Global analysis of shared T cell specificities in human non-small cell lung cancer enables HLA inference and antigen discovery.

To identify disease-relevant T cell receptors (TCRs) with shared antigen specificity, we analyzed 778,938 TCRß chain sequences from 178 non-small cell lung cancer patients using the GLIPH2 (grouping of lymphocyte interactions with paratope hotspots 2) algorithm. We identified over 66,000 shared specificity groups, of which 435 were clonally expanded and enriched in tumors compared to adjacent lung. The antigenic epitopes of one such tumor-enriched specificity group were identified using a yeast peptide-HLA A∗02:01 display library. These included a peptide from the epithelial protein TMEM161A, which is overexpressed in tumors and cross-reactive epitopes from Epstein-Barr virus and E. coli. Our findings suggest that this cross-reactivity may underlie the presence of virus-specific T cells in tumor infiltrates and that pathogen cross-reactivity may be a feature of multiple cancers. The approach and analytical pipelines generated in this work, as well as the specificity groups defined here, present a resource for understanding the T cell response in cancer.

Study on the regulatory mechanism of latent membrane protein 2A on GCNT3 expression in nasopharyngeal carcinoma.

O-Glycan synthesis enzyme glucosaminyl (N-acetyl) transferase 3 (GCNT3) is closely related to the occurrence and development of various cancers. However, the regulatory mechanism and function of GCNT3 in nasopharyngeal carcinoma (NPC) are still poorly understood. This study aims to explore the regulatory mechanism of EBV-encoded latent membrane protein 2A (LMP2A) on GCNT3 and the biological role of GCNT3 in NPC. The results show that LMP2A can activate GCNT3 through the mTORC1 pathway, and there is a positive feedback between the mTORC1 and GCNT3. GCNT3 regulates EMT progression by forming a complex with ZEB1 to promote cell migration. GCNT3 can also promote cell proliferation. These findings indicate that targeting the LMP2A-mTORC1-GCNT3 axis may represent a novel therapeutic target in NPC.

Epstein-Barr Virus Latent Membrane Protein 2A (LMP2A) Enhances ATP Production in B Cell Tumors through mTOR and HIF-1α.

Epstein-Barr Virus (EBV) exists in a latent state in 90% of the world's population and is linked to numerous cancers, such as Burkitt's Lymphoma, Hodgkin's, and non-Hodgkin's Lymphoma. One EBV latency protein, latency membrane protein 2A (LMP2A), is expressed in multiple latency phenotypes. LMP2A signaling has been extensively studied and one target of LMP2A is the mammalian target of rapamycin (mTOR). Since mTOR has been linked to reprogramming tumor metabolism and increasing levels of hypoxia-inducible factor 1 α (HIF-1α), we hypothesized that LMP2A would increase HIF-1α levels to enhance ATP generation in B lymphoma cell lines. Our data indicate that LMP2A increases ATP generation in multiple Burkitt lymphoma cell lines that were dependent on HIF-1α. Subsequent studies indicate that the addition of the mTOR inhibitor, rapamycin, blocked the LMP2A-dependent increase in HIF-1α. Further studies demonstrate that LMP2A does not increase HIF-1α levels by increasing HIF-1α RNA or STAT3 activation. In contrast, LMP2A and mTOR-dependent increase in HIF-1α required mTOR-dependent phosphorylation of p70 S6 Kinase and 4E-BP1. These findings implicate the importance of LMP2A in promoting B cell lymphoma survival by increasing ATP generation and identifying potential pharmaceutical targets to treat EBV-associated tumors.

Nuclear respiratory factor 1 promotes the progression of EBV-associated gastric cancer and maintains EBV latent infection.

