus ◽

Viral Protein Kinase ◽

Epstein Barr ◽

Productive Phase

ABSTRACTHerpesviruses are ubiquitous, and infection by some, like Epstein-Barr virus (EBV), is nearly universal. To persist, EBV must periodically switch from a latent to a replicative/lytic phase. This productive phase is responsible for most herpesvirus-associated diseases. EBV encodes a latency-to-lytic switch protein which, upon activation, sets off a vectorially constrained cascade of gene expression that results in production of infectious virus. While triggering expression of the switch protein ZEBRA is essential to lytic cycle entry, sustaining its expression is equally important to avoid premature termination of the lytic cascade. We report that the viral protein kinase (vPK), encoded by a gene that is kinetically downstream of the lytic switch, sustains expression of ZEBRA, amplifies the lytic cascade, increasing virus production, and, importantly, prevents the abortive lytic cycle. We find that vPK, through a noncanonical site phosphorylation, activates the cellular phosphatidylinositol 3-kinase-related kinase ATM to cause phosphorylation of the heterochromatin enforcer KAP1/TRIM28 even in the absence of EBV genomes or other EBV proteins. Phosphorylation of KAP1 renders it unable to restrain ZEBRA, thereby further derepressing and sustaining its expression to culminate in virus production. This partnership with a host kinase and a transcriptional corepressor enables retrograde regulation by vPK of ZEBRA, an observation that is counter to the unidirectional regulation of gene expression reminiscent of most DNA viruses.IMPORTANCEHerpesviruses infect nearly all humans and persist quiescently for the life of the host. These viruses intermittently activate into the lytic phase to produce infectious virus, thereby causing disease. To ensure that lytic activation is not prematurely terminated, expression of the virally encoded lytic switch protein needs to be sustained. In studying Epstein-Barr virus, one of the most prevalent human herpesviruses that also causes cancer, we have discovered that a viral kinase activated by the viral lytic switch protein partners with a cellular kinase to deactivate a silencer of the lytic switch protein, thereby providing a positive feedback loop to ensure successful completion of the viral productive phase. Our findings highlight key nodes of interaction between the host and virus that could be exploited to treat lytic phase-associated diseases by terminating the lytic phase or kill cancer cells harboring herpesviruses by accelerating the completion of the lytic cascade.

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Epstein-Barr virus inactivates the transcriptome and disrupts the chromatin architecture of its host cell in the first phase of lytic reactivation

10.1101/573659 ◽

2019 ◽

Cited By ~ 2

Author(s):

Alexander Buschle ◽

Paulina Mrozek-Gorska ◽

Stefan Krebs ◽

Helmut Blum ◽

Filippo M. Cernilogar ◽

...

Keyword(s):

Host Cell ◽

Epstein Barr Virus ◽

De Novo ◽

Human Tumor ◽

Regulatory Elements ◽

Lytic Cycle ◽

Host Cells ◽

Dependent Manner ◽

Barr Virus ◽

Epstein Barr

ABSTRACTEpstein-Barr virus (EBV), a herpes virus also termed HHV 4 and the first identified human tumor virus, establishes a stable long-term latent infection in human B cells, its preferred host. Upon induction of EBV’s lytic phase the latently infected cells turn into a virus factory, a process, that is governed by EBV. In the lytic, productive phase all herpesviruses ensure the efficient induction of all lytic viral genes to produce progeny, but certain of these genes also repress the ensuing antiviral responses of the virally infected host cells, regulate their apoptotic death or control the cellular transcriptome. We now find that EBV causes previously unknown massive and global alterations in the chromatin of its host cell upon induction of the viral lytic phase and prior to the onset of viral DNA replication. The viral initiator protein of the lytic cycle, BZLF1, binds to >105binding sites with different sequence motifs in cellular chromatin and in a concentration dependent manner. Concomitant with DNA binding, silent chromatin opens locally as shown by ATAC-seq experiments, while previously wide-open cellular chromatin becomes inaccessible on a global scale within hours. While viral transcripts increase drastically, the induction of the lytic phase results in a massive reduction of cellular transcripts and a loss of chromatin-chromatin interactions of cellular promoters with their distal regulatory elements as shown in Capture-C experiments. Our data document that EBV’s lytic cycle induces discrete early processes that disrupt the architecture of host cellular chromatin and repress the cellular epigenome and transcriptome likely supporting the efficientde novosynthesis of this herpesvirus.

