ABSTRACT
After Epstein-Barr virus (EBV) genome replication and encapsidation in the nucleus, nucleocapsids are translocated into the cytoplasm for subsequent tegumentation and maturation. The EBV BGLF4 kinase, which induces partial disassembly of the nuclear lamina, and the nuclear egress complex BFRF1/BFLF2 coordinately facilitate the nuclear egress of nucleocapsids. Here, we demonstrate that within EBV reactivated epithelial cells, viral capsids, tegument proteins, and glycoproteins are clustered in the juxtanuclear concave region, accompanied by redistributed cytoplasmic organelles and the cytoskeleton regulator IQ-domain GTPase-activation protein 1 (IQGAP1), close to the microtubule-organizing center (MTOC). The assembly compartment (AC) structure was diminished in BGLF4-knockdown TW01-EBV cells and BGLF4-knockout bacmid-carrying TW01 cells, suggesting that the formation of AC structure is BGLF4-dependent. Notably, glycoprotein gp350/220 was observed by confocal imaging to be distributed in the perinuclear concave region and surrounded by the endoplasmic reticulum (ER) membrane marker calnexin, indicating that the AC may be located within a globular structure derived from ER membranes, adjacent to the outer nuclear membrane. Moreover, the viral capsid protein BcLF1 and tegument protein BBLF1 were co-localized with IQGAP1 near the cytoplasmic membrane in the late stage of replication. Knockdown of IQGAP1 did not affect the AC formation but decreased virion release from both TW01-EBV and Akata+ cells, suggesting IQGAP1-mediated trafficking regulates EBV virion release. The data presented here show that BGLF4 is required for cytoskeletal rearrangement, coordination with the redistribution of cytoplasmic organelles and IQGAP1 for virus maturation, and subsequent IQGAP1-dependent virion release.IMPORTANCEEBV genome is replicated and encapsidated in the nucleus, and the resultant nucleocapsids are translocated to the cytoplasm for subsequent virion maturation. We show that a cytoplasmic AC, containing viral proteins, markers of the endoplasmic reticulum, Golgi, and endosomes, is formed in the juxtanuclear region of epithelial and B cells during EBV reactivation. The viral BGLF4 kinase contributes to the formation of the AC. The cellular protein IQGAP1 is also recruited to the AC and partially co-localizes with the virus capsid protein BcLF1 and tegument protein BBLF1 in EBV-reactivated cells, dependent on the BGLF4-induced cytoskeletal rearrangement. In addition, virion release was attenuated in IQGAP1-knockdown epithelial and B cells after reactivation, suggesting that IQGAP1-mediated trafficking may regulate the efficiency of virus maturation and release.
Subject(s)
Cytoplasm , Herpesvirus 4, Human , Protein Serine-Threonine Kinases , Viral Proteins , Virion , Virus Assembly , Virus Release , ras GTPase-Activating Proteins , Humans , Capsid Proteins/metabolism , Cytoplasm/metabolism , Cytoplasm/virology , Epstein-Barr Virus Infections/metabolism , Epstein-Barr Virus Infections/virology , Herpesvirus 4, Human/chemistry , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/growth & development , Herpesvirus 4, Human/metabolism , Membrane Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , ras GTPase-Activating Proteins/metabolism , Viral Proteins/metabolism , Virion/chemistry , Virion/growth & development , Virion/metabolism , Virus Assembly/physiology , Endoplasmic Reticulum/metabolism , Endosomes/metabolism , Golgi Apparatus/metabolismABSTRACT
The strongest evidence of the oncogenicity of Epstein-Barr virus (EBV) in vitro is its ability to immortalize human primary B lymphocytes into lymphoblastoid cell lines (LCLs). Yet the underlying mechanisms explaining how the virus tempers the growth program of the host cells have not been fully elucidated. The mitogen-activated protein kinases (MAPKs) are implicated in many cellular processes and are constitutively activated in LCLs. We questioned the expression and regulation of the dual-specificity phosphatases (DUSPs), the main negative regulator of MAPKs, during EBV infection and immortalization. Thirteen DUSPs, including 10 typical and 3 atypical types of DUSPs, were tested. Most of them were downregulated after EBV infection. Here, a role of viral oncogene latent membrane protein 1 (LMP1) in limiting DUSP6 and DUSP8 expression was identified. Using MAPK inhibitors, we found that LMP1 activates extracellular signal-regulated kinase (ERK) or p38 to repress the expression of DUSP6 and DUSP8, with corresponding substrate specificity. Morphologically, overexpression of DUSP6 and DUSP8 attenuates the ability of EBV-immortalized LCL cells to clump together. Mechanistically, apoptosis induced by restoring DUSP6 and DUSP8 in LCLs indicated a novel mechanism for LMP1 to provide a survival signal during EBV immortalization. Collectively, this report provides the first description of the interplay between EBV genes and DUSPs and contributes considerably to the interpretation of MAPK regulation in EBV immortalization.IMPORTANCE Infections by the ubiquitous Epstein-Barr virus (EBV) are associated with a wide spectrum of lymphomas and carcinomas. It has been well documented that activation levels of MAPKs are found in cancer cells to translate various external or intrinsic stimuli into cellular responses. Physiologically, the dual-specificity phosphates (DUSPs) exhibit great ability in regulating MAPK activities with respect to their capability of dephosphorylating MAPKs. In this study, we found that DUSPs were generally downregulated after EBV infection. EBV oncogenic latent membrane protein 1 (LMP1) suppressed DUSP6 and DUSP8 expression via MAPK pathway. In this way, LMP1-mediated MAPK activation was a continuous process. Furthermore, DUSP downregulation was found to contribute greatly to prevent apoptosis of EBV-infected cells. To sum up, this report sheds light on a novel molecular mechanism explaining how EBV maintains the unlimited proliferation status of the immortalized cells and provides a new link to understand EBV-induced B cell survival.
