Epstein-Barr Virus Lytic Replication Research Articles

A new compound, phomaherbarine A, induces cytolytic reactivation in epstein-barr virus-positive B cell lines

Epstein-Barr virus (EBV), the first virus found to induce cancer in humans, has been frequently detected in various types of B cell lymphomas. During its latent phase, EBV expresses a limited set of proteins crucial for its persistence. Induction of the lytic phase of EBV has shown promise in the treatment of EBV-associated malignancies. The present study assessed the ability of phomaherbarine A, a novel compound derived from the endophytic fungus Phoma herbarum DBE-M1, to stimulate lytic replication of EBV in B95-8 cells. Phomaherbarine A was found to efficiently initiate the expression of both early and late EBV lytic genes in B95-8 cells, with this initiation being further heightened by the addition of phorbol myristate acetate and sodium butyrate. Moreover, phomaherbarine A demonstrated notable cytotoxicity against the EBV-associated B cell lymphoma cell lines B95-8 and Raji. Mechanistically, phomaherbarine A induces apoptosis in these cells through the activation of caspase-3/7. When combined with ganciclovir, phomaherbarine A does not interfere with the reduction of viral replication by ganciclovir and sustains its apoptosis induction. In conclusion, these findings indicate that phomaherbarine A may be a promising candidate for therapeutic intervention in patients with EBV-associated B cell lymphomas.

Ceramide promotes lytic reactivation of Epstein-Barr virus in gastric carcinoma.

Epstein-Barr virus (EBV) has a lifelong latency period after initial infection. Rarely, however, when the EBV immediate early gene BZLF1 is expressed by a specific stimulus, the virus switches to the lytic cycle to produce progeny viruses. We found that EBV infection reduced levels of various ceramide species in gastric cancer cells. As ceramide is a bioactive lipid implicated in the infection of various viruses, we assessed the effect of ceramide on the EBV lytic cycle. Treatment with C6-ceramide (C6-Cer) induced an increase in the endogenous ceramide pool and increased production of the viral product as well as BZLF1 expression. Treatment with the ceramidase inhibitor ceranib-2 induced EBV lytic replication with an increase in the endogenous ceramide pool. The glucosylceramide synthase inhibitor Genz-123346 inhibited C6-Cer-induced lytic replication. C6-Cer induced extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB phosphorylation, c-JUN expression, and accumulation of the autophagosome marker LC3B. Treatment with MEK1/2 inhibitor U0126, siERK1&2, or siCREB suppressed C6-Cer-induced EBV lytic replication and autophagy initiation. In contrast, siJUN transfection had no impact on BZLF1 expression. The use of 3-methyladenine (3-MA), an inhibitor targeting class III phosphoinositide 3-kinases (PI3Ks) to inhibit autophagy initiation, resulted in reduced beclin-1 expression, along with suppressed C6-Cer-induced BZLF1 expression and LC3B accumulation. Chloroquine, an inhibitor of autophagosome-lysosome fusion, increased BZLF1 protein intensity and LC3B accumulation. However, siLC3B transfection had minimal effect on BZLF1 expression. The results suggest the significance of ceramide-related sphingolipid metabolism in controlling EBV latency, highlighting the potential use of drugs targeting sphingolipid metabolism for treating EBV-positive gastric cancer.IMPORTANCEEpstein-Barr virus remains dormant in the host cell but occasionally switches to the lytic cycle when stimulated. However, the exact molecular mechanism of this lytic induction is not well understood. In this study, we demonstrate that Epstein-Barr virus infection leads to a reduction in ceramide levels. Additionally, the restoration of ceramide levels triggers lytic replication of Epstein-Barr virus with increase in phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) and CREB. Our study suggests that the Epstein-Barr virus can inhibit lytic replication and remain latent through reduction of host cell ceramide levels. This study reports the regulation of lytic replication by ceramide in Epstein-Barr virus-positive gastric cancer.

Viral and host factors drive a type 1 Epstein-Barr virus spontaneous lytic phenotype.

Epstein-Barr virus (EBV) infects over 95% of adults worldwide. Given its connection to various cancers and autoimmune disorders, it is important to understand the mechanisms by which infection with EBV can lead to these diseases. In this study, we describe an unusual spontaneous lytic phenotype in EBV strains isolated from Kenyan endemic Burkitt lymphoma patients. Because lytic replication of EBV has been linked to the pathogenesis of various diseases, these data could illuminate viral and host factors involved in this process.