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Epstein-Barr Virus Transforming Protein LMP1 Plays a Critical Role in Virus Production

Journal of Virology ◽

10.1128/jvi.79.7.4415-4424.2005 ◽

2005 ◽

Vol 79 (7) ◽

pp. 4415-4424 ◽

Cited By ~ 31

Author(s):

Nazmul Ahsan ◽

Teru Kanda ◽

Kazuo Nagashima ◽

Kenzo Takada

Keyword(s):

Epstein Barr Virus ◽

Critical Role ◽

Biological Significance ◽

Virus Production ◽

Lytic Replication ◽

Lytic Cycle ◽

Virus Release ◽

Entire Genome ◽

Barr Virus ◽

Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1), which is critical for EBV-induced B-cell transformation, is also abundantly expressed during the lytic cycle of viral replication. However, the biological significance of this strong LMP1 induction remains unknown. We engineered a bacterial artificial chromosome clone containing the entire genome of Akata strain EBV to specifically disrupt the LMP1 gene. Akata cell clones harboring the episomes of LMP1-deleted EBV were established, and the effect of LMP1 loss on virus production was investigated. We found that the degree of viral DNA amplification and the expression levels of viral late gene products were unaffected by LMP1 loss, demonstrating that the LMP1-deleted EBV entered the lytic replication cycle as efficiently as the wild-type counterpart. This was confirmed by our electron microscopic observation that nucleocapsid formation inside nuclei occurred even in the absence of LMP1. By contrast, loss of LMP1 severely impaired virus release into culture supernatants, resulting in poor infection efficiency. The expression of truncated LMP1 in Akata cells harboring LMP1-deleted EBV rescued the virus release into the culture supernatant and the infectivity, and full-length LMP1 partially rescued the infectivity. These results indicate that inducible expression of LMP1 during the viral lytic cycle plays a critical role in virus production.

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Protein Kinase C-Independent Activation of the Epstein-Barr Virus Lytic Cycle

Journal of Virology ◽

10.1128/jvi.76.11.5612-5626.2002 ◽

2002 ◽

Vol 76 (11) ◽

pp. 5612-5626 ◽

Cited By ~ 62

Author(s):

Lyndle Gradoville ◽

David Kwa ◽

Ayman El-Guindy ◽

George Miller

Keyword(s):

Phorbol Ester ◽

Epstein Barr Virus ◽

Hdac Inhibitors ◽

Lytic Cycle ◽

Early Gene ◽

Immediate Early ◽

Barr Virus ◽

Kinase C ◽

Epstein Barr

ABSTRACT The protein kinase C (PKC) pathway has been considered to be essential for activation of latent Epstein-Barr virus (EBV) into the lytic cycle. The phorbol ester tetradecanoyl phorbol acetate (TPA), a PKC agonist, is one of the best understood activators of EBV lytic replication. Zp, the promoter of the EBV immediate-early gene BZLF1, whose product, ZEBRA, drives the lytic cycle, contains several phorbol ester response elements. We investigated the role of the PKC pathway in lytic cycle activation in prototype cell lines that differed dramatically in their response to inducing agents. We determined whether PKC was involved in lytic cycle induction by histone deacetylase (HDAC) inhibitors. Consistent with prevailing views, B95-8 cells were activated into the lytic cycle by the phorbol ester TPA, via a PKC-dependent mechanism. B95-8 was not inducible by HDAC inhibitors such as n-butyrate and trichostatin A (TSA). Bisindolylmaleimide I, a selective PKC inhibitor, blocked lytic cycle activation in B95-8 cells in response to TPA. In marked contrast, in HH514-16 cells, the immediate-early promoters Zp and Rp were simultaneously activated by the HDAC inhibitors; TPA by itself failed to activate lytic gene expression. Inhibition of PKC activity by bisindolylmaleimide I did not block lytic cycle activation in HH514-16 cells by n-butyrate or TSA. In an extensive exploration of the mechanism underlying these different responses we found that the variable role of the PKC pathway in the two cell lines could not be accounted for by significant polymorphisms in the promoters of the immediate-early genes, by differences in the start sites of immediate-early gene transcription, or by differences in the nucleosomal organization of EBV DNA in the region of Zp or Rp. While B95-8 cells contained more total PKC activity than did HH514-16 cells in an in vitro assay, another EBV-transformed marmoset lymphoblastoid cell line, FF41, in which the lytic cycle was not inducible by TPA, contained comparably high levels of PKC activity. Moreover, two marmoset lymphoblastoid cells lines in which the lytic cycle could not be triggered by TPA maintained the same profile of EBV latency proteins as B95-8 cells. Thus, the profile of EBV latency proteins did not account for susceptibility to induction by PKC agonists. PKC activation is neither obligatory nor sufficient for the switch between latency and lytic cycle gene expression of EBV in many cell backgrounds. Lytic cycle induction by HDAC inhibitors proceeds by a PKC-independent mechanism.

