Editorial: Signal transduction by viral factors: critical interface between the virus and its host cell with implications for the viral life cycle and disease development

Mosquito Salivary Gland Extracts Induce EBV-Infected NK Cell Oncogenesis Via CD4+ T Cells in Patients with Hypersensitivity to Mosquito Bites

Post-transplant lymphoproliferative disorder (PTLD) is a potentially fatal complication of organ transplantation characterized by abnormal proliferation of lymphoid cells in the setting of immunosuppression after transplantation. Epstein-Barr virus (EBV) is responsible for abnormal lymphocyte proliferation in 50–80% of PTLDs, particularly in early-onset disease. Latent membrane protein 1 (LMP1) is the chief oncogenic protein of EBV and has been shown to be critical in the transformation of human B cells by the virus. LMP1 may activate several cellular signal transduction pathways including MAPK/ERK, p38/JNK, PI3K/AKT and NF-kB/AP-1. The ability to usurp and dysregulate signaling pathways may explain how LMP1 promotes cell transformation, survival, and proliferation. Our laboratory has shown that LMP1 isolated from EBV-associated B cell lymphoma lines of PTLD patients contains gain-of-function mutations at AA212 (G-S) and AA366 (S-T) that result in sustained ERK signaling, c-Fos activation, and AP-1 activity. In this study, we asked whether these mutations, or other genetic alterations, are present in primary EBV+ PTLD tumors themselves. DNA was isolated from formalin-fixed paraffin-embedded tissue sections of EBV+ PTLD tumors (n=8). Nested PCR was used to amplify LMP1, and the PCR products generated were cloned and sequenced. The presence or absence of gain-of-function mutations at AA212 (G-S) and AA366 (S-T) were assessed. 7 out of the 8 EBV+ PTLD tumors demonstrated both gain-of-function mutations in LMP1. Furthermore, 6 out of the 8 tumors contained an extra repeat of 8 amino acids within the LMP1 signaling tail corresponding to a putative JAK3 binding motif. We have previously identified this repeat in 3 of 6 EBV+ B cell lymphoma lines from PTLD patients. LMP1 was also cloned from the whole blood of a pediatric small bowel PTLD patient. In addition to these 2 mutations being present, the 8 amino acid repeat was also present but in triplicate, suggesting this motif may be crucial to the oncogenic activity of LMP1. In order to assess host cell mutations in EBV+ PTLD tumors, a qBiomarker Mutation PCR array was performed for the PI3K/AKT/mTOR pathway, known to be important in human malignancies. While mutations in PI3K/AKT/mTOR were identified in EBV+ B cell lymphoma lines from PTLD patients, there were significantly more mutations in the primary EBV+ PTLD tumors, with 25 distinct mutations found within the PTEN, PI3K, and STK11 molecules. 3 distinct mutations were identified in more than 1 tumor, and 4 distinct mutations were shared between a cell line and tumor. Our findings clearly demonstrate that key gain-of-functions mutations in LMP1 detected in blood and cell lines are also detected in the primary tumor, suggesting a role in tumorigenesis and great potential as biomarkers of EBV+ PTLD. Moreover, host cell mutations may also contribute to dysregulation of key signal transduction pathways in EBV+ PTLD.

Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1) is oncogenic and indispensable for EBV-mediated B cell transformation. LMP1 is capable of activating several intracellular signaling pathways including the NF-kappaB pathway, which contributes to the EBV-mediated cell transformation. Two regions in the cytoplasmic carboxyl tail of LMP1, namely C-terminal activating regions 1 and 2 (CTAR1 and CTAR2), are responsible for NF-kappaB activation, with CTAR2 being the main NF-kappaB activator. Although the CTAR1-mediated NF-kappaB activation was previously shown to be TRAF3-dependent, we showed here that the CTAR2-mediated NF-kappaB activation is mainly TRAF6-dependent but TRAF2/5-independent. In contrast to the interleukin-1 receptor/toll-like receptor-mediated NF-kappaB pathways, the CTAR2-mediated NF-kappaB pathway does not require MyD88, IRAK1, or IRAK4 for TRAF6 engagement. Furthermore, we showed that TAK1 is required for NF-kappaB activation by LMP1. Thus, LMP1 utilizes two distinct pathways to activate NF-kappaB: a major one through CTAR2/TRAF6/TAK1/IKKbeta (canonical pathway) and a minor one through CTAR1/TRAF3/NIK/IKKalpha (noncanonical pathway).

