The Epstein-Barr virus replication and transcription activator, Rta/BRLF1, induces cellular senescence in epithelial cells

Autophagy is an intracellular degradation pathway that provides a host defense mechanism against intracellular pathogens. However, many viruses exploit this mechanism to promote their replication. This study shows that lytic induction of EpsteinBarr virus (EBV) increases the membrane-bound form of LC3 (LC3-II) and LC3-containing punctate structures in EBV-positive cells. Transfecting 293T cells with a plasmid that expresses Rta also induces autophagy, revealing that Rta is responsible for autophagic activation. The activation involves Atg5, a key component of autophagy, but not the mTOR pathway. The expression of Rta also activates the transcription of the genes that participate in the formation of autophagosomes, including LC3A, LC3B, and ATG9B genes, as well as those that are involved in the regulation of autophagy, including the genes TNF, IRGM, and TRAIL. Additionally, treatment with U0126 inhibits the Rta-induced autophagy and the expression of autophagy genes, indicating that the autophagic activation is caused by the activation of extracellular signal-regulated kinase (ERK) signaling by Rta. Finally, the inhibition of autophagic activity by an autophagy inhibitor, 3-methyladenine, or Atg5 small interfering RNA, reduces the expression of EBV lytic proteins and the production of viral particles, revealing that autophagy is critical to EBV lytic progression. This investigation reveals how an EBV-encoded transcription factor promotes autophagy to affect viral lytic development. IMPORTANCEAutophagy is a cellular process that degrades and recycles nutrients under stress conditions to promote cell survival. Although autophagy commonly serves as a defense mechanism against viral infection, many viruses exploit this mechanism to promote their replication. This study finds that a transcription factor that is encoded by Epstein-Barr virus (EBV), Rta, activates autophagy, and the inhibition of autophagy reduces the ability of the virus to express viral lytic proteins and to generate progeny. Unlike other virus-encoded proteins that modulate autophagy by interacting with proteins that are involved in the autophagic pathway, Rta activates the transcription of the autophagy-related genes via the ERK pathway. The results of this study reveal how the virus manipulates autophagy to promote its lytic development.

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confidence: 99%

Regulation of Autophagic Activation by Rta of Epstein-Barr Virus via the Extracellular Signal-Regulated Kinase Pathway

Hung

Chen

Wang

et al. 2014

“…These two proteins are activators of viral and cellular gene expression, play essential roles in lytic viral DNA replication, and impinge on many host cell processes, such as signal transduction, cell cycle control, and cellular senescence (10,27,30,31). The bzlf1 and brlf1 genes are not expressed during latency, but all external stimuli known to activate the EBV lytic cascade induce their expression (3,16,28,39,48).…”

mentioning

confidence: 99%

Cellular Immediate-Early Gene Expression Occurs Kinetically Upstream of Epstein-Barr Virus bzlf1 and brlf1 following Cross-Linking of the B Cell Antigen Receptor in the Akata Burkitt Lymphoma Cell Line

Gradoville²,

Miller

2010

The Epstein-Barr virus (EBV) lytic activator genes bzlf1 and brlf1 are conventionally referred to as immediate-early (IE) genes. However, previous studies showed that the earliest expression of these genes was blocked by cycloheximide when the EBV lytic cycle was induced by histone deacetylase (HDAC) inhibitors and protein kinase C agonists. Anti-IgG activates a complex signal transduction pathway that leads to EBV lytic activation in the Akata cell line. Here we demonstrate that in Akata cells, where lytic cycle activation occurs very rapidly after anti-IgG treatment, de novo protein synthesis is also required for induction of bzlf1 and brlf1 expression. New protein synthesis is required up to 1.25 h after application of anti-IgG; bzlf1 and brlf1 mRNAs can be detected 1.5 h after anti-IgG. Five cellular IE genes were shown to be expressed by 1 h after addition of anti-IgG, and their expression preceded that of bzlf1 and brlf1. These include early growth response genes (egr1, egr2, and egr3) and nuclear orphan receptors (nr4a1 and nr4a3). These genes were activated by anti-IgG treatment of Akata cells with and without the EBV genome; therefore, their expression was not dependent on expression of any EBV gene product. EGR1, EGR2, and EGR3 proteins were kinetically upstream of ZEBRA and Rta proteins. Expression of EGR1, ZEBRA, and Rta proteins were inhibited by bisindolylmaleimide X, a selective inhibitor of PKC. The findings suggest a revised model in which the signal transduction cascade activated by cross-linking of the B cell receptor induces expression of cellular IE genes, such as early growth response and nuclear orphan receptor genes, whose products, in turn, regulate bzlf1 and brlf1 expression.Two Epstein-Barr virus (EBV) genes, bzlf1 and brlf1, encode protein products, ZEBRA and Rta, that control the transition from viral latency to lytic replication (12,13,23,34,46). These two proteins are activators of viral and cellular gene expression, play essential roles in lytic viral DNA replication, and impinge on many host cell processes, such as signal transduction, cell cycle control, and cellular senescence (10,27,30,31). The bzlf1 and brlf1 genes are not expressed during latency, but all external stimuli known to activate the EBV lytic cascade induce their expression (3,16,28,39,48). Therefore, a central question is, "What controls the expression of these two EBV lytic activator genes?" We refer to this question as the "upstream problem" in lytic activation (29,44).A full understanding of the upstream events has been elusive for several reasons. Many mechanically heterogeneous stimuli activate the lytic cascade in cultured lymphoid cells, where the molecular events can be readily analyzed. Each cell line seems to respond to the inducing stimuli in an idiosyncratic fashion (3,16,28,39,48). The lytic cycle-inducing agents vary in the duration of exposure required to elicit a response. Moreover, the cells vary in their response time (11). It is not yet known whether the many pathways engaged by the diverse sti...