This study aimed to investigate the association of Epstein-Barr virus (EBV) with nuclear respiratory factor 1 (NRF1) and the biological function of NRF1 in EBV-associated gastric cancer (EBVaGC). Western blot and qRT-PCR were used to assess the effect of latent membrane protein 2A (LMP2A) on NRF1 expression after transfection with LMP2A plasmid or siLMP2A. The effects of NRF1 on the migration and apoptosis ability of GC cells were investigated by transwell assay and flow cytometry apoptosis analysis in vitro, respectively. In addition, we determined the regulatory role of NRF1 in EBV latent infection by western blot and droplet digital PCR (ddPCR). LMP2A upregulated NRF1 expression by activating the NF-κB pathway. Moreover, NRF1 upregulated the expression of N-Cadherin and ZEB1 to promote cell migration. NRF1 promoted the expression of Bcl-2 to increase the anti-apoptotic ability of cells. In addition, NRF1 maintained latent infection of EBV by promoting the expression of the latent protein Epstein-Barr nuclear antigen 1 (EBNA1) and inhibiting the expression of the lytic proteins. Our data indicated the role of NRF1 in EBVaGC progression and the maintenance of EBV latent infection. This provided a new theoretical basis for further NRF1-based anti-cancer therapy.

Transcriptome sequencing of LMP2A-transfected gastric cancer cells identifies potential biomarkers in EBV-associated gastric cancer.

Epstein-barr virus (EBV) is a well-known human oncogenic virus. However, its molecular mechanisms in the initiation and development of EBV-associated gastric cancer (EBVaGC) remain poorly understood. Latent membrane protein 2A (LMP2A) is an EBV latency-associated protein expressed in part of EBVaGC cases. This study analyzed the effect of LMP2A on the gene expression of gastric cancer cells by transcriptome sequencing on the gastric cancer cell line SGC7901 that expresses LMP2A. The study monitored a total of 238 genes with significant differences in expression, including 101 upregulated genes and 137 downregulated genes. Using the KEGG pathway analysis, it was found that more genes were enriched in the Steroid biosynthesis, Axon guidance, and Terpenoid backbone biosynthesis pathway, and there were 5 genes each enriched in PI3K-Akt and AMPK signaling pathway, all of which were significant. This indicates that LMP2A may be involved in cell biosynthesis, and affects downstream genes and cell biological behavior through AKT and AMPK signaling pathway. Further evaluation confirmed that LMP2A induces ETV5 transcription, but repress GATA6 and NOTCH3 expression. ETV5, GATA6 and NOTCH3 are the candidate targets of LMP2A in gastric cancer.

Virus-Mediated Inhibition of Apoptosis in the Context of EBV-Associated Diseases: Molecular Mechanisms and Therapeutic Perspectives.

Epstein-Barr virus (EBV), the representative of the Herpesviridae family, is a pathogen extensively distributed in the human population. One of its most characteristic features is the capability to establish latent infection in the host. The infected cells serve as a sanctuary for the dormant virus, and therefore their desensitization to apoptotic stimuli is part of the viral strategy for long-term survival. For this reason, EBV encodes a set of anti-apoptotic products. They may increase the viability of infected cells and enhance their resistance to chemotherapy, thereby contributing to the development of EBV-associated diseases, including Burkitt's lymphoma (BL), Hodgkin's lymphoma (HL), gastric cancer (GC), nasopharyngeal carcinoma (NPC) and several other malignancies. In this paper, we have described the molecular mechanism of anti-apoptotic actions of a set of EBV proteins. Moreover, we have reviewed the pro-survival role of non-coding viral transcripts: EBV-encoded small RNAs (EBERs) and microRNAs (miRNAs), in EBV-carrying malignant cells. The influence of EBV on the expression, activity and/or intracellular distribution of B-cell lymphoma 2 (Bcl-2) protein family members, has been presented. Finally, we have also discussed therapeutic perspectives of targeting viral anti-apoptotic products or their molecular partners.

Latent membrane protein 2A inhibits expression level of Smad2 through regulating miR-155-5p in EBV-associated gastric cancer cell lines.