Activation of the JAK/STAT Pathway in Epstein Barr Virus+-Associated Posttransplant Lymphoproliferative Disease: Role of Interferon-γ

21099 Background: Epstein Barr virus (EBV), a human oncogenic virus, has two types, EBV1 and 2, and several polymorphic markers allow individual variants to be distinguished. Variations in EBV latent membrane protein 1 (LMP1) gene could define a more oncogenic strain or reflect geographic EBV origin. By sequence analysis of C-ter, N-ter and promoter (prom) regions, 4 distinct EBV groups (A to D) have been defined. Aim: To analyze LMP1 molecular variability of two different groups of Hodgkin`s lymphoma (HL) from the same geographic area. Methods: EBV association by EBERs in situ hibridization and LMP1 immunohistochemistry was analyzed in 27 HL from Argentina (Arg) and 36 HL from Brazil (Br). EBV type was assessed by EBNA PCR. C-ter LMP1 PCR was done in all cases. Sequencing of C-ter, N-ter and prom regions was done in 16 patients. Results: 21/27 (78%) Arg HL and 31/36 (86%) Br HL were EBV1, while 6/27 (22%) and 4/36 (11%) were EBV2, respectively (p= 0.29). Coinfection was observed in 1 (3%) Br HL. LMP1 deleted (del) variant was observed in 20/27 (74%) Arg HL and 20/36 (55%) Br HL (p= 0.18), as well as in non-neoplastic controls, 4/11 and 3/10 (36 and 30%) from Arg and Br respectively. Most LMP1 del displayed high number (5–7) of 33bp repeats (86% LMP1 del Br HL and 77% LMP1 del Arg HL) compared with LMP1 wt that exhibited low number (3–4) 33bp repeats (68% LMP1 wt Br HL and 100% LMP1 wt Arg HL). Analysis of LMP1 sequences showed that: LMP1 wt C-ter regions had unmutated N-ter and prom as B95.8, A and B groups (31%), except for a case showing new mutations. LMP1 del C-ter regions showed molecular identity with C group, but they showed new, undescribed mutations in prom and N-ter (63%). Conclusions: We found high frequency of LMP1 del variants in HL from Argentina and Brazil, which was associated with high number of 33bp repeats in C-ter region. This suggests a role for this variant in lymphomagenesis. A new molecular variant with characteristic promoter and N-ter mutations was identified in LMP1 del cases, which could be proposed as a regional South American variant. No significant financial relationships to disclose.

Simple SummaryEpstein-Barr Virus (EBV) infection is associated with various lymphomas and carcinomas as well as other diseases in humans. The transmembrane protein LMP1 plays versatile roles in EBV life cycle and pathogenesis, by perturbing, reprograming, and regulating a large range of host cellular mechanisms and functions, which have been increasingly disclosed but not fully understood so far. We summarize recent research progress on LMP1 signaling, including the novel components LIMD1, p62, and LUBAC in LMP1 signalosome and LMP1 novel functions, such as its induction of p62-mediated selective autophagy, regulation of metabolism, induction of extracellular vehicles, and activation of NRF2-mediated antioxidative defense. A comprehensive understanding of LMP1 signal transduction and functions may allow us to leverage these LMP1-regulated cellular mechanisms for clinical purposes. The Epstein–Barr Virus (EBV) principal oncoprotein Latent Membrane Protein 1 (LMP1) is a member of the Tumor Necrosis Factor Receptor (TNFR) superfamily with constitutive activity. LMP1 shares many features with Pathogen Recognition Receptors (PRRs), including the use of TRAFs, adaptors, and kinase cascades, for signal transduction leading to the activation of NFκB, AP1, and Akt, as well as a subset of IRFs and likely the master antioxidative transcription factor NRF2, which we have gradually added to the list. In recent years, we have discovered the Linear UBiquitin Assembly Complex (LUBAC), the adaptor protein LIMD1, and the ubiquitin sensor and signaling hub p62, as novel components of LMP1 signalosome. Functionally, LMP1 is a pleiotropic factor that reprograms, balances, and perturbs a large spectrum of cellular mechanisms, including the ubiquitin machinery, metabolism, epigenetics, DNA damage response, extracellular vehicles, immune defenses, and telomere elongation, to promote oncogenic transformation, cell proliferation and survival, anchorage-independent cell growth, angiogenesis, and metastasis and invasion, as well as the development of the tumor microenvironment. We have recently shown that LMP1 induces p62-mediated selective autophagy in EBV latency, at least by contributing to the induction of p62 expression, and Reactive Oxygen Species (ROS) production. We have also been collecting evidence supporting the hypothesis that LMP1 activates the Keap1-NRF2 pathway, which serves as the key antioxidative defense mechanism. Last but not least, our preliminary data shows that LMP1 is associated with the deregulation of cGAS-STING DNA sensing pathway in EBV latency. A comprehensive understanding of the LMP1 signaling landscape is essential for identifying potential targets for the development of novel strategies towards targeted therapeutic applications.