T cell-mediated immunity during Epstein-Barr virus infections in children.

Epstein-Barr virus (EBV) infection is extremely common worldwide, with approximately 90% of adults testing positive for EBV antibodies. Human are susceptible to EBV infection, and primary EBV infection typically occurs early in life. EBV infection can cause infectious mononucleosis (IM) as well as some severe non-neoplastic diseases, such as chronic active EBV infection (CAEBV) and EBV-associated hemophagocytic lymphohistiocytosis (EBV-HLH), which can have a heavy disease burden. After primary EBV infection, individuals develop robust EBV-specific T cell immune responses, with EBV-specific CD8+ and part of CD4+ T cells functioning as cytotoxic T cells, defending against virus. Different proteins expressed during EBV's lytic replication and latent proliferation can cause varying degrees of cellular immune responses. Strong T cell immunity plays a key role in controlling infection by decreasing viral load and eliminating infected cells. However, the virus persists as latent infection in EBV healthy carriers even with robust T cell immune response. When reactivated, it undergoes lytic replication and then transmits virions to a new host. Currently, the relationship between the pathogenesis of lymphoproliferative diseases and the adaptive immune system is still not fully clarified and needs to be explored in the future. Investigating the T cell immune responses evoked by EBV and utilizing this knowledge to design promising prophylactic vaccines are urgent issues for future research due to the importance of T cell immunity.

Effect of Malaria Infection on Epstein-Barr Virus Persistence in Kenyan Children.

The 2 cofactors in the etiology of Burkitt lymphoma (BL) are Epstein-Barr virus (EBV) and repeated Plasmodium falciparum malaria infections. This study evaluated EBV loads in mucosal and systemic compartments of children with malaria and controls. Age was analyzed as a covariate because immunity to malaria in endemic regions is age dependent. Children (2-10 years) with clinical malaria from Western Kenya and community controls without malaria were enrolled. Saliva and blood samples were collected, EBV viral load was assessed by quantitative polymerase chain reaction, and EpiTYPER MassARRAY was used to assess methylation of 3 different EBV genes. Regardless of the compartment, we detected EBV more frequently in malaria cases compared to controls, although the difference was not significant. When EBV was detected, there were no differences in viral load between cases and controls. However, EBV methylation was significantly lower in the malaria group compared to controls in both plasma and saliva (P < .05), indicating increased EBV lytic replication. In younger children before development of immunity to malaria, there was a significant effect of malaria on EBV load in peripheral blood mononuclear cells (P = .04). These data suggest that malaria can directly modulate EBV persistence in children, increasing their risk for BL.

Role of Oxidative Stress in the Epstein–Barr Virus Lifecycle and Tumorigenicity

The Epstein–Barr virus (EBV) is an oncogenic virus with both latent and lytic states during its lifecycle. EBV employs a latency period as a strategy to avoid host immune surveillance and achieve lifelong persistent infection. However, the latent state may be interrupted and EBV may reactivate into a lytic replication cycle, exacerbating transmission and pathogenicity. The balance and transition between these two phases in the EBV lifecycle are complex, and reactive oxygen species play an important role. We reviewed the relationship between oxidative stress and lytic replication of EBV, and the role of oxidative stress in the development of EBV-related tumors. Further research is required to explore and develop tumor antioxidant therapy.

Retinoblastoma Protein Is Required for Epstein-Barr Virus Replication in Differentiated Epithelia.