Epstein-Barr virus (EBV) infection is one of the causes of gastric cancer (GC). Besides, previous studies have demonstrated that EBV-encoded latent membrane protein 2A (LMP2A) influences the pathogenesis of EBV-associated gastric cancer (EBVaGC) through regulating several key pathways. In this study, the expression level of Smad2 was observed, which was reduced in EBVaGC cell lines, especially in the presence of LMP2A. Meanwhile, we found that LMP2A promoted the expression of miR-155-5p by activated nuclear factor-κB (NF-κB) signaling. After being treated with NF-κB inhibitor (BAY 11-7082), miR-155-5p sharply decreased. Western blot analysis proved that the overexpression of miR-155-5p could inhibit Smad2. Functional studies showed that the role of miR-155-5p might lead to good prognosis in EBV-positive GC through promoting cell apoptosis and cell cycle arrest, as well as inhibiting tumor proliferation. In addition, p-Smad2 protein was also reduced or induced by overexpression or knockdown, respectively, of miR-155-5p. Immunofluorescence analysis further indicated that LMP2A prevented p-Smad2 from transferring to the nucleus, which played a crucial role in transforming growth factor-ß (TGF-ß) signaling. In summary, our findings confirmed the relationship between LMP2A and Smad2 and provided a potential regulation of the TGF-ß pathway in EBVaGC.

Mouse model of Epstein-Barr virus LMP1- and LMP2A-driven germinal center B-cell lymphoproliferative disease.

Epstein-Barr virus (EBV) is a major cause of immunosuppression-related B-cell lymphomas and Hodgkin lymphoma (HL). In these malignancies, EBV latent membrane protein 1 (LMP1) and LMP2A provide infected B cells with surrogate CD40 and B-cell receptor growth and survival signals. To gain insights into their synergistic in vivo roles in germinal center (GC) B cells, from which most EBV-driven lymphomas arise, we generated a mouse model with conditional GC B-cell LMP1 and LMP2A coexpression. LMP1 and LMP2A had limited effects in immunocompetent mice. However, upon T- and NK-cell depletion, LMP1/2A caused massive plasmablast outgrowth, organ damage, and death. RNA-sequencing analyses identified EBV oncoprotein effects on GC B-cell target genes, including up-regulation of multiple proinflammatory chemokines and master regulators of plasma cell differentiation. LMP1/2A coexpression also up-regulated key HL markers, including CD30 and mixed hematopoietic lineage markers. Collectively, our results highlight synergistic EBV membrane oncoprotein effects on GC B cells and provide a model for studies of their roles in immunosuppression-related lymphoproliferative diseases.

Mouse model for acute Epstein-Barr virus infection.

Epstein-Barr Virus (EBV) infects human B cells and drives them into continuous proliferation. Two key viral factors in this process are the latent membrane proteins LMP1 and LMP2A, which mimic constitutively activated CD40 receptor and B-cell receptor signaling, respectively. EBV-infected B cells elicit a powerful T-cell response that clears the infected B cells and leads to life-long immunity. Insufficient immune surveillance of EBV-infected B cells causes life-threatening lymphoproliferative disorders, including mostly germinal center (GC)-derived B-cell lymphomas. We have modeled acute EBV infection of naive and GC B cells in mice through timed expression of LMP1 and LMP2A. Although lethal when induced in all B cells, induction of LMP1 and LMP2A in just a small fraction of naive B cells initiated a phase of rapid B-cell expansion followed by a proliferative T-cell response, clearing the LMP-expressing B cells. Interfering with T-cell activity prevented clearance of LMP-expressing B cells. This was also true for perforin deficiency, which in the human causes a life-threatening EBV-related immunoproliferative syndrome. LMP expression in GC B cells impeded the GC reaction but, upon loss of T-cell surveillance, led to fatal B-cell expansion. Thus, timed expression of LMP1 together with LMP2A in subsets of mouse B cells allows one to study major clinically relevant features of human EBV infection in vivo, opening the way to new therapeutic approaches.

Latent Membrane Protein 1 (LMP1) and LMP2A Collaborate To Promote Epstein-Barr Virus-Induced B Cell Lymphomas in a Cord Blood-Humanized Mouse Model but Are Not Essential.