The Epstein-Barr virus (EBV)-encoded protein latent membrane protein 1 (LMP1) is essential for EBV-mediated B cell transformation and plays a critical role in the development of post-transplant B cell lymphomas. LMP1 also contributes to the exacerbation of autoimmune diseases such as systemic lupus erythematosus (SLE). LMP1 is a functional mimic of the tumor necrosis factor receptor (TNFR) superfamily member CD40, and relies on TNFR-associated factor (TRAF) adaptor proteins to mediate signaling. However, LMP1 activation signals to the B cell are amplified and sustained compared to CD40 signals. We previously demonstrated that LMP1 and CD40 use TRAF molecules differently. Although associating with CD40 and LMP1 via separate mechanisms, TRAF6 plays a significant role in signal transduction by both. It is unknown whether TRAF6 mediates CD40 versus LMP1 functions via distinct or shared pathways. In this study, we tested the hypothesis that TRAF6 uses the kinase TAK1 to trigger important signaling pathways following both CD40 and LMP1 stimulation. We determined that TAK1 was required for JNK activation and interleukin-6 (IL-6) production mediated by CD40 and LMP1, in both mouse and human B cells. Additionally, TRAF3 negatively regulated TRAF6-dependent, CD40-mediated TAK1 activation by limiting TRAF6 recruitment. This mode of regulation was not observed for LMP1 and may contribute to the dysregulation of LMP1 compared to CD40 signals.

Infection by the Epstein-Barr virus (EBV) is associated with a variety of human cancers, notably Burkitt lymphoma and nasopharyngeal carcinoma (NPC). Most malignant cells within EBV-associated cancers are latently infected, and some viral products expressed during the latent phase of the viral cycle have well-described carcinogenicity, including the EBV latent membrane protein 1 (LMP1). Distinct viral genotypes described so far include the B95.8, the most studied viral strain originally found in an elderly patient with transfusion-induced infectious mononucleosis, and the M81 strain, isolated from a NPC case from a Chinese patient. Compared to B95.8, the EBV M81 strain infects epithelial cells more readily, and it is more likely to induce spontaneous viral lytic reactivation. However, whether these EBV strains have distinct biological features relevant for the progression of EBV-associated cancers remains to be elucidated. Thus, this study aimed to evaluate whether the expression of B95.1 and M81 variants of EBV LMP1 have different impact on cell migration and invasiveness of human cells lines in vitro, as well as on the expression of selected endogenous microRNAs (miRs) that were previously reported to have a role on cancer progression. Expression of EBV LMP1 was achieved in 293 and NP69 cells transfected with vectors encoding its B95.8 or M81 variants. In vitro migration and invasion rates were assessed by the scratch wound healing assay and the Boyden chamber assay, respectively. The expression of 91 selected miRs were evaluated by a customized qPCR-array approach. Cells expressing EBV LMP1 showed increased migration and invasion rates compared with mock-transfected cells. However, the differences observed between B95.8 and M81 variants did not achieve statistical significance. Conversely, 293 cells expressing the M81 variant of LMP1 showed hyperexpression of human miRs 497-5p, 17-3p, and 34a-5p, compared to B95.8. The in silico target prediction for these miRs showed enrichment of gene targets involved in the MAPK, NF-κB, and PI3K/AKT intracellular signaling pathways. This would reflect in negative regulation of cell proliferation and cell motility/adhesion, as well as higher levels of apoptosis. Based on these data, it is plausible to suggest that EBV LMP1 from M81 viral strain is somewhat less effective in terms of carcinogenicity, and this might be a reflect of the improved ability of EBV M81 to induce lytic viral infection compared to the B95.8 strain, as previously reported. In conclusion, the results indicate that cells expressing LMP1 from EBV isolates B95.8 and M81 showed differences in terms of miR expression signatures, which ultimately might impact the pathogenesis and progression of EBV-associated cancers. Citation Format: Bárbara Grasiele Müller-Coan, Ethel Cesarman, Deilson Elgui De Oliveira. Effects of Epstein-Barr virus latent membrane protein 1 (LMP1) on cell invasiveness and expression of endogenous microRNAs in human cells in vitro [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5756. doi:10.1158/1538-7445.AM2017-5756