Coinfection of human papillomavirus (HPV) and Epstein-Barr virus (EBV) has been detected in oropharyngeal squamous cell carcinoma. Although HPV and EBV replicate in differentiated epithelial cells, we previously reported that HPV epithelial immortalization reduces EBV replication within organotypic raft culture and that the HPV16 oncoprotein E7 was sufficient to inhibit EBV replication. A well-established function of HPV E7 is the degradation of the retinoblastoma (Rb) family of pocket proteins (pRb, p107, and p130). Here, we show that pRb knockdown in differentiated epithelia and EBV-positive Burkitt lymphoma (BL) reduces EBV lytic replication following de novo infection and reactivation, respectively. In differentiated epithelia, EBV immediate early (IE) transactivators were expressed, but loss of pRb blocked expression of the early gene product, EA-D. Although no alterations were observed in markers of epithelial differentiation, DNA damage, and p16, increased markers of S-phase progression and altered p107 and p130 levels were observed in suprabasal keratinocytes after pRb knockdown. In contrast, pRb interference in Akata BX1 Burkitt lymphoma cells showed a distinct phenotype from differentiated epithelia with no significant effect on EBV IE or EA-D expression. Instead, pRb knockdown reduced the levels of the plasmablast differentiation marker PRDM1/Blimp1 and increased the abundance of c-Myc protein in reactivated Akata BL with pRb knockdown. c-Myc RNA levels also increased following the loss of pRb in epithelial rafts. These results suggest that pRb is required to suppress c-Myc for efficient EBV replication in BL cells and identifies a mechanism for how HPV immortalization, through degradation of the retinoblastoma pocket proteins, interferes with EBV replication in coinfected epithelia. IMPORTANCE Terminally differentiated epithelium is known to support EBV genome amplification and virion morphogenesis following infection. The contribution of the cell cycle in differentiated tissues to efficient EBV replication is not understood. Using organotypic epithelial raft cultures and genetic interference, we can identify factors required for EBV replication in quiescent cells. Here, we phenocopied HPV16 E7 inhibition of EBV replication through knockdown of pRb. Loss of pRb was found to reduce EBV early gene expression and viral replication. Interruption of the viral life cycle was accompanied by increased S-phase gene expression in postmitotic keratinocytes, a process also observed in E7-positive epithelia, and deregulation of other pocket proteins. Together, these findings provide evidence of a global requirement for pRb in EBV lytic replication and provide a mechanistic framework for how HPV E7 may facilitate a latent EBV infection through its mediated degradation of pRb in copositive epithelia.

Rare POLN mutations confer risk for familial nasopharyngeal carcinoma through weakened Epstein-Barr virus lytic replication.

SummaryBackgroundNasopharyngeal carcinoma (NPC) exhibits significant familial aggregation; however, its susceptibility genes are largely unknown. Thus, this study aimed to identify germline mutations that might contribute to the risk of familial NPC, and explore their biological functions.MethodsWhole-exome sequencing was performed in 13 NPC pedigrees with multiple cases. Mutations co-segregated with disease status were further validated in a cohort composed of 563 probands from independent families, 2,953 sporadic cases, and 3,175 healthy controls. Experimental studies were used to explore the functions of susceptibility genes and their disease-related mutations.FindingsThe three rare missense mutations in POLN (DNA polymerase nu) gene, P577L, R303Q, and F545C, were associated with familial NPC risk (5/576 [0·87%] in cases vs. 2/3374 [0·059%] in healthy controls with an adjusted OR of 44·84 [95% CI:3·91-514·34, p = 2·25 × 10−3]). POLN was involved in Epstein-Barr virus (EBV) lytic replication in NPC cells in vitro. POLN promoted viral DNA replication, immediate-early and late lytic gene expression, and progeny viral particle production, ultimately affecting the proliferation of host cells. The three mutations were located in two pivotal functional domains and were predicted to alter the protein stability of POLN in silico. Further assays demonstrated that POLN carrying any of the three mutations displayed reduced protein stability and decreased expression levels, thereby impairing its ability to promote complete EBV lytic replication and facilitate cell survival.InterpretationWe identified a susceptibility gene POLN for familial NPC and elucidated its function.FundingThis study was funded by the National Key Research and Development Program of China (2021YFC2500400); the National Key Research and Development Program of China (2020YFC1316902); the Basic and Applied Basic Research Foundation of Guangdong Province, China (2021B1515420007); the National Natural Science Foundation of China (81973131); the National Natural Science Foundation of China (82003520); the National Natural Science Foundation of China (81903395).