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Negative Autoregulation of Epstein-Barr Virus (EBV) Replicative Gene Expression by EBV SM Protein

Journal of Virology ◽

10.1128/jvi.00382-09 ◽

2009 ◽

Vol 83 (16) ◽

pp. 8041-8050 ◽

Cited By ~ 11

Author(s):

Dinesh Verma ◽

Chen Ling ◽

Eric Johannsen ◽

Tirumuru Nagaraja ◽

Sankar Swaminathan

Keyword(s):

Epstein Barr Virus ◽

Feedback Regulation ◽

Lytic Replication ◽

Lytic Cycle ◽

Immediate Early ◽

Mrna Levels ◽

Barr Virus ◽

Infected Cells ◽

Epstein Barr ◽

Sm Protein

ABSTRACT The Epstein-Barr virus (EBV) SM protein is essential for lytic EBV DNA replication and virion production. When EBV replication is induced in cells infected with an SM-deleted recombinant EBV, approximately 50% of EBV genes are expressed inefficiently. When EBV replication is rescued by transfection of SM, SM enhances expression of these genes by direct and indirect mechanisms. While expression of most EBV genes is either unaffected or enhanced by SM, expression of several genes is decreased in the presence of SM. Expression of BHRF1, a homolog of cellular bcl-2, is particularly decreased in the presence of SM. Investigation of the mechanism of BHRF1 downregulation revealed that SM downregulates expression of the immediate-early EBV transactivator R. In EBV-infected cells, R-responsive promoters, including the BHRF1 and SM promoters, were less active in the presence of SM, consistent with SM inhibition of R expression. SM decreased spliced R mRNA levels, supporting a posttranscriptional mechanism of R inhibition. R and BHRF1 expression were also found to decrease during later stages of EBV lytic replication in EBV-infected lymphoma cells. These data indicate that feedback regulation of immediate-early and early genes occurs during the lytic cycle of EBV regulation.

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Valpromide Inhibits Lytic Cycle Reactivation of Epstein-Barr Virus

mBio ◽

10.1128/mbio.00113-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 7

Author(s):

Kelly L. Gorres ◽

Derek Daigle ◽

Sudharshan Mohanram ◽

Grace E. McInerney ◽

Danielle E. Lyons ◽

...

Keyword(s):

Gene Expression ◽

Valproic Acid ◽

Hdac Inhibitor ◽

Epstein Barr Virus ◽

Lytic Cycle ◽

Immediate Early ◽

Barr Virus ◽

Early Genes ◽

Lytic Reactivation ◽

Epstein Barr

ABSTRACTReactivation of Epstein-Barr virus (EBV) from latency into the lytic phase of its life cycle allows the virus to spread among cells and between hosts. Valproic acid (VPA) inhibits initiation of the lytic cycle in EBV-infected B lymphoma cells. While VPA blocks viral lytic gene expression, it induces expression of many cellular genes, because it is a histone deacetylase (HDAC) inhibitor. Here we show, using derivatives of VPA, that blockade of EBV reactivation is separable from HDAC inhibition. Valpromide (VPM), an amide derivative of valproic acid that is not an HDAC inhibitor, prevented expression of two EBV genes, BZLF1 and BRLF1, that mediate lytic reactivation. VPM also inhibited expression of a viral late gene, but not early genes, when BZLF1 was exogenously expressed. Unlike VPA, VPM did not activate lytic expression of Kaposi’s sarcoma-associated herpesvirus. Expression of cellular immediate-early genes, such as FOS and EGR1, is kinetically upstream of the EBV lytic cycle. VPM did not activate expression of these cellular immediate-early genes but decreased their level of expression when induced by butyrate, an HDAC inhibitor. VPM did not alter expression of several other cellular immediate-early genes, including STAT3, which were induced by the HDAC inhibitors in cells refractory to lytic induction. Therefore, VPM selectively inhibits both viral and cellular gene expression. VPA and VPM represent a new class of antiviral agents. The mechanism by which VPA and VPM block EBV reactivation may be related to their anticonvulsant activity.IMPORTANCEEpstein-Barr virus, (EBV), a human tumor virus, establishes a life-long latent infection. Reactivation of EBV into the lytic phase of its life cycle allows the virus to spread. Previously, we showed that EBV reactivation was blocked by valproic acid (VPA), an inhibitor of cellular histone deacetylases (HDACs). VPA alters the expression of thousands of cellular genes. In this study, we demonstrate that valpromide (VPM), an amide derivative of valproic acid that is not an HDAC inhibitor, prevented initiation of the EBV lytic cycle. VPA induced lytic reactivation of Kaposi’s sarcoma-associated herpesvirus (KSHV), but VPM did not. Unlike VPA, VPM did not activate cellular immediate-early gene expression. VPM is a new type of antiviral agent. VPM will be useful in probing the mechanism of EBV lytic reactivation and may have therapeutic application.