The Epstein-Barr virus (EBV) oncoprotein LMP1 (latent membrane protein 1) mimics a constitutively active receptor molecule. It contributes to viral cell transformation by the activation of NF-kappaB, JNK/AP1, MAPK, JAK/STAT and PI3-kinase signaling. LMP1 recruits TRAF1-3, 5 and 6, TRADD and RIP1, which are also known as signaling mediators of Toll-like and tumor necrosis factor-receptors. Here, we established a functional proteomics approach to identify novel interaction partners of the LMP1 signaling domain. This approach led to the characterization of the tyrosine phosphatase SHP1 as a direct binding partner of LMP1. Interaction of SHP1 with LMP1 was verified in primary human B-cells, which had been transformed with a recombinant EBV carrying a HA-tagged LMP1 allele. The SHP1 binding site of LMP1 is located within the membrane-proximal region of the LMP1 signaling domain and shows no overlap with known protein interaction domains of LMP1. The unique sequence of this site does not resemble known SHP1 interaction motifs of cellular proteins. Mutation of the SHP1 site caused the loss of SHP1 binding to LMP1 in EBV-transformed human B-cells. SHP1 has previously been described as a negative regulator of growth factor or immune receptor signaling by dephosphorylating e.g. tyrosine kinases such as JAKs or SRC kinases. LMP1 induction of the NF-kappaB pathway was greatly enhanced in SHP1-knockout DT40 B-cells as compared to wildtype cells. This effect was reverted by reconstitution of SHP1 expression in the SHP1-KO cells. Also mutation of the SHP1 interaction site or the co-expression of a dominant-negative SHP1 caused hyperactivation of NF-kappaB signaling and JAK3 hyperphosphorylation by LMP1. Because the SHP1 interaction site of LMP1 mediates inhibitory effects on LMP1 signaling, we named this region CTIR1 (C-terminal inhibitory region 1). In summary, the proteomic analysis of the LMP1 complex revealed a novel autoregulatory mechanism of oncogenic LMP1 signaling, which limits its own activity through the recruitment of a tyrosine phosphatase. This mechanism might be of high relevance for the survival of EBV-transformed cells because LMP1 hyperactivity is known to be toxic for the target cells.

Aims: Epstein-Barr virus (EBV) immortalises B cells in vitro and is associated with several malignancies. Most phenotypic effects of EBV are mediated by latent membrane protein 1 (LMP1), which interacts...

P20: Does LMP1 oncogene expression pattern reflect specific pathology or geographic origin of the EPSTEIN–BARR virus?