Cardiac glycosides from the roots of Streblus asper Lour. with activity against Epstein-Barr virus lytic replication

Cardiac glycosides (CGs) show potential broad-spectrum antiviral activity by targeting cellular host proteins. Herein are reported the isolation of five new (1–5) and eight known (7–13) CGs from the roots of Streblus asper Lour. Of these compounds 1 and 7 exhibited inhibitory action against EBV early antigen (EA) expression, with half-maximal effective concentration values (EC50) being less than 60 nM, and they also showed selectivity, with selectivity index (SI) values being 56.80 and 103.17, respectively. Preliminary structure activity relationships indicated that the C-10 substituent, C-5 hydroxy groups, and C-3 sugar unit play essential roles in the mediation of the inhibitory activity of CGs against EBV. Further enzyme experiments demonstrated that these compounds might inhibit ion pump function and thereby change the intracellular signal transduction pathway by binding to Na+/K+-ATPase, as validated by simulated molecular docking. This study is the first report that CGs can effectively limit EBV lytic replication, and the observations made in this study may be of value for lead compound development.

Epstein-Barr virus lytic replication activates and is dependent upon MAPK-interacting kinase 1/2 in a cell-type dependent manner.

Epstein-Barr virus (EBV) is known to manipulate its cellular environment to enhance viral replication, which can lead to dysregulation of cellular machinery, setting the stage for potential future disease. Previous research showed that under rapamycin-mediated inhibition of mTORC1, EBV lytic protein production was altered in a cell-type specific manner, suggesting that EBV differentially activates or utilizes signaling pathways in B versus epithelial cells. Here we correlated activation of the mTORC1, ERK1/2, and p38 pathways in relation to EBV lytic replication and discovered that activation of MAPK-interacting kinase 1/2 (Mnk1/2) was strongly associated with EBV lytic replication. Inhibition studies revealed that Mnk1/2 activated lytic replication in epithelial cells yet acted as an inhibitor of lytic replication in B cells. The ability of lytic epithelial cells to migrate, a potentially important aspect of metastasis, was also demonstrated to be dependent upon Mnk1/2 activity.

Replication Compartments-The Great Survival Strategy for Epstein-Barr Virus Lytic Replication.

During Epstein–Barr virus (EBV) lytic replication, viral DNA synthesis is carried out in viral replication factories called replication compartments (RCs), which are located at discrete sites in the nucleus. Viral proteins constituting the viral replication machinery are accumulated in the RCs to amplify viral genomes. Newly synthesized viral DNA is stored in a subdomain of the RC termed the BMRF1-core, matured by host factors, and finally packed into assembled viral capsids. Late (L) genes are transcribed from DNA stored in the BMRF1-core through a process that is mainly dependent on the viral pre-initiation complex (vPIC). RC formation is a well-regulated system and strongly advantageous for EBV survival because of the following aspects: (1) RCs enable the spatial separation of newly synthesized viral DNA from the cellular chromosome for protection and maturation of viral DNA; (2) EBV-coded proteins and their interaction partners are recruited to RCs, which enhances the interactions among viral proteins, cellular proteins, and viral DNA; (3) the formation of RCs benefits continuous replication, leading to L gene transcription; and (4) DNA storage and maturation leads to efficient progeny viral production. Here, we review the state of knowledge of this important viral structure and discuss its roles in EBV survival.

Prognostic Value of Oral Epstein-Barr Virus DNA Load in Locoregionally Advanced Nasopharyngeal Carcinoma.

Background: Plasma Epstein–Barr virus (EBV) DNA load has been widely used for nasopharyngeal carcinoma (NPC) prognostic risk stratification. However, oral EBV DNA load, a non-invasive biomarker that reflects the EBV lytic replication activity, has not been evaluated for its prognostic value in NPC yet. Methods: A total number of 1,194 locoregionally advanced NPC (LA-NPC) patients from south China were included from a prospective observational cohort (GARTC) with a median follow-up of 107.3 months. Pretreatment or mid-treatment mouthwashes were collected for EBV DNA detection by quantitative polymerase chain reaction (qPCR). The difference of pre- and mid-treatment oral EBV DNA load was tested by the Wilcoxon signed-rank test. The associations of oral EBV DNA load with overall survival (OS), progression-free survival (PFS), distant metastasis–free survival (DMFS), and locoregional relapse-free survival (LRFS) were assessed using the log-rank test and multivariate Cox regression. Results: The high level of the oral EBV DNA load (>2,100 copies/mL) was independently associated with worse OS (HR = 1.45, 95% CI: 1.20–1.74, p < 0.001), PFS (HR = 1.38, 95% CI: 1.16–1.65, p < 0.001), DMFS (HR = 1.66, 95% CI: 1.25–2.21, p = 0.001), and LRFS (HR = 1.43, 95% CI: 1.05–1.96, p = 0.023). Similar and robust associations between oral EBV DNA load and prognosis were observed for patients in both the pretreatment and mid-treatment stages. The detection rate (71.7 vs. 48.6%, p < 0.001) and the median load of oral EBV DNA (13,368 vs. 382 copies/mL, p < 0.001) for patients in the pretreatment stage were significantly higher than those in the mid-treatment stage. The combination of the oral EBV DNA load and TNM staging provided a more precise risk stratification for the LA-NPC patients. Conclusion: Oral EBV DNA load was an alternative non-invasive predictor of prognosis and may facilitate risk stratification for the LA-NPC patients.