“…Latent LMP1 attenuates senescence during latent EBV infection (30,31), whereas during lytic EBV infection, BRLF1 induces intrinsic senescence in epithelial cells (32). Here, we reveal that latent EBV infection transmits inflammatory paracrine senescence to neighboring epithelial cells through TNF-␣ and elevated oxygen free radicals, whereas lytic EBV infection attenuates transmission via BZLF1-mediated reductions in both TNF-␣ secretion and consequent oxidative stress.…”

Section: Discussionmentioning

confidence: 74%

“…However, the EBV lytic life cycle suppresses and escapes from the inflammatory response and paracrine senescence to facilitate viral propagation. In addition to replicative stress and oncogene-induced senescence experienced during the EBV lytic cycle, BZLF1 and BRLF1 also induce the accumulation of p21 CIP1 and G 0 /G 1 arrest (32,33,52), which are components of the premature state of cellular senescence. These findings indicate that lytic EBV-replicating cells generate intrinsic senescent stress but prohibit transmitting it to neighboring cells via the inhibition of inflammatory factors by BZLF1.…”

Section: Discussionmentioning

confidence: 99%

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BZLF1 Attenuates Transmission of Inflammatory Paracrine Senescence in Epstein-Barr Virus-Infected Cells by Downregulating Tumor Necrosis Factor Alpha

Long

Yang

et al. 2016

Recent studies have shown that inflammatory responses trigger and transmit senescence to neighboring cells and activate the senescence-associated secretory phenotype (SASP). Latent Epstein-Barr virus (EBV) infection induces increasedC ellular senescence, an irreversible arrest of the cell cycle with major hallmarks of senescence-associated heterochromatic foci and DNA segments, is induced by genotoxic or oncogenic stress (1, 2). Oncogene-induced senescence (OIS) is triggered by excessive expression of oncogenes or oncogene-induced replicative stress and acts as an efficient barrier against malignancy (3, 4). However, tumors develop ways to evade OIS during early tumorigenesis (5). Interestingly, senescent cells also secrete proinflammatory factors that are important for tumor progression; this phenotype is called the senescence-associated secretory phenotype (SASP) (6). Recent studies have shown that inflammatory responses trigger and transmit cellular senescence to neighboring cells (7-9), indicating that profound cross talk and signal integration occur between senescent cells and the inflammatory microenvironment and that this communication may promote either tumor progression or suppression.Herpesviruses produce few transcripts during latent infection. In contrast, during lytic infection, transcripts of the entire herpesvirus genome are produced and cellular machinery and multiple signaling pathways are exploited to facilitate replication and spread (10-12). Host defenses against viral infection include the activation of innate immune and inflammatory responses; however, herpesviruses employ multiple strategies and multiple viral products to evade host defenses (13)(14)(15)(16). In addition to being involved in antiviral defenses during acute infection, inflammatory factors are also involved in the progression of persistent infection, cancers, and other inflammatory disorders (10,(17)(18)(19).Studies have identified several inflammatory factors involved in infectious diseases caused by Epstein-Barr virus (EBV) infection that are mediated by both lytic and latent viral gene products (20)(21)(22)(23)(24)(25). Levels of these inflammatory factors are elevated during EBV infection, and they elicit chronic inflammation, which leads