BackgroundA strong association between Epstein-Barr virus (EBV) infection and nasopharyngeal carcinoma (NPC) has been widely recognized in recent decades. The aim of the present study was to investigate latent membrane protein 1 (LMP1) regulation of nasopharyngeal carcinoma (NPC) CNE-2 cell growth and then examine the effects of LMP1-knockout with CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9on Epstein-Barr virus (EBV) infection and CNE-2 cell growth.MethodsHuman NPC CNE-2 cells were infected with the recombinant LMP1- and LMP2A-carrying lentivirus, and then examined for cell growth with the colony forming assay as well as for the activation of transcription of eukaryotic translation initiation factor 4E (eIF4E) with reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) and western blot. CRISPR/Cas9-mediated knockout of LMP1 or LMP2A was performed with a single-guide RNA (sgRNA) targeting sequences within LMP1 or LMP2A. The knockout effect and the EBV proliferation were examined with RT-qPCR, western blot and cell growth assay.ResultsLMP1 overexpression promoted CNE-2 cell growth, compared to LMP2A overexpression. Loss-of-function experiments confirmed that eukaryotic translation initiation factor 4E (eIF4E) upregulation mediated this effect. LMP1 knockout significantly inhibited EBV proliferation in CNE-2 cells and markedly inhibited LMP1-mediated promotion of cell growth. The knockout of either LMP1 or LMP2A blocked the eIF4E activation, which is induced either by the EBV infection or by the overexpression of LMP1 or LMP2A.ConclusionWe confirmed the LMP1-mediated promotion of NPC cell growth. Such promotion can be effectively blocked by CRISPR/Cas9-mediated LMP1 knockout. Precise LMP1 knockout might be a promising method for targeted inhibition of EBV infection and NPC cell growth.