Subject(s)
Dual-Specificity Phosphatases/genetics , Herpesvirus 4, Human/metabolism , Viral Matrix Proteins/metabolism , Apoptosis/genetics , B-Lymphocytes/virology , Cell Line, Tumor , Dual-Specificity Phosphatases/metabolism , Epstein-Barr Virus Infections/virology , Extracellular Signal-Regulated MAP Kinases/metabolism , Genes, Viral/genetics , Humans , Mitogen-Activated Protein Kinases/metabolism , NF-kappa B/metabolism , Primary Cell Culture , Viral Matrix Proteins/physiology , Viral Proteins/metabolism , Virus Latency/genetics , Virus Latency/physiology , p38 Mitogen-Activated Protein Kinases/metabolismABSTRACT
Epstein-Barr virus (EBV) genomic DNA is replicated and packaged into procapsids in the nucleus to form nucleocapsids, which are then transported into the cytoplasm for tegumentation and final maturation. The process is facilitated by the coordination of the viral nuclear egress complex (NEC), which consists of BFLF2 and BFRF1. By expression alone, BFLF2 is distributed mainly in the nucleus. However, it colocalizes with BFRF1 at the nuclear rim and in cytoplasmic nuclear envelope-derived vesicles in coexpressing cells, suggesting temporal control of the interaction between BFLF2 and BFRF1 is critical for their proper function. The N-terminal sequence of BFLF2 is less conserved than that of alpha- and betaherpesvirus homologs. Here, we found that BFLF2 amino acids (aa) 2 to 102 are required for both nuclear targeting and its interaction with BFRF1. Coimmunoprecipitation and confocal analysis indicated that aa 82 to 106 of BFLF2 are important for its interaction with BFRF1. Three crucial amino acids (R47, K50, and R52) and several noncontinuous arginine and histidine residues within aa 59 to 80 function together as a noncanonical nuclear localization signal (NLS), which can be transferred onto yellow fluorescent protein (YFP)-LacZ for nuclear targeting in an importin ß-dependent manner. Virion secretion is defective in 293 cells harboring a BFLF2 knockout EBV bacmid upon lytic induction and is restored by trans-complementation of wild-type BFLF2, but not NLS or BFRF1-interacting defective mutants. In addition, multiple domains of BFRF1 were found to bind BFLF2, suggesting multiple contact regions within BFRF1 and BFLF2 are required for proper nuclear egress of EBV nucleocapsids.IMPORTANCE Although Epstein-Barr virus (EBV) BFRF1 and BFLF2 are homologs of conserved viral nuclear egress complex (NEC) in all human herpesviruses, unique amino acid sequences and functions were identified in both proteins. In this study, the nuclear targeting and BFRF1-interacting domains were found within the N terminus of BFLF2. We showed that amino acids (aa) 82 to 106 are the major region required for BFLF2 to interact with BFRF1. However, the coimmunoprecipitation (Co-IP) data and glutathione transferase (GST) pulldown experiments revealed that multiple regions of both proteins contribute to reciprocal interactions. Different from the canonical nuclear localization signal (NLS) in other herpes viral homologs, BFLF2 contains a novel importin-dependent nuclear localization signal, including R47, K50, and R52 and several neighboring discontinuous arginine and histidine residues. Using a bacmid complementation system, we show that both the nuclear targeting and the novel nuclear localization signal within aa 82 to 106 of BFLF2 are required for virion secretion.
Subject(s)
Cell Nucleus/virology , Herpesvirus 4, Human/genetics , Viral Proteins/metabolism , Virus Release/physiology , Amino Acid Sequence , Cell Line , Cytoplasm/metabolism , Glutathione Transferase/metabolism , HEK293 Cells , HeLa Cells , Humans , Membrane Proteins/chemistry , Membrane Proteins/genetics , Membrane Proteins/metabolism , Models, Molecular , Nuclear Envelope , Nuclear Localization Signals/metabolism , Protein Conformation , Sequence Analysis, Protein , Viral Proteins/chemistry , Viral Proteins/genetics , Virion/metabolism , Virus Release/genetics , beta KaryopherinsABSTRACT
The role of the epithelial-mesenchymal transition (EMT) in lung epithelial cells is increasingly being recognized as a key stage in the development of COPD, fibrosis, and lung cancers, which are all highly associated with cigarette smoking and with exposure to second-hand smoke. Using the exposure of human lung cancer epithelial A549 cells and non-cancerous Beas-2B cells to sidestream cigarette smoke extract (CSE) as a model, we studied the protective effects of adipose-derived stem cell-conditioned medium (ADSC-CM) against CSE-induced cell death and EMT. CSE dose-dependently induced cell death, decreased epithelial markers, and increased the expression of mesenchymal markers. Upstream regulator analysis of differentially expressed genes after CSE exposure revealed similar pathways as those observed in typical EMT induced by TGF-ß1. CSE-induced cell death was clearly attenuated by ADSC-CM but not by other control media, such as a pass-through fraction of ADSC-CM or A549-CM. ADSC-CM effectively inhibited CSE-induced EMT and was able to reverse the gradual loss of epithelial marker expression associated with TGF-ß1 treatment. CSE or TGF-ß1 enhanced the speed of A549 migration by 2- to 3-fold, and ADSC-CM was effective in blocking the cell migration induced by either agent. Future work will build on the results of this in vitro study by defining the molecular mechanisms through which ADSC-CM protects lung epithelial cells from EMT induced by toxicants in second-hand smoke.
Subject(s)
Cigarette Smoking/adverse effects , Lung Neoplasms/prevention & control , Lung/drug effects , Mesenchymal Stem Cells/cytology , Cell Death/drug effects , Cell Line , Cell Line, Tumor , Culture Media, Conditioned , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Epithelial Cells/pathology , Epithelial-Mesenchymal Transition , Humans , Lung/metabolism , Lung/pathology , Lung Neoplasms/metabolism , Lung Neoplasms/pathology , Mesenchymal Stem Cells/metabolism , Pulmonary Disease, Chronic Obstructive/metabolism , Pulmonary Disease, Chronic Obstructive/pathology , Pulmonary Disease, Chronic Obstructive/prevention & control , Signal Transduction , Smoke/adverse effectsABSTRACT
Polyethylene glycol (PEG) coating of gold nanoparticles (AuNPs) improves AuNP distribution via blood circulation. The use of PEG-coated AuNPs was shown to result in acute injuries to the liver, kidney, and spleen, but long-term toxicity has not been well studied. In this study, we investigated reporter induction for up to 90 days in NF-κB transgenic reporter mice following intravenous injection of PEG-coated AuNPs. The results of different doses (1 and 4 µg AuNPs per gram of body weight), particle sizes (13 nm and 30 nm), and PEG surfaces (methoxyl- or carboxymethyl-PEG 5 kDa) were compared. The data showed up to 7-fold NF-κB reporter induction in mouse liver from 3 h to 7 d post PEG-AuNP injection compared to saline-injected control mice, and gradual reduction to a level similar to control by 90 days. Agglomerates of PEG-AuNPs were detected in liver Kupffer cells, but neither gross pathological abnormality in liver sections nor increased activity of liver enzymes were found at 90 days. Injection of PEG-AuNPs led to an increase in collagen in liver sections and elevated total serum cholesterol, although still within the normal range, suggesting that inflammation resulted in mild fibrosis and affected hepatic function. Administrating PEG-AuNPs inevitably results in nanoparticles entrapped in the liver; thus, further investigation is required to fully assess the long-term impacts by PEG-AuNPs on liver health.
