Activation of the ERK signal transduction pathway by Epstein–Barr virus immediate-early protein Rta

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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.

Section: Resultsmentioning

confidence: 99%

mentioning

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

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“…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).…”

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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...

“…BZLF1 activates p38 and JNK to turn on the ATF2 transcription factor (22). BRLF1, another EBV immediate early protein, activates the AP-1 pathway by increasing the levels of phosphorylated p38, JNK, and ERK (22,23) and induces phosphorylation of Akt through the PI3K pathway (24). Activation of Akt, ERK, and p38 signaling is required for EBV reactivation from latency (25)(26)(27)(28).…”

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confidence: 99%

Epstein-Barr Virus (EBV) Tegument Protein BGLF2 Promotes EBV Reactivation through Activation of the p38 Mitogen-Activated Protein Kinase

Liu

Cohen

2016

IMPORTANCE Epstein-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. E pstein-Barr virus (EBV) is a cause of infectious mononucleosis and is associated with both B lymphocyte and epithelial cell malignancies. EBV encodes a number of proteins that trigger cell signaling pathways, such as AP-1, JAK-STAT, NF-B, and phosphatidylinositol 3-kinase (PI3K)/Akt, which are critical for cell survival, virus latency, and growth transformation (1-6). For example, EBV latent membrane protein 1 (LMP1) mimics CD40 signaling and is important for EBV-induced B cell growth proliferation, inhibition of apoptosis, and EBV transformation. LMP1 interacts with tumor necrosis factor receptor-associated factors (TRAFs), leading to activation of NF-B, c-Jun N-terminal kinase (JNK), p38 mitogen-activated protein kinase (MAPK), PI3K/Akt, and STAT3 (7-12). EBV LMP2A mimics B cell receptor signaling and contributes to the long-term survival of B cells (13-16). LMP2A activates extracellular signal-related protein kinase (ERK), PI3K/Akt, and JNK (17) and downregulates NF-B and STAT signaling pathways (18). The EBV immediate early protein BZLF1 activates virus reactivation from latency (19-21). BZLF1 activates p38 and JNK to turn on the ATF2 transcription factor (22). BRLF1, another EBV immediate early protein, activates the AP-1 pathway by increasing the levels of phosphorylated p38, JNK, and ERK (22, 23) and induces phosphorylation of Akt through the PI3K pathway (24). Activation of Akt, ERK, and p38 signaling is required for EBV reactivation from latency (25-28).To identify additional EBV proteins important for regulating cell signaling, we used a proteomic approach to screen viral proteins for AP-1 promoter activity in AP-1-luciferase reporter assays. We found that the EBV tegument protein BGLF2 induced AP-1 reporter activity and activated p38 and JNK. BGLF2 promoted EBV reactivation by enhancing BZLF1 expression and EBV production, and p38 activation by BGLF2 was required for these activities. Citation Liu X, Cohen JI. 2016. Epstein-Barr virus (EBV) tegument protein BGLF2 promotes EBV reactivation through activation of the p38 mitogen-activated protein kinase.