Epstein-Barr virus (EBV) infection is associated with B cell lymphomas in humans. The ability of EBV to convert human B cells into long-lived lymphoblastoid cell lines (LCLs) in vitro requires the collaborative effects of EBNA2 (which hijacks Notch signaling), latent membrane protein 1 (LMP1) (which mimics CD40 signaling), and EBV-encoded nuclear antigen 3A (EBNA3A) and EBNA3C (which inhibit oncogene-induced senescence and apoptosis). However, we recently showed that an LMP1-deleted EBV mutant induces B cell lymphomas in a newly developed cord blood-humanized mouse model that allows EBV-infected B cells to interact with CD4 T cells (the major source of CD40 ligand). Here we examined whether the EBV LMP2A protein, which mimics constitutively active B cell receptor signaling, is required for EBV-induced lymphomas in this model. We find that the deletion of LMP2A delays the onset of EBV-induced lymphomas but does not affect the tumor phenotype or the number of tumors. The simultaneous deletion of both LMP1 and LMP2A results in fewer tumors and a further delay in tumor onset. Nevertheless, the LMP1/LMP2A double mutant induces lymphomas in approximately half of the infected animals. These results indicate that neither LMP1 nor LMP2A is absolutely essential for the ability of EBV to induce B cell lymphomas in the cord blood-humanized mouse model, although the simultaneous loss of both LMP1 and LMP2A decreases the proportion of animals developing tumors and increases the time to tumor onset. Thus, the expression of either LMP1 or LMP2A may be sufficient to promote early-onset EBV-induced tumors in this model.IMPORTANCE EBV causes human lymphomas, but few models are available for dissecting how EBV causes lymphomas in vivo in the context of a host immune response. We recently used a newly developed cord blood-humanized mouse model to show that EBV can cooperate with human CD4 T cells to cause B cell lymphomas even when a major viral transforming protein, LMP1, is deleted. Here we examined whether the EBV protein LMP2A, which mimics B cell receptor signaling, is required for EBV-induced lymphomas in this model. We find that the deletion of LMP2A alone has little effect on the ability of EBV to cause lymphomas but delays tumor onset. The deletion of both LMP1 and LMP2A results in a smaller number of lymphomas in infected animals, with an even more delayed time to tumor onset. These results suggest that LMP1 and LMP2A collaborate to promote early-onset lymphomas in this model, but neither protein is absolutely essential.

Evasion of affinity-based selection in germinal centers by Epstein-Barr virus LMP2A.

Epstein-Barr virus (EBV) infects germinal center (GC) B cells and establishes persistent infection in memory B cells. EBV-infected B cells can cause B-cell malignancies in humans with T- or natural killer-cell deficiency. We now find that EBV-encoded latent membrane protein 2A (LMP2A) mimics B-cell antigen receptor (BCR) signaling in murine GC B cells, causing altered humoral immune responses and autoimmune diseases. Investigation of the impact of LMP2A on B-cell differentiation in mice that conditionally express LMP2A in GC B cells or all B-lineage cells found LMP2A expression enhanced not only BCR signals but also plasma cell differentiation in vitro and in vivo. Conditional LMP2A expression in GC B cells resulted in preferential selection of low-affinity antibody-producing B cells despite apparently normal GC formation. GC B-cell-specific LMP2A expression led to systemic lupus erythematosus-like autoimmune phenotypes in an age-dependent manner. Epigenetic profiling of LMP2A B cells found increased H3K27ac and H3K4me1 signals at the zinc finger and bric-a-brac, tramtrack domain-containing protein 20 locus. We conclude that LMP2A reduces the stringency of GC B-cell selection and may contribute to persistent EBV infection and pathogenesis by providing GC B cells with excessive prosurvival effects.

A human Fab exclusively binding to the extracellular domain of LMP2A.