Subject(s)
Gold/chemistry , Inflammation/pathology , Liver/pathology , Metal Nanoparticles/toxicity , NF-kappa B/genetics , Polyethylene Glycols/chemistry , Animals , Inflammation/chemically induced , Inflammation/metabolism , Liver/drug effects , Liver/metabolism , Luciferases , Mice , Mice, Inbred C57BL , Mice, Transgenic , NF-kappa B/metabolismABSTRACT
During the lytic phase of Epstein-Barr virus (EBV), binding of the transactivator Zta to the origin of lytic replication (oriLyt) and the BHLF1 transcript, forming a stable RNA-DNA hybrid, is required to initiate viral DNA replication. EBV-encoded viral DNA replication proteins form complexes to amplify viral DNA. BMRF1, the viral DNA polymerase accessory factor, is essential for lytic DNA replication and also known as a transcriptional regulator of the expression of BHLF1 and BALF2 (single-stranded DNA [ssDNA]-binding protein). In order to determine systematically how BMRF1 regulates viral transcription, a BMRF1 knockout bacmid was generated to analyze viral gene expression using a viral DNA microarray. We found that a subset of Rta-responsive late genes, including BcLF1, BLLF1, BLLF2, and BDLF3, were downregulated in cells harboring a BMRF1 knockout EBV bacmid (p2089ΔBMRF1). In reporter assays, BMRF1 appears to transactivate a subset of viral late promoters through distinct pathways. BMRF1 activates the BDLF3 promoter in an SP1-dependent manner. Notably, BMRF1 associates with the transcriptional regulator BRG1 in EBV-reactivated cells. BMRF1-mediated transactivation activities on the BcLF1 and BLLF1 promoters were attenuated by knockdown of BRG1. In BRG1-depleted EBV-reactivated cells, BcLF1 and BLLF1 transcripts were reduced in number, resulting in reduced virion secretion. BMRF1 and BRG1 bound to the adjacent upstream regions of the BcLF1 and BLLF1 promoters, and depletion of BRG1 attenuated the recruitment of BMRF1 onto both promoters, suggesting that BRG1 is involved in BMRF1-mediated regulation of these two genes. Overall, we reveal a novel pathway by which BMRF1 can regulate viral promoters through interaction with BRG1.IMPORTANCE The cascade of viral gene expression during Epstein-Barr virus (EBV) replication is exquisitely regulated by the coordination of the viral DNA replication machinery and cellular factors. Upon lytic replication, the EBV immediate early proteins Zta and Rta turn on the expression of early proteins that assemble into viral DNA replication complexes. The DNA polymerase accessory factor, BMRF1, also is known to transactivate early gene expression through its interaction with SP1 or Zta on specific promoters. Through a global analysis, we demonstrate that BMRF1 also turns on a subset of Rta-regulated, late structural gene promoters. Searching for BMRF1-interacting cellular partners revealed that the SWI/SNF chromatin modifier BRG1 contributes to BMRF1-mediated transactivation of a subset of late promoters through protein-protein interaction and viral chromatin binding. Our findings indicate that BMRF1 regulates the expression of more viral genes than thought previously through distinct viral DNA replication-independent mechanisms.
Subject(s)
Antigens, Viral/genetics , DNA Helicases/genetics , Gene Expression Regulation, Viral/genetics , Herpesvirus 4, Human/metabolism , Immediate-Early Proteins/genetics , Membrane Glycoproteins/genetics , Nuclear Proteins/genetics , Trans-Activators/genetics , Transcription Factors/genetics , Transcriptional Activation/genetics , Viral Proteins/genetics , Antigens, Viral/metabolism , Cell Line, Tumor , Chromatin/metabolism , DNA Replication/genetics , DNA, Viral/genetics , Gene Knockout Techniques , HEK293 Cells , HeLa Cells , Herpesvirus 4, Human/genetics , Humans , Membrane Glycoproteins/metabolism , Promoter Regions, Genetic , RNA Interference , RNA, Small Interfering/genetics , Transcription, Genetic , Viral Proteins/metabolism , Virus Replication/geneticsABSTRACT
Epstein-Barr virus (EBV), an oncogenic human virus, is associated with several lymphoproliferative disorders, including Burkitt lymphoma, Hodgkin disease, diffuse large B-cell lymphoma (DLBCL), and posttransplant lymphoproliferative disorder (PTLD). In vitro, EBV transforms primary B cells into lymphoblastoid cell lines (LCLs). Recently, several studies have shown that receptor tyrosine kinases (RTKs) play important roles in EBV-associated neoplasia. However, details of the involvement of RTKs in EBV-regulated B-cell neoplasia and malignancies remain largely unclear. Here, we found that erythropoietin-producing hepatocellular receptor A4 (EphA4), which belongs to the largest RTK Eph family, was downregulated in primary B cells post-EBV infection at the transcriptional and translational levels. Overexpression and knockdown experiments confirmed that EBV-encoded latent membrane protein 1 (LMP1) was responsible for this EphA4 suppression. Mechanistically, LMP1 triggered the extracellular signal-regulated kinase (ERK) pathway and promoted Sp1 to suppress EphA4 promoter activity. Functionally, overexpression of EphA4 prevented LCLs from proliferation. Pathologically, the expression of EphA4 was detected in EBV(-) tonsils but not in EBV(+) PTLD. In addition, an inverse correlation of EphA4 expression and EBV presence was verified by immunochemical staining of EBV(+) and EBV(-) DLBCL, suggesting EBV infection was associated with reduced EphA4 expression. Analysis of a public data set showed that lower EphA4 expression was correlated with a poor survival rate of DLBCL patients. Our findings provide a novel mechanism by which EphA4 can be regulated by an oncogenic LMP1 protein and explore its possible function in B cells. The results provide new insights into the role of EphA4 in EBV(+) PTLD and DLBCL.