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The Epstein-Barr Virus Rta Protein Activates Lytic Cycle Genes and Can Disrupt Latency in B Lymphocytes

Journal of Virology ◽

10.1128/jvi.72.10.7978-7984.1998 ◽

1998 ◽

Vol 72 (10) ◽

pp. 7978-7984 ◽

Cited By ~ 164

Author(s):

Tobias Ragoczy ◽

Lee Heston ◽

George Miller

Keyword(s):

Epithelial Cells ◽

B Lymphocytes ◽

Epstein Barr Virus ◽

Lytic Cycle ◽

Immediate Early ◽

Late Gene Expression ◽

Barr Virus ◽

Viral Genes ◽

Specific Manner ◽

Epstein Barr

ABSTRACT The transition of Epstein-Barr virus (EBV) from latency into the lytic cycle is associated with the expression of two immediate-early viral genes, BZLF1 and BRLF1. Overexpression of ZEBRA, the product of BZLF1, is sufficient to disrupt latency in B lymphocytes and epithelial cells by stimulating expression of lytic cycle genes, including BRLF1. The BRLF1 product Rta functions as a transcriptional activator in both B lymphocytes and epithelial cells. However, Rta has recently been reported to disrupt latency in an epithelial specific manner (S. Zalani, E. Holley-Guthrie, and S. Kenney, Proc. Natl. Acad. Sci. USA 93:9194–9199, 1996). Here we demonstrate that expression of Rta is also sufficient for disruption of latency in a permissive B-cell line. In HH514-16 cells, transfection of Rta leads to synthesis of ZEBRA, viral DNA replication, and late gene expression. However, Rta by itself is less potent than ZEBRA in the ability to activate most early and late lytic cycle genes. In light of previous work implicating ZEBRA in the activation of Rta, we suggest a cooperative model for EBV entry into the lytic cycle. Expression of either BZLF1 or BRLF1 triggers expression of the other immediate-early factor, and together these activators act individually or in synergy on downstream targets to activate the viral lytic cycle.

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Epstein-Barr virus lytic cycle activation alters proteasome subunit expression in Burkitt's lymphoma cells

Biological Chemistry ◽

10.1515/bc.2010.107 ◽

2010 ◽

Vol 391 (9) ◽

Cited By ~ 4

Author(s):

Alessandra De Leo ◽

Giulia Matusali ◽

Giuseppe Arena ◽

Livia Di Renzo ◽

Elena Mattia

Keyword(s):

Epstein Barr Virus ◽

Burkitt’S Lymphoma ◽

Lytic Cycle ◽

Host Cells ◽

Burkitt's Lymphoma ◽

Lymphoma Cells ◽

Proteasome Subunit ◽

Barr Virus ◽

Subunit Expression ◽

Epstein Barr

AbstractWe have shown that Epstein-Barr virus (EBV) lytic cycle activation in Burkitt's lymphoma (BL) cells down-regulates chymotrypsin- and caspase-like activities of the proteasome. The aim of the present study was to evaluate whether EBV activation might also affect proteasome subunit composition. Our results indicate that, independently of the latency program established in the host cells, induction of the EBV lytic cycle reduces the expression of the proteasomal components β5, β1 and β2i, whereas it increases that of β2, β1i, PA28α and PA28β. The modulation of the composition and enzymatic activities of the proteolytic complex are indicative of a less efficient generation of viral immunoepitopes.

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The Epstein-Barr Virus Immediate-Early Protein BZLF1 Induces both a G2 and a Mitotic Block

Journal of Virology ◽

10.1128/jvi.76.19.10030-10037.2002 ◽

2002 ◽

Vol 76 (19) ◽

pp. 10030-10037 ◽

Cited By ~ 32

Author(s):

Amy Mauser ◽

Elizabeth Holley-Guthrie ◽

Dennis Simpson ◽

William Kaufmann ◽

Shannon Kenney

Keyword(s):

Epstein Barr Virus ◽

Cyclin B1 ◽

Lytic Infection ◽

Host Cells ◽

Immediate Early ◽

Barr Virus ◽

Early Protein ◽

Mitotic Block ◽

Epstein Barr ◽

Mitotic Defect

ABSTRACT The Epstein-Barr virus immediate-early protein BZLF1 is a transcriptional activator that mediates the switch from latent to lytic infection. Here we demonstrate that BZLF1 induces both a G2 block and a mitotic block in HeLa cells and inhibits chromosome condensation. While the G2 block is associated with decreased cyclin B1 in host cells and can be rescued by overexpression of cyclin B1, the mechanism for the mitotic defect is as yet undetermined.

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