In the areas of North Africa, Southeast Asia as well as South China, Nasopharyngeal carcinoma (NPC) is among the most widespread cancers. Plenty of research findings confirmed that Epstein-Barr virus (EBV) played a crucial role in NPC. EBV-encoded Latent membrane protein 2A (LMP2A) which continuously expressed in cell membrane protein induced an epithelial-mesenchymal transition and increased the number of side population stem-like cancer cells in NPC. This reveals that LMP2A could contribute to the development and recurrence in NPC. Above evidences suggest that LMP2A could be the potential target molecule in the treatment of NPC. In the current study, a novel human antibody Fab (Fab29) against the extracellular domain of LMP2A was produced with success. Through immunofluorescence experiment it was proved that human antibody Fab29 exclusively combined the surface of SUNE cells (LMP2A-positive). Then flow cytometry result exhibited that the fluorescent intensities of SUNE cells and CNE cells were distinct (96.89% and 0.02% respectively). After that, it was shown by affinity test that the Fab29 fragment had high affinity (KD (M) 1.79E-09) with LMP2A. It was also revealed by immunohistochemical analysis that the Fab29 fragment could combine with LMP2A-positive human NPC tissues in comparison with the control group. Finally, the MTT result indicated that the Fab29 fragment could inhibit the proliferation of LMP2A-positive NPC cells. The inhibiting rate to SUNE cell proliferation reached a peak by Fab29 (19.67%) compared with unrelated Fab and CNE with Fab29 at a concentration of 500 µg/L in first 24 h and in the next 24 h the inhibition rate grew to 22.54%. In brief, it was shown that Fab29, a characteristic human antibody, could recognize LMP2A protein and inhibit the proliferation of LMP2A-expressing NPC cells in vitro.

S100A4 hypomethylation affects epithelial-mesenchymal transition partially induced by LMP2A in nasopharyngeal carcinoma.

To identify cellular target genes involved in NPC cell invasion and metastasis, gene expression profiles of CNE-1 cells with or without ectopic LMP2A expression were compared by using the metastatic gene array. S100 calcium binding protein A4 (S100A4) was the highest increased one among these genes both in mRNA and protein levels of NPC cells. Moreover, S100A4 was upregulated in LMP2A-positive NPC tissues. We found that CNE-1-S100A4 showed significantly increased invasion ability as compared to the controls both in vitro and in vivo, which indicated that S100A4 induced EMT occurrence and promoted metastasis. Notably, the DNA hypomethylation of S100A4 was found in LMP2A-positive NPC tissues. Besides, inhibition of DNA methyltransferases via 5-Aza-dC stimulated the expression of S100A4 in the cells without ectopic LMP2A expression. The methylation changes were confirmed by methylation specific PCR (MSP), suggesting that LMP2A ectopic expression led to the demethylation of S100A4 promoter. These results demonstrated that LMP2A-induced hypomethylation participated in regulating S100A4 expression in NPC. Our findings provide an evidence for the emerging notion that hypomethylation and activation of correlated genes are crucial for metastasis progression in cancer. © 2015 Wiley Periodicals, Inc.

Latent Membrane Protein 2 (LMP2).

LMP2A is an EBV-encoded protein with three domains: (a) an N-terminal cytoplasmic domain, which has PY motifs that bind to WW domain-containing E3 ubiquitin ligases and an ITAM that binds to SH2 domain-containing proteins, (b) a transmembrane domain with 12 transmembrane segments that localizes LMP2A in cellular membranes, and (c) a 27-amino acid C-terminal domain which mediates homodimerization and heterodimerization of LMP2 protein isoforms. The most prominent two isoforms of the protein are LMP2A and LMP2B. The LMP2B isoform lacks the 19-amino acid N-terminal domain found in LMP2A, which modulates cellular signaling resulting in a baseline activation of B cells and degradation of cellular kinases leading to the downregulation of normal B cell signaling pathways. These two seemingly contradictory processes allow EBV to establish and maintain latency. LMP2 is expressed in many EBV-associated malignancies. While its antigenic properties may be useful in developing LMP2-specific immunity, the LMP2A N-terminal motifs also provide a basis to target LMP2A-modulated cellular kinases for the development of treatment strategies.