Subject(s)
Epstein-Barr Virus Infections/complications , Lymphoma, Large B-Cell, Diffuse/mortality , Lymphoproliferative Disorders/mortality , Receptor, EphA4/metabolism , Viral Matrix Proteins/metabolism , Cells, Cultured , Down-Regulation , Epstein-Barr Virus Infections/virology , Extracellular Signal-Regulated MAP Kinases/genetics , Extracellular Signal-Regulated MAP Kinases/metabolism , Herpesvirus 4, Human , Humans , Lymphoma, Large B-Cell, Diffuse/metabolism , Lymphoma, Large B-Cell, Diffuse/virology , Lymphoproliferative Disorders/metabolism , Lymphoproliferative Disorders/virology , Prognosis , Receptor, EphA4/genetics , Signal Transduction , Survival Rate , Viral Matrix Proteins/geneticsABSTRACT
UNLABELLED: Epstein-Barr virus (EBV) expresses few viral proteins in nasopharyngeal carcinoma (NPC) but high levels of BamHI-A rightward transcripts (BARTs), which include long noncoding RNAs (lncRNAs) and BART microRNAs (miRNAs). It is hypothesized that the mechanism for regulation of BARTs may relate to EBV pathogenesis in NPC. We showed that nuclear factor-κB (NF-κB) activates the BART promoters and modulates the expression of BARTs in EBV-infected NPC cells but that introduction of mutations into the putative NF-κB binding sites abolished activation of BART promoters by NF-κB. Binding of p50 subunits to NF-κB sites in the BART promoters was confirmed in electrophoretic mobility shift assays (EMSA) and further demonstrated in vivo using chromatin immunoprecipitation (ChIP) analysis. Expression of BART miRNAs and lncRNAs correlated with NF-κB activity in EBV-infected epithelial cells, while treatment of EBV-harboring NPC C666-1 cells with aspirin (acetylsalicylic acid [ASA]) and the IκB kinase inhibitor PS-1145 inhibited NF-κB activity, resulting in downregulation of BART expression. Expression of EBV LMP1 activates BART promoters, whereas an LMP1 mutant which cannot induce NF-κB activation does not activate BART promoters, further supporting the idea that expression of BARTs is regulated by NF-κB signaling. Expression of LMP1 is tightly regulated in NPC cells, and this study confirmed that miR-BART5-5p downregulates LMP1 expression, suggesting a feedback loop between BART miRNA and LMP1-mediated NF-κB activation in the NPC setting. These findings provide new insights into the mechanism underlying the deregulation of BARTs in NPC and identify a regulatory loop through which BARTs support EBV latency in NPC. IMPORTANCE: Nasopharyngeal carcinoma (NPC) cells are ubiquitously infected with Epstein-Barr virus (EBV). Notably, EBV expresses very few viral proteins in NPC cells, presumably to avoid triggering an immune response, but high levels of EBV BART miRNAs and lncRNAs which exhibit complex functions associated with EBV pathogenesis. The mechanism for regulation of BARTs is critical for understanding NPC oncogenesis. This study provides multiple lines of evidence to show that expression of BARTs is subject to regulation by NF-κB signaling. EBV LMP1 is a potent activator of NF-κB signaling, and we demonstrate that LMP1 can upregulate expression of BARTs through NF-κB signaling and that BART miRNAs are also able to downregulate LMP1 expression. It appears that aberrant NF-κB signaling and expression of BARTs form an autoregulatory loop for maintaining EBV latency in NPC cells. Further exploration of how targeting NF-κB signaling interrupts EBV latency in NPC cells may reveal new options for NPC treatment.
Subject(s)
Deoxyribonuclease BamHI/genetics , Epstein-Barr Virus Infections/virology , Gene Expression Regulation, Viral , MicroRNAs/genetics , NF-kappa B/metabolism , Nasopharyngeal Neoplasms/genetics , RNA, Long Noncoding/genetics , Base Sequence , Carcinoma , Herpesvirus 4, Human/pathogenicity , Humans , NF-kappa B/genetics , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms/pathology , Nasopharyngeal Neoplasms/virology , Promoter Regions, Genetic/genetics , RNA, Viral/genetics , Signal Transduction , Tumor Cells, Cultured , Viral Matrix Proteins/genetics , Viral Matrix Proteins/metabolism , Virus LatencyABSTRACT
UNLABELLED: The cellular endosomal sorting complex required for transport (ESCRT) was recently found to mediate important morphogenesis processes at the nuclear envelope (NE). We previously showed that the Epstein-Barr virus (EBV) BFRF1 protein recruits the ESCRT-associated protein Alix to modulate NE structure and promote EBV nuclear egress. Here, we uncover new cellular factors and mechanisms involved in this process. BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. BFRF1 is ubiquitinated, and elimination of possible ubiquitination by either lysine mutations or fusion of a deubiquitinase hampers NE-derived vesicle formation and virus maturation. While it interacts with multiple Nedd4-like ubiquitin ligases, BFRF1 preferentially binds Itch ligase. We show that Itch associates with Alix and BFRF1 and is required for BFRF1-induced NE vesicle formation. Our data demonstrate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE and EBV maturation, uncovering novel regulatory mechanisms of nuclear egress of viral nucleocapsids. IMPORTANCE: The nuclear envelope (NE) of eukaryotic cells not only serves as a transverse scaffold for cellular processes, but also as a natural barrier for most DNA viruses that assemble their nucleocapsids in the nucleus. Previously, we showed that the cellular endosomal sorting complex required for transport (ESCRT) machinery is required for the nuclear egress of EBV. Here, we further report the molecular interplay among viral BFRF1, the ESCRT adaptor Alix, and the ubiquitin ligase Itch. We found that BFRF1-induced NE vesicles are similar to those observed following EBV reactivation. The lysine residues and the ubiquitination of BFRF1 regulate the formation of BFRF1-induced NE-derived vesicles and EBV maturation. During the process, a ubiquitin ligase, Itch, preferably associates with BFRF1 and is required for BFRF1-induced NE vesicle formation. Therefore, our data indicate that Itch, ubiquitin, and Alix control the BFRF1-mediated modulation of the NE, suggesting novel regulatory mechanisms for ESCRT-mediated NE modulation.