Structural basis of DNA methylation-dependent site selectivity of the Epstein-Barr virus lytic switch protein ZEBRA/Zta/BZLF1.

In infected cells, Epstein–Barr virus (EBV) alternates between latency and lytic replication. The viral bZIP transcription factor ZEBRA (Zta, BZLF1) regulates this cycle by binding to two classes of ZEBRA response elements (ZREs): CpG-free motifs resembling the consensus AP-1 site recognized by cellular bZIP proteins and CpG-containing motifs that are selectively bound by ZEBRA upon cytosine methylation. We report structural and mutational analysis of ZEBRA bound to a CpG-methylated ZRE (meZRE) from a viral lytic promoter. ZEBRA recognizes the CpG methylation marks through a ZEBRA-specific serine and a methylcytosine-arginine-guanine triad resembling that found in canonical methyl-CpG binding proteins. ZEBRA preferentially binds the meZRE over the AP-1 site but mutating the ZEBRA-specific serine to alanine inverts this selectivity and abrogates viral replication. Our findings elucidate a DNA methylation-dependent switch in ZEBRA’s transactivation function that enables ZEBRA to bind AP-1 sites and promote viral latency early during infection and subsequently, under appropriate conditions, to trigger EBV lytic replication by binding meZREs.

Epstein-Barr Virus miR-BHRF1-3 Targets the BZLF1 3'UTR and Regulates the Lytic Cycle.

Suppression of lytic viral gene expression is a key aspect of the Epstein-Barr virus (EBV) life cycle to facilitate the establishment of latent infection. Molecular mechanisms regulating transitions between EBV lytic replication and latency are not fully understood. Here, we investigated the impact of viral microRNAs on the EBV lytic cycle. Through functional assays, we found that miR-BHRF1-3 attenuates EBV lytic gene expression following reactivation. To understand the miRNA targets contributing to this activity, we performed Ago PAR-CLIP analysis on EBV-positive, reactivated Burkitt's lymphoma cells and identified multiple miR-BHRF1-3 interactions with viral transcripts. Using luciferase reporter assays, we confirmed a miRNA interaction site within the 3'UTR of BZLF1 which encodes the essential immediate early (IE) transactivator Zta. Comparison of >850 published EBV genomes identified sequence polymorphisms within the miR-BHRF1-3 locus that deleteriously affect miRNA expression and function. Molecular interactions between the homologous viral miRNA, miR-rL1-17, and IE transcripts encoded by rhesus lymphocryptovirus were further identified. Our data demonstrate that regulation of IE gene expression by a BHRF1 miRNA is conserved among lymphocryptoviruses, and further reveal virally-encoded genetic elements that orchestrate viral antigen expression during the lytic cycle. IMPORTANCE Epstein-Barr virus infection is predominantly latent in healthy individuals, while periodic cycles of reactivation are thought to facilitate persistent lifelong infection. Lytic infection has been linked to development of certain EBV-associated diseases. Here, we demonstrate that EBV miR-BHRF1-3 can suppress lytic replication by directly inhibiting Zta expression. Moreover, we identify nucleotide variants that impact the function of miR-BHRF1-3, which may contribute to specific EBV pathologies.

()-Epigallocatechin-3-Gallate Inhibits EBV Lytic Replication via Targeting LMP1-Mediated MAPK Signal Axes.