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A)-mediated changes in Fas expression and Fas-dependent apoptosis: Role of Lyn/Syk activation.

Epstein-Barr virus Latent Membrane Protein 2A (LMP2A) is expressed in EBV-infected B cells in the germinal center, a site of significant apoptosis induced by engagement of Fas on activated B cells. Signals from the B cell receptor (BCR) protect germinal center B cells from Fas-mediated apoptosis, and since LMP2A is a BCR mimic, we hypothesized that LMP2A would also protect B cells from Fas-mediated apoptosis. Surprisingly, latently-infected human and murine B cell lines expressing LMP2A were more sensitive to Fas-mediated apoptosis, as determined by increases in Annexin-V staining, and cleavage of caspase-8, -3 and PARP. Additional studies show that LMP2A-expressing B cell lines demonstrate a Lyn- and Syk-dependent increase in sensitivity to Fas-mediated apoptosis, due to an LMP2A-dependent enhancement in Fas expression. These findings demonstrate the ability for LMP2A to directly increase a pro-apoptotic molecule and have implications for EBV latency as well as the treatment of EBV-associated malignancies.

Skewed B cell differentiation affects lymphoid organogenesis but not T cell-mediated autoimmunity.

B cell receptor (BCR) signalling determines B cell differentiation and may potentially alter T cell-mediated immune responses. In this study we used two transgenic strains of BCR-deficient mice expressing Epstein-Barr virus latent membrane protein (LMP)2A in B cells, where either follicular and marginal zone differentiation (D(H)LMP2A mice) or B-1 cell development (V(H)LMP2A mice) were supported, and evaluated the effects of skewed B lymphocyte differentiation on lymphoid organogenesis and T cell responses in vivo. Compared to wild-type animals, both transgenic strains displayed alterations in the composition of lymphoid organs and in the dynamics of distinct immune cell subsets following immunization with the self-antigen PLP185₋206. However, ex-vivo T cell proliferation to PLP185₋206 peptide measured in immunized D(H)LMP2A and V(H)LMP2A mice was similar to that detected in immunized control mice. Further, clinical expression of experimental autoimmune encephalitis in both LMP2A strains was identical to that of wild-type mice. In conclusion, mice with skewed B cell differentiation driven by LMP2A expression in BCR-negative B cells do not show changes in the development of a T cell mediated disease model of autoimmunity, suggesting that compensatory mechanisms support the generation of T cell responses.

EBV and multiple sclerosis: expression of LMP2A in MS patients.

Several evidences, including increased serum titers of Epstein-Barr virus (EBV)-specific antibodies and the presence of EBV DNA in brain of patients suggest a possible role of this virus in the pathogenesis of Multiple Sclerosis (MS), a chronic neurodegenerative disease with an unknown etiopathology. Aim of the present study is to verify if the expression of LMP2A and EBNA-1, two EBV genes, is altered in MS patients. EBV viral load, LMP2A and EBNA-1 gene expression and EBNA-1 antibodies titers were evaluated in blood of EBV-seropositive MS patients (n = 57; 31 relapsing remitting -RRMS- and 26 progressive -PMS-patients) and age- and sex-matched healthy controls (HC, n = 49). Results showed that EBNA-1 and VCA antibodies titers are significantly augmented in MS patients compared to HC (p < 0.05 for both antibodies); detection of EBV DNA was more frequent as well in MS patients compared to HC, although without reaching statistical significance. Regarding viral gene expression, LMP2A was significantly more frequently detected and more expressed in MS patients compared to HC (p < 0.005) whereas no differences were observed for EBNA-1. Considering patients alone, EBNA-1 was significantly more frequent in PMS compared to RRMS (p < 0.05), whereas no differences were observed for LMP2A. Increased expression of the LMP2A latency-associated gene in MS patients supports the hypothesis that EBV plays a role in disease etiopathology.

WW domain-containing oxidoreductase is involved in upregulation of matrix metalloproteinase 9 by Epstein-Barr virus latent membrane protein 2A.