Subject(s)
Herpesvirus 4, Human/physiology , Host-Pathogen Interactions , Membrane Proteins/metabolism , Nuclear Envelope/metabolism , Repressor Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Viral Proteins/metabolism , Virus Assembly , Virus Replication , HeLa Cells , HumansABSTRACT
BACKGROUND: The burden of diabetes-related chronic kidney disease (CKD) on individuals and society is increasing, shifting attention toward improving the quality of care for patients with CKD and diabetes. We assessed the quality of CKD care and its association with long-term dialysis, acute kidney injury (AKI), and death. STUDY DESIGN: Retrospective cohort study (2004-2011). SETTING & PARTICIPANTS: Adults in Taiwan with incident CKD enrolled in the Longitudinal Cohort of Diabetes Patients. PREDICTORS: 3 CKD-care quality indicators based on medical and pharmacy claims data were studied: prescription of renin-angiotensin system inhibitors, testing for proteinuria, and nutritional guidance. Each was examined individually, and all were summed into an overall quality score. OUTCOMES: The primary outcome was initiation of long-term dialysis therapy. Secondary outcomes were hospitalization due to AKI and death from any cause. MEASUREMENTS: Using instrumental variables related to the quality indicators to minimize both unmeasured and measured confounding, we fit a 2-stage residual inclusion model to estimate HRs and 95% CIs for each outcome. RESULTS: Among the 63,260 patients enrolled, 43.9% were prescribed renin-angiotensin system inhibitors, 60.6% were tested for proteinuria, and 13.4% received nutritional guidance. During a median follow-up of 37.9 months, 1,471 patients started long-term dialysis therapy, 2,739 patients were hospitalized due to AKI, and 4,407 patients died. Higher overall quality scores were associated with lower hazards for long-term dialysis in instrumental variable analyses (HR of 0.62 [95% CI, 0.40-0.98] per 1-point greater score) and hospitalization due to AKI (HR of 0.69 [95% CI, 0.50-0.96] per 1-point greater score). The hazard for all-cause death was nonsignificantly lower (HR of 0.80 [95% CI, 0.62-1.03] per 1-point greater score). LIMITATIONS: Potential misclassification and uncontrolled confounding by indication. CONCLUSIONS: Our findings suggest potential opportunities to improve long-term outcomes among patients with diabetes and CKD by improving the quality of their CKD care.
Subject(s)
Acute Kidney Injury/epidemiology , Angiotensin Receptor Antagonists/therapeutic use , Angiotensin-Converting Enzyme Inhibitors/therapeutic use , Diabetes Mellitus/therapy , Diabetic Nephropathies/therapy , Mortality , Nutrition Therapy/statistics & numerical data , Proteinuria/diagnosis , Quality of Health Care , Renal Dialysis , Renal Insufficiency, Chronic/therapy , Aged , Cause of Death , Comorbidity , Databases, Factual , Diabetes Mellitus/epidemiology , Diabetic Nephropathies/epidemiology , Female , Hospitalization/statistics & numerical data , Humans , Longitudinal Studies , Male , Middle Aged , Proportional Hazards Models , Quality Indicators, Health Care , Renal Insufficiency, Chronic/epidemiology , Taiwan/epidemiologyABSTRACT
Oncogenic Epstein-Barr virus (EBV) uses various approaches to escape host immune responses and persist in B cells. Such persistent infections may provide the opportunity for this virus to initiate tumor formation. Using EBV-immortalized lymphoblastoid cell lines (LCLs) as a model, we found that the expression of major histocompatibility complex (MHC) class II and CD74 in B cells is repressed after EBV infection. Class II transactivator (CIITA) is the master regulator of MHC class II-related genes. As expected, CIITA was downregulated in LCLs. We showed that downregulation of CIITA is caused by EBV latent membrane protein 2A (LMP2A) and driven by the CIITA-PIII promoter. Furthermore, we demonstrated that LMP2A-mediated E47 and PU.1 reduction resulted in CIITA suppression. Mechanistically, the LMP2A immunoreceptor tyrosine-based activation motif was critical for the repression of E47 and PU.1 promoter activity via Syk, Src, and the phosphatidylinositol 3-kinase/Akt pathway. Elimination of LMP2A in LCLs using a shLMP2A approach showed that the expression levels of E47, PU.1, CIITA, MHC class II, and CD74 are reversed. These data indicated that the LMP2A may reduce MHC class II expression through interference with the E47/PU.1-CIITA pathway. Finally, we demonstrated that MHC class II may be detected in tonsils and EBV-negative Hodgkin disease but not in EBV-associated posttransplant lymphoproliferative disease and Hodgkin disease.
Subject(s)
Gene Expression Regulation , Histocompatibility Antigens Class II/chemistry , Nuclear Proteins/metabolism , Proto-Oncogene Proteins/genetics , Trans-Activators/genetics , Trans-Activators/metabolism , Transcription Factor 3/genetics , Viral Matrix Proteins/metabolism , B-Lymphocytes/immunology , B-Lymphocytes/metabolism , B-Lymphocytes/pathology , Blotting, Western , Cells, Cultured , Down-Regulation , Epstein-Barr Virus Infections/immunology , Epstein-Barr Virus Infections/metabolism , Epstein-Barr Virus Infections/virology , Herpesvirus 4, Human/physiology , Histocompatibility Antigens Class II/genetics , Histocompatibility Antigens Class II/metabolism , Hodgkin Disease/immunology , Hodgkin Disease/metabolism , Hodgkin Disease/pathology , Humans , Immunoenzyme Techniques , Lymphoproliferative Disorders/immunology , Lymphoproliferative Disorders/metabolism , Lymphoproliferative Disorders/pathology , Nuclear Proteins/genetics , Promoter Regions, Genetic/genetics , Proto-Oncogene Proteins/metabolism , RNA, Messenger/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Transcription Factor 3/metabolism , Transcriptional Activation , Viral Matrix Proteins/geneticsABSTRACT