Epstein–Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) plays an important oncogenic role in the viral latent infection. Recently, increasing evidence indicates that the high expression of LMP1 during EBV lytic cycle is related to the viral lytic replication. However, the mechanism by which LMP1 regulates EBV lytic replication remains unclear. (−)-Epigallocatechin-3-gallate (EGCG) prevents carcinogenesis by directly targeting numerous membrane proteins and effectively inhibits EBV lytic cascade. Here, we demonstrated that LMP1 promotes EBV lytic replication through the downstream signal molecules MAPKs, including ERKs, p38, and JNKs. LMP1 induces the phosphorylation of p53 through MAPKs to enhance the ability of wild-type p53 (wt-p53) to activate expression of BZLF1 gene, while the JNKs/c-Jun signal axis appears to be involved in EBV lytic replication induced by LMP1 in p53 mutant manner. We provided the first evidence that EGCG directly targets the viral membrane LMP1 (K d = 0.36 μM, n = 1) using fluorescence quenching, isothermal titration calorimetry (ITC) assay, and CNBR-activated Sepharose 4B pull-down affinity chromatography. Furthermore, we revealed that EGCG inhibits EBV lytic replication via suppressing LMP1 and thus blocking the downstream MAPKs/wt-p53 signal axis in AGS-EBV cells and JNKs/c-Jun signal axis in p53 mutant B95.8 cells. Our study, for the first time, reports the binding and inhibitory efficacy of EGCG to the LMP1, which is a key oncoprotein encoded by EBV. These findings suggest the novel function of LMP1 in the regulation of EBV lytic cycle and reveal the new role of EGCG in EBV-associated malignancies through suppressing viral reactivation.

PIAS1 potentiates the anti-EBV activity of SAMHD1 through SUMOylation

BackgroundSterile alpha motif and HD domain 1 (SAMHD1) is a deoxynucleotide triphosphohydrolase (dNTPase) that restricts the infection of a variety of RNA and DNA viruses, including herpesviruses. The anti-viral function of SAMHD1 is associated with its dNTPase activity, which is regulated by several post-translational modifications, including phosphorylation, acetylation and ubiquitination. Our recent studies also demonstrated that the E3 SUMO ligase PIAS1 functions as an Epstein-Barr virus (EBV) restriction factor. However, whether SAMHD1 is regulated by PIAS1 to restrict EBV replication remains unknown.ResultsIn this study, we showed that PIAS1 interacts with SAMHD1 and promotes its SUMOylation. We identified three lysine residues (K469, K595 and K622) located on the surface of SAMHD1 as the major SUMOylation sites. We demonstrated that phosphorylated SAMHD1 can be SUMOylated by PIAS1 and SUMOylated SAMHD1 can also be phosphorylated by viral protein kinases. We showed that SUMOylation-deficient SAMHD1 loses its anti-EBV activity. Furthermore, we demonstrated that SAMHD1 is associated with EBV genome in a PIAS1-dependent manner.ConclusionOur study reveals that PIAS1 synergizes with SAMHD1 to inhibit EBV lytic replication through protein–protein interaction and SUMOylation.

BRLF1-dependent viral and cellular transcriptomes and transcriptional regulation during EBV primary infection in B lymphoma cells

The immediate-early protein BRLF1 plays important roles in lytic infection of Epstein-Barr virus (EBV), in which it activates lytic viral transcription and replication. However, knowledge of the influence of BRLF1 on cellular gene expression and transcriptional reprogramming during the early lytic cycle remains limited. In the present study, deep RNA-sequencing analysis identified all differentially expressed genes (DEGs) and alternative splicing in B lymphoma cells subjected to wild-type and BRLF1-deficient EBV primary infection. The BRLF1-dependent cellular DEGs were annotated, and major differentially enriched pathways were related to DNA replication and transcription, immune and inflammatory responses, cytokine-receptor interactions and chemokine signaling and metabolic processes. Furthermore, analysis of BRLF1-binding proteins by mass spectrometry shows that BRLF1 binds to and cooperates with several transcription factors and components of the spliceosome and then influences both RNA polymerase II-dependent transcription and pre-mRNA splicing. The RTA-binding RRE motifs or specific motifs of unique cooperative transcription factors in viral and cellular DEG promoter regions indicate that BRLF1 employs different strategies for regulating viral and cellular transcription. Thus, our study characterized BRLF1-dependent cellular and viral transcriptional profile during primary infection and then revealed the comprehensive virus-cell interaction and alterations of transcription during EBV primary infection and lytic replication.