WW domain-containing oxidoreductase (WOX1) participates in tumor suppression and many other biologic functions, but its molecular and functional interactions with viral proteins remain largely unknown. This study reveals that WOX1 is physically associated with latent membrane protein 2A (LMP2A), an oncoprotein of Epstein-Barr virus. The molecular interaction involves the tyrosine residue 33 of WOX1 and the proline-rich motifs of LMP2A. Interestingly, endogenous WOX1 is required for some LMP2A-triggered, cancer-promoting effects, including activation of extracellular signal-regulated kinase-1/2, upregulation of matrix metalloproteinase 9 (MMP9) and promotion of cell invasion. Upon knockdown of endogenous WOX1, LMP2A-triggered MMP9 induction is restored by exogenous wild-type WOX1, but not by a WOX1 mutant defective in LMP2A binding. These results indicate that, through interaction with LMP2A, WOX1 is involved in MMP9 induction, suggesting a novel role of WOX1 in Epstein-Barr virus-associated cancer progression.

Diverse Expression Patterns of EBV Oncogenes (LMP2A, EBV-Encoded microRNA, and EBV-encoded dUTPase) in EBV Associated Gastric Carcinoma and their Association with Viral Loads.

The chromogenic in situ hybridization (CISH) test is the gold standard for detecting Epstein-Barr virus (EBV)-associated gastric carcinoma (GC). Real-time (RT) PCR method is also a sensitive test that can detect the viral load in samples. As such, three EBV oncogenes were investigated in this study. RNA extraction and cDNA synthesis were performed on GC tissues of nine patients, who were previously confirmed to have EBVGC subtype. In addition, 44 patients that had positive RT-PCR but negative CISH results were also included as the control group. TaqMan RT-PCR analysis was performed to determine the expression of EBV-encoded microRNAs, and the expression of EBV-encoded dUTPase, as well as LMP2A, was analyzed by SYBR Green RT-PCR. EBV-encoded microRNAs and LMP2A were identified in 2 out of 9 (22%) EBVGC subtypes. In addition, EBV-encoded dUTPase was detected in 4 out of 9 (44.5%) EBVGC subtypes. EBV-encoded dUTPase was also expressed in a sample of the control group. The expression of LMP2A, EBV-encoded microRNAs, and EBV-encoded dUTPase viral oncogenes in patients with high EBV viral loads indicates that these expressions correlate with viral loads. Our findings indicate that the EBV-encoded dUTPase gene may have a role in EBVGC patients' non-response to treatment and might be considered a Biomarker-targeted therapy.

A Mouse Model to Study the Pathogenesis of γ-herpesviral Infections in Germinal Center B Cells.

CD30-positive germinal center (GC)-derived B cell lymphomas are frequently linked to Epstein-Barr Virus (EBV) infection. However, a suitable animal model for the investigation of the interplay between γ-herpesvirus and host cells in B cell pathogenesis is currently lacking. Here, we present a novel in vivo model enabling the analysis of genetically modified viruses in combination with genetically modified GC B cells. As a murine γ-herpesvirus, we used MHV-68 closely mirroring the biology of EBV. Our key finding was that Cre-mediated recombination can be successfully induced by an MHV-68 infection in GC B cells from Cγ1-Cre mice allowing for deletion or activation of loxP-flanked cellular genes. The implementation of PrimeFlow RNA assay for MHV-68 demonstrated the enrichment of MHV-68 in GC and isotype-switched B cells. As illustrations of virus and cellular modifications, we inserted the EBV gene LMP2A into the MHV-68 genome and induced constitutively active CD30-signaling in GC B cells through MHV-68 infections, respectively. While the LMP2A-expressing MHV-68 behaved similarly to wildtype MHV-68, virally induced constitutively active CD30-signaling in GC B cells led to the expansion of a pre-plasmablastic population. The findings underscore the potential of our novel tools to address crucial questions about the interaction between herpesviral infections and deregulated cellular gene-expression in future studies.