UNLABELLED: BGLF4 kinase, the only Ser/Thr protein kinase encoded by the Epstein-Barr virus (EBV) genome, phosphorylates multiple viral and cellular substrates to optimize the cellular environment for viral DNA replication and the nuclear egress of nucleocapsids. Previously, we found that nuclear targeting of BGLF4 is through direct interaction with the FG repeat-containing nucleoporins (FG-Nups) Nup62 and Nup153 independently of cytosolic transport factors. Here, we investigated the regulatory effects of BGLF4 on the structure and biological functions of the nuclear pore complex (NPC). In EBV-positive NA cells, the distribution of FG-Nups was modified during EBV reactivation. In transfected cells, BGLF4 changed the staining pattern of Nup62 and Nup153 in a kinase activity-dependent manner. Detection with anti-phospho-Ser/Thr-Pro MPM-2 antibody demonstrated that BGLF4 induced the phosphorylation of Nup62 and Nup153. The nuclear targeting of importin ß was attenuated in the presence of BGLF4, leading to inhibition of canonical nuclear localization signal (NLS)-mediated nuclear import. An in vitro nuclear import assay revealed that BGLF4 induced the nuclear import of larger molecules. Notably, we found that BGLF4 promoted the nuclear import of several non-NLS-containing EBV proteins, including the viral DNA-replicating enzymes BSLF1, BBLF2/3, and BBLF4 and the major capsid protein (VCA), in cotransfected cells. The data presented here suggest that BGLF4 interferes with the normal functions of Nup62 and Nup153 and preferentially helps the nuclear import of viral proteins for viral DNA replication and assembly. In addition, the nuclear import-promoting activity was found in cells expressing the BGLF4 homologs of another two gammaherpesviruses but not those from alpha- and betaherpesviruses. IMPORTANCE: During lytic replication, many EBV genome-encoded proteins need to be transported into the nucleus, not only for viral DNA replication but also for the assembly of nucleocapsids. Because nuclear pore complexes are effective gateways that control nucleocytoplasmic traffic, most EBV proteins without canonical NLSs are retained in the cytoplasm until they form complexes with their NLS-containing partners for nuclear targeting. In this study, we found that EBV BGLF4 protein kinase interacts with the Nup62 and Nup153 and induces the redistribution of FG-Nups. BGLF4 modulates the function of the NPC to inhibit the nuclear import of host NLS-containing proteins. Simultaneously, the nuclear import of non-NLS-containing EBV lytic proteins was enhanced, possibly through phosphorylation of Nup62 and Nup153, nuclear pore dilation, or microtubule reorganization. Overall, our data suggest that BGLF4-induced modification of nuclear pore transport may block nuclear targeting of cellular proteins and increase the import of viral proteins to promote viral lytic replication.
Subject(s)
Active Transport, Cell Nucleus , Herpesvirus 4, Human/physiology , Membrane Glycoproteins/metabolism , Nuclear Pore Complex Proteins/metabolism , Protein Serine-Threonine Kinases/metabolism , Viral Proteins/metabolism , Virus Assembly , Virus Replication , Cell Line , Host-Pathogen Interactions , Humans , Nuclear Pore/metabolism , Phosphorylation , Protein Processing, Post-TranslationalABSTRACT
UNLABELLED: Epstein-Barr virus (EBV), an oncogenic herpesvirus, has the potential to immortalize primary B cells into lymphoblastoid cell lines (LCLs) in vitro. During immortalization, several EBV products induce cytokines or chemokines, and most of these are required for the proliferation of LCLs. Interleukin-32 (IL-32), a recently discovered proinflammatory cytokine, is upregulated after EBV infection, and this upregulation is detectable in all LCLs tested. EBV latent membrane protein 1 (LMP1) is responsible for inducing IL-32 expression at the mRNA and protein levels. Mechanistically, we showed that this LMP1 induction is provided by the p65 subunit of NF-κB, which binds to and activates the IL-32 promoter. Furthermore, the short hairpin RNA (shRNA)-mediated depletion of endogenous LMP1 and p65 in LCLs suppressed IL-32 expression, further suggesting that LMP1 is the key factor that stimulates IL-32 in LCLs via the NF-κB p65 pathway. Functionally, knockdown of IL-32 in LCLs elicits viral reactivation and affects cytokine expression, but it has no impact on cell proliferation and apoptosis. Of note, we reveal the mechanism whereby IL-32 is involved in the maintenance of EBV viral latency by inactivation of Zta promoter activity. This atypical cytoplasmic IL-32 hijacks the Zta activator protein kinase Cδ (PKCδ) and inhibits its translocation from the cytoplasm to the nucleus, where PKCδ binds to the Zta promoter and activates lytic cycle progression. These novel findings reveal that IL-32 is involved in the maintenance of EBV latency in LCLs. This finding may provide new information to explain how EBV maintains latency, in addition to viral chromatin structure and epigenetic modification. IMPORTANCE: EBV persists in two states, latency and lytic replication, which is a unique characteristic of human infections. So far, little is known about how herpesviruses maintain latency in particular tissues or cell types. EBV is an excellent model to study this question because more than 90% of people are latently infected. EBV can immortalize primary B cells into lymphoblastoid cell lines in vitro. Expression of IL-32, a novel atypical cytoplasmic proinflammatory cytokine, increased after infection. The expression of IL-32 was controlled by LMP1. In investigating the regulatory mechanism, we demonstrated that the p65 subunit of NF-κB is required for this upregulation. Of note, the important biological activity of IL-32 was to trap protein kinase Cδ in the cytoplasm and prevent it from binding to the Zta promoter, which is the key event for EBV reaction. So, the expression of LMP1-induced IL-32 plays a role in the maintenance of EBV latency.