Roles of Lytic Viral Replication and Co-Infections in the Oncogenesis and Immune Control of the Epstein-Barr Virus.

Simple SummaryThe Epstein–Barr virus (EBV) colonizes more than 95% of the adult human population. Its cancer-forming potential is usually contained by lifelong immune control. Genetic alterations and immune modulation by co-infection point towards cytotoxic lymphocytes, such as natural killer and CD8+ T cells, as the main pillars of this immune protection. In this review, we discuss how the EBV infection program that leads to infectious virion production and co-infections, such as with malaria parasites, the human immunodeficiency virus (HIV) and the Kaposi sarcoma-associated herpesvirus (KSHV), modulate this immune control.Epstein–Barr virus (EBV) is the prototypic human tumor virus whose continuous lifelong immune control is required to prevent lymphomagenesis in the more than 90% of the human adult population that are healthy carriers of the virus. Here, we review recent evidence that this immune control has not only to target latent oncogenes, but also lytic replication of EBV. Furthermore, genetic variations identify the molecular machinery of cytotoxic lymphocytes as essential for this immune control and recent studies in mice with reconstituted human immune system components (humanized mice) have begun to provide insights into the mechanistic role of these molecules during EBV infection. Finally, EBV often does not act in isolation to cause disease. Some of EBV infection-modulating co-infections, including human immunodeficiency virus (HIV) and Kaposi sarcoma-associated herpesvirus (KSHV), have been modeled in humanized mice. These preclinical in vivo models for EBV infection, lymphomagenesis, and cell-mediated immune control do not only promise a better understanding of the biology of this human tumor virus, but also the possibility to explore vaccine candidates against it.

Conquering the Nuclear Envelope Barriers by EBV Lytic Replication.

The nuclear envelope (NE) of eukaryotic cells has a highly structural architecture, comprising double lipid-bilayer membranes, nuclear pore complexes, and an underlying nuclear lamina network. The NE structure is held in place through the membrane-bound LINC (linker of nucleoskeleton and cytoskeleton) complex, spanning the inner and outer nuclear membranes. The NE functions as a barrier between the nucleus and cytoplasm and as a transverse scaffold for various cellular processes. Epstein–Barr virus (EBV) is a human pathogen that infects most of the world’s population and is associated with several well-known malignancies. Within the nucleus, the replicated viral DNA is packaged into capsids, which subsequently egress from the nucleus into the cytoplasm for tegumentation and final envelopment. There is increasing evidence that viral lytic gene expression or replication contributes to the pathogenesis of EBV. Various EBV lytic proteins regulate and modulate the nuclear envelope structure in different ways, especially the viral BGLF4 kinase and the nuclear egress complex BFRF1/BFRF2. From the aspects of nuclear membrane structure, viral components, and fundamental nucleocytoplasmic transport controls, this review summarizes our findings and recently updated information on NE structure modification and NE-related cellular processes mediated by EBV.

The Epstein-Barr Virus Lytic Protein BMLF1 Induces Upregulation of GRP78 Expression through ATF6 Activation.

The unfolded protein response (UPR) is an intracellular signaling pathway essential for alleviating the endoplasmic reticulum (ER) stress. To support the productive infection, many viruses are known to use different strategies to manipulate the UPR signaling network. However, it remains largely unclear whether the UPR signaling pathways are modulated in the lytic cycle of Epstein-Barr virus (EBV), a widely distributed human pathogen. Herein, we show that the expression of GRP78, a central UPR regulator, is up-regulated during the EBV lytic cycle. Our data further revealed that knockdown of GRP78 in EBV-infected cell lines did not substantially affect lytic gene expression; however, GRP78 knockdown in these cells markedly reduced the production of virus particles. Importantly, we identified that the early lytic protein BMLF1 is the key regulator critically contributing to the activation of the grp78 gene promoter. Mechanistically, we found that BMLF1 can trigger the proteolytic cleavage and activation of the UPR senor ATF6, which then transcriptionally activates the grp78 promoter through the ER stress response elements. Our findings therefore provide evidence for the connection between the EBV lytic cycle and the UPR, and implicate that the BMLF1-mediated ATF6 activation may play critical roles in EBV lytic replication.