Subject(s)
Herpesvirus 4, Human/physiology , Host-Pathogen Interactions , Interleukins/biosynthesis , Protein Kinase C-delta/metabolism , Viral Matrix Proteins/metabolism , Virus Latency , B-Lymphocytes/virology , Cells, Cultured , Herpesviridae , Humans , Transcription Factor RelA/metabolismABSTRACT
UNLABELLED: Epstein-Barr virus (EBV) BKRF3 shares sequence homology with members of the uracil-N-glycosylase (UNG) protein family and has DNA glycosylase activity. Here, we explored how BKRF3 participates in the DNA replication complex and contributes to viral DNA replication. Exogenously expressed Flag-BKRF3 was distributed mostly in the cytoplasm, whereas BKRF3 was translocated into the nucleus and colocalized with the EBV DNA polymerase BALF5 in the replication compartment during EBV lytic replication. The expression level of BKRF3 increased gradually during viral replication, coupled with a decrease of cellular UNG2, suggesting BKRF3 enzyme activity compensates for UNG2 and ensures the fidelity of viral DNA replication. In immunoprecipitation-Western blotting, BKRF3 was coimmuno-precipitated with BALF5, the polymerase processivity factor BMRF1, and the immediate-early transactivator Rta. Coexpression of BMRF1 appeared to facilitate the nuclear targeting of BKRF3 in immunofluorescence staining. Residues 164 to 255 of BKRF3 were required for interaction with Rta and BALF5, whereas residues 81 to 166 of BKRF3 were critical for BMRF1 interaction in glutathione S-transferase (GST) pulldown experiments. Viral DNA replication was defective in cells harboring BKRF3 knockout EBV bacmids. In complementation assays, the catalytic mutant BKRF3(Q90L,D91N) restored viral DNA replication, whereas the leucine loop mutant BKRF3(H213L) only partially rescued viral DNA replication, coupled with a reduced ability to interact with the viral DNA polymerase and Rta. Our data suggest that BKRF3 plays a critical role in viral DNA synthesis predominantly through its interactions with viral proteins in the DNA replication compartment, while its enzymatic activity may be supplementary for uracil DNA glycosylase (UDG) function during virus replication. IMPORTANCE: Catalytic activities of both cellular UDG UNG2 and viral UDGs contribute to herpesviral DNA replication. To ensure that the enzyme activity executes at the right time and the right place in DNA replication forks, complex formation with other components in the DNA replication machinery provides an important regulation for UDG function. In this study, we provide the mechanism for EBV UDG BKRF3 nuclear targeting and the interacting domains of BKRF3 with viral DNA replication proteins. Through knockout and complementation approaches, we further demonstrate that in addition to UDG activity, the interaction of BKRF3 with viral proteins in the replication compartment is crucial for efficient viral DNA replication.
Subject(s)
DNA Replication/genetics , DNA, Viral/genetics , Herpesvirus 4, Human/genetics , Uracil-DNA Glycosidase/genetics , Uracil-DNA Glycosidase/metabolism , Viral Proteins/genetics , Viral Proteins/metabolism , Cell Line , Cell Line, Tumor , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cytoplasm/genetics , Cytoplasm/metabolism , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , DNA-Directed DNA Polymerase/genetics , DNA-Directed DNA Polymerase/metabolism , Epstein-Barr Virus Infections/genetics , Epstein-Barr Virus Infections/metabolism , Epstein-Barr Virus Infections/virology , Genome, Viral/genetics , Glutathione Transferase/genetics , Glutathione Transferase/metabolism , HEK293 Cells , HeLa Cells , Herpesvirus 4, Human/metabolism , Humans , Virus Replication/geneticsABSTRACT
UNLABELLED: Epstein-Barr virus (EBV) lytic replication involves complex processes, including DNA synthesis, DNA cleavage and packaging, and virion egress. These processes require many different lytic gene products, but the mechanisms of their actions remain unclear, especially for DNA cleavage and packaging. According to sequence homology analysis, EBV BALF3, encoded by the third leftward open reading frame of the BamHI-A fragment in the viral genome, is a homologue of herpes simplex virus type 1 UL28. This gene product is believed to possess the properties of a terminase, such as nucleolytic activity on newly synthesized viral DNA and translocation of unit length viral genomes into procapsids. In order to characterize EBV BALF3, the protein was produced by and purified from recombinant baculoviruses and examined in an enzymatic reaction in vitro, which determined that EBV BALF3 acts as an endonuclease and its activity is modulated by Mg(2+), Mn(2+), and ATP. Moreover, in EBV-positive epithelial cells, BALF3 was expressed and transported from the cytoplasm into the nucleus following induction of the lytic cycle, and gene silencing of BALF3 caused a reduction of DNA packaging and virion release. Interestingly, suppression of BALF3 expression also decreased the efficiency of DNA synthesis. On the basis of these results, we suggest that EBV BALF3 is involved simultaneously in DNA synthesis and packaging and is required for the production of mature virions. IMPORTANCE: Virus lytic replication is essential to produce infectious virions, which is responsible for virus survival and spread. This work shows that an uncharacterized gene product of the human herpesvirus Epstein-Barr virus (EBV), BALF3, is expressed during the lytic cycle. In addition, BALF3 mediates an endonucleolytic reaction and is involved in viral DNA synthesis and packaging, leading to influence on the production of mature virions. According to sequence homology and physical properties, the lytic gene product BALF3 is considered a terminase in EBV. These findings identify a novel viral gene with an important role in contributing to a better understanding of the EBV life cycle.
Subject(s)
Endodeoxyribonucleases/metabolism , Endonucleases/metabolism , Herpesvirus 4, Human/enzymology , Herpesvirus 4, Human/physiology , Viral Proteins/metabolism , Virus Assembly , Virus Replication , Cations, Divalent/metabolism , Enzyme Activators/metabolism , Magnesium/metabolism , Manganese/metabolismABSTRACT
Nuclear export is an important process that not only regulates the functions of cellular factors but also facilitates the assembly of viral nucleoprotein complexes. Chromosome region maintenance 1 (CRM1) that mediates the transport of proteins bearing the classical leucine-rich nuclear export signal (NES) is the best-characterized nuclear export receptor. Recently, several CRM1-independent nuclear export pathways were also identified. The nuclear export of the large form of hepatitis delta antigen (HDAg-L), a nucleocapsid protein of hepatitis delta virus (HDV), which contains a CRM1-independent proline-rich NES, is mediated by the host NES-interacting protein (NESI). The mechanism of the NESI protein in mediating nuclear export is still unknown. In this study, NESI was characterized as a highly glycosylated membrane protein. It interacted and colocalized well in the nuclear envelope with lamin A/C and nucleoporins. Importantly, HDAg-L could be coimmunoprecipitated with lamin A/C and nucleoporins. In addition, binding of the cargo HDAg-L to the C terminus of NESI was detected for the wild-type protein but not for the nuclear export-defective HDAg-L carrying a P205A mutation [HDAg-L(P205A)]. Knockdown of lamin A/C effectively reduced the nuclear export of HDAg-L and the assembly of HDV. These data indicate that by forming complexes with lamin A/C and nucleoporins, NESI facilitates the CRM1-independent nuclear export of HDAg-L.
Subject(s)
Hepatitis Delta Virus/physiology , Hepatitis delta Antigens/metabolism , Host-Pathogen Interactions , Karyopherins/metabolism , Lamin Type A/metabolism , Nuclear Pore Complex Proteins/metabolism , Nucleocytoplasmic Transport Proteins/metabolism , Receptors, Cytoplasmic and Nuclear/metabolism , Active Transport, Cell Nucleus , Humans , Immunoprecipitation , Protein Multimerization , Virus Assembly , Exportin 1 ProteinABSTRACT
Epstein-Barr virus (EBV) alters the regulation and expression of a variety of cytokines in its host cells to modulate host immune surveillance and facilitate viral persistence. Using cytokine antibody arrays, we found that, in addition to the cytokines reported previously, two chemotactic cytokines, CCL3 and CCL4, were induced in EBV-infected B cells and were expressed at high levels in all EBV-immortalized lymphoblastoid cell lines (LCLs). Furthermore, EBV latent membrane protein 1 (LMP1)-mediated Jun N-terminal protein kinase activation was responsible for upregulation of CCL3 and CCL4. Inhibition of CCL3 and CCL4 in LCLs using a short hairpin RNA approach or by neutralizing antibodies suppressed cell proliferation and caused apoptosis, indicating that autocrine CCL3 and CCL4 are required for LCL survival and growth. Importantly, significant amounts of CCL3 were detected in EBV-positive plasma from immunocompromised patients, suggesting that EBV modulates this chemokine in vivo. This study reveals the regulatory mechanism and a novel function of CCL3 and CCL4 in EBV-infected B cells. CCL3 might be useful as a therapeutic target in EBV-associated lymphoproliferative diseases and malignancies.
Subject(s)
B-Lymphocytes/virology , Cell Proliferation , Chemokine CCL3/biosynthesis , Chemokine CCL4/biosynthesis , Herpesvirus 4, Human/pathogenicity , Host-Pathogen Interactions , Viral Matrix Proteins/metabolism , B-Lymphocytes/metabolism , B-Lymphocytes/physiology , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Signal TransductionABSTRACT
The cellular endosomal sorting complex required for transport (ESCRT) machinery participates in membrane scission and cytoplasmic budding of many RNA viruses. Here, we found that expression of dominant negative ESCRT proteins caused a blockade of Epstein-Barr virus (EBV) release and retention of viral BFRF1 at the nuclear envelope. The ESCRT adaptor protein Alix was redistributed and partially colocalized with BFRF1 at the nuclear rim of virus replicating cells. Following transient transfection, BFRF1 associated with ESCRT proteins, reorganized the nuclear membrane and induced perinuclear vesicle formation. Multiple domains within BFRF1 mediated vesicle formation and Alix recruitment, whereas both Bro and PRR domains of Alix interacted with BFRF1. Inhibition of ESCRT machinery abolished BFRF1-induced vesicle formation, leading to the accumulation of viral DNA and capsid proteins in the nucleus of EBV-replicating cells. Overall, data here suggest that BFRF1 recruits the ESCRT components to modulate nuclear envelope for the nuclear egress of EBV.
Subject(s)
Cell Nucleus/metabolism , Endosomal Sorting Complexes Required for Transport/metabolism , Herpesvirus 4, Human/physiology , Membrane Proteins/metabolism , Nuclear Envelope/metabolism , Viral Proteins/metabolism , Virus Assembly/physiology , Calcium-Binding Proteins/metabolism , Calcium-Binding Proteins/physiology , Cell Cycle Proteins/metabolism , Cell Cycle Proteins/physiology , Cell Line, Tumor , Endosomal Sorting Complexes Required for Transport/physiology , Gene Expression Regulation, Viral/physiology , Herpesvirus 4, Human/genetics , Herpesvirus 4, Human/metabolism , Humans , Membrane Proteins/genetics , Membrane Proteins/physiology , Protein Binding/genetics , Protein Transport , Tissue Distribution , Transfection , Viral Proteins/genetics , Viral Proteins/physiology , Virus Assembly/genetics , Virus Release/genetics , Virus Release/physiologyABSTRACT
BACKGROUND: Stromal cell derived factor-1 (SDF-1) plays a pivotal role in the recruitment of stem cells to injured livers. However, the changes of SDF-l in patients with hepatitis B virus (HBV)-related acute-on-chronic liver failure (ACLF) have yet to be elucidated. AIM: To study the SDF-1 changes in patients with HBV-related ACLF. METHODS: 30 patients with HBV-related ACLF, 27 patients with chronic hepatitis B and 20 healthy individuals are involved in our study. The SDF-l mRNA expression in liver tissue was detected by quantitative real-time polymerase chain reaction. Immunohistochemical staining was performed to illustrate the expression of SDF-l, CXC receptor 4 (CXCR4) and Ki67. The serum SDF-l concentrations were also detected by enzyme-linked immunosorbent assays. RESULTS: The expression of SDF-1 mRNA from ACLF patients was remarkably higher than that from other patients (both P < 0.05). The expression of SDF-l, CXCR4 and Ki67 from ACLF were the highest among the three groups (all P < 0.01). The serum SDF-l levels in ACLF patients were significantly lower than that in other patients (both P < 0.01). Moreover, in ACLF patients, the serum SDF-1 Levels were positively correlated with serum total bilirubin and international normalized ratio. In addition, the serum SDF-l levels in survival were significantly lower compared with the non-survivals (P < 0.05). The area under the curve for the serum SDF-1 level in predicting 28-d mortality was 0.722 (P < 0.05). CONCLUSION: This study provides the SDF-1 changes in patients with HBV-related ACLF. The SDF-1 Level at admission may serve as a promising prognostic marker for predicting short-term prognosis.
ABSTRACT
The unique virus-cell interaction in Epstein-Barr virus (EBV)-associated malignancies implies targeting the viral latent-lytic switch is a promising therapeutic strategy. However, the lack of specific and efficient therapeutic agents to induce lytic cycle in these cancers is a major challenge facing clinical implementation. We develop a synthetic transcriptional activator that specifically activates endogenous BZLF1 and efficiently induces lytic reactivation in EBV-positive cancer cells. A lipid nanoparticle encapsulating nucleoside-modified mRNA which encodes a BZLF1-specific transcriptional activator (mTZ3-LNP) is synthesized for EBV-targeted therapy. Compared with conventional chemical inducers, mTZ3-LNP more efficiently activates EBV lytic gene expression in EBV-associated epithelial cancers. Here we show the potency and safety of treatment with mTZ3-LNP to suppress tumor growth in EBV-positive cancer models. The combination of mTZ3-LNP and ganciclovir yields highly selective cytotoxic effects of mRNA-based lytic induction therapy against EBV-positive tumor cells, indicating the potential of mRNA nanomedicine in the treatment of EBV-associated epithelial cancers.