Epstein-Barr virus (EBV) is an ubiquitous human g-herpesvirus and is associated withnseveral lymphoid and epithelial malignancies, like Burkitt's lymphoma (BL),nnasopharyngeal carcinoma (NPC), Hodgkin's disease (HD), B- and T-cell non-Hodgkin's lymphoma and gastric carcinomas. EBV is transcriptionally active in thenmalignant cells of these cancers, leading to abundant expression of a restricted set ofnEBV encoded proteins. These gene expression patterns can be divided into 3 differentnlatency patterns. Latency II type is characterised by the expression of Epstein-Barrnnuclear antigen 1 (EBNA1), latent membrane protein 1 (LMP1) and latent membranenprotein (LMP2). This latency pattern is found in HD and NPC and these type nnmalignancies characteristically occur in apparently immunocompetent individuals. Ofnthe three EBV proteins expressed in HD and NPC, EBNA1 is not processed. Thenglycine-alanine repeat (GAr) sequences within this protein inhibit proteasomalnprocessing and thus this protein is not an option for immunotherapy. Thus LMP1 andnLMP2 are considered to be the only suitable targets for immunotherapy.n In recent years CD8+ cytotoxic T-cell (CTL) responses to multiple LMP2nepitopes have been described, but only a limited number of epitopes have beenndescribed for LMP1. Little is known about the CD4+T-cell responses directed againstnLMPl and LMP2. In the present study, the ex vivo T-cell response directed againstnLMPl was analysed using interferon-g-(IFN-g)- based enzyme linked immunospotn(ELISPOT) assays in a large panel of healthy EBV carriers of diverse ethnic originnand NPC patients. By comparing the frequencies of T cells specific for overlappingnpeptides spanning LMP1, 18 novel CD8 and CD4 restricted LMP1 T-cell epitopesnwere identified. Of the 7 new T-cell epitopes with defined HLA restrictions, 5 werenHLA A2-restricted and one is restricted through both HLA-B57 and HLA-B58 whilenanother epitope showed potential CD4+ T-cell reactivity. More importantly, extensivensequence analysis of LMP1 revealed that the majority of the T-cell epitopes are highlynconserved in EBV isolates from Caucasian, Papua New Guinean, African andnSoutheast Asian populations.nn In the next phase of this study, the efficacy of delivering LMP1 epitopes as antherapeutic vaccine was examined to test whether this strategy would potentiallynprovide a long-term benefit to EBV-associated HD and NPC patients. Hence anpolyepitope vaccine comprising 6 HLA A2-restricted LMP1 epitopes expressed wasndeveloped using a recombinant vaccinia virus vector. Human cells infected with thisnrecombinant polyepitope construct were efficiently recognised by LMP1-specificnCTL lines from HLA A2 healthy individuals. Furthermore, immunisation of HLAnA/Kb mice with this polyepitope vaccine consistently generated strong LMP1 specificnCTL responses to 5 of the 6 epitopes which were readily detected by both exnvivo and in vitro assays. More importantly, this polyepitope vaccine successfullynreversed the outgrowth of LMP1 expressing tumours in HLA A2/Kb mice.n Furthermore, another polyepitope vaccine was made to provide coverage overna broad range of HLA types with particular emphasis on HLA types present in NPCnendemic regions (HLA A2, Al 1, A24, B27, B40) and both LMP1 and LMP2 epitopesnwere included. This polyepitope was inserted into a clinical grade replication-incompetentnadenovirus vector. Immunisation with this adenovirus vector expressingnthe LMP polyepitope was capable of inducing multiple independent MHC-restrictednCTL responses. These epitopes were not only efficiently processed endogenously bynthe human cells but also recalled memory CTL responses specific for LMP antigensnin healthy virus carriers. In vivo studies showed that this polyepitope vaccine wasncapable of inducing a primary T-cell response which was shown to be therapeutic in antumour challenge model by reversing LMP1-expressing tumours in HLA-A2/Kb mice.n The final phase of this study was aimed to profile LMP memory response innprimary (newly diagnosed and relapsed) and long-term HD patients. The HD patientsnwere all serologically positive but importantly in about half of the patients thenmalignant Hodgkin-Reed-Stemberg (H-RS) cells were LMP positive. The resultsnshow that the memory response was significantly weak or undetectable in primary HDnpatients when compared to long-term HD patients although the latter group were notnas strong as that seen in healthy immune individuals. These results indicate that HDnpatients with active disease suffer a reduced LMP-specific CTL memory response.nImportantly, this reduction of LMP-specific T-cell response was independent of thenLMP status of the H-RS cells. The activation protocol includes a number of novelnaspects designed to minimise the period required to achieve significant levels of lysisnin bulk cultures from patients.n The results of these studies are important in terms of developing therapeuticnstrategies against HD and NPC. On the one hand, the adenovirus-polyepitope mightnbe used as a therapeutic vaccine to raise the CTL response to LMP1 and LMP2nepitopes. On the other hand, the in vitro activation and expansion of these memorynresponses to these proteins offers the potential of their use in autologous adoptiventransfer. In this application, the adenovirus-polyepitope would be used to activatenautologous LMP1- and 2-specific bulk cultures and to adoptively transfer them backninto HD and NPC patients. Both of these protocols might be used as either stand-alonentherapy or in concert.

High prevalence of a 30-base pair deletion in the Epstein-Barr virus (EBV) latent membrane protein 1 gene and of strain type B EBV in Mexican classical Hodgkin's disease and reactive lymphoid tissue

Almost exactly twenty years after the discovery of Epstein-Barr virus (EBV), the latent membrane protein 1 (LMP1) entered the EBV stage, and soon thereafter, it was recognized as the primary transforming gene product of the virus. LMP1 is expressed in most EBV-associated lymphoproliferative diseases and malignancies, and it critically contributes to pathogenesis and disease phenotypes. Thirty years of LMP1 research revealed its high potential as a deregulator of cellular signal transduction pathways leading to target cell proliferation and the simultaneous subversion of cell death programs. However, LMP1 has multiple roles beyond cell transformation and immortalization, ranging from cytokine and chemokine induction, immune modulation, the global alteration of gene and microRNA expression patterns to the regulation of tumor angiogenesis, cell-cell contact, cell migration, and invasive growth of tumor cells. By acting like a constitutively active receptor, LMP1 recruits cellular signaling molecules associated with tumor necrosis factor receptors such as tumor necrosis factor receptor-associated factor (TRAF) proteins and TRADD to mimic signals of the costimulatory CD40 receptor in the EBV-infected B lymphocyte. LMP1 activates NF-κB, mitogen-activated protein kinase (MAPK), phosphatidylinositol 3-kinase (PI3-K), IRF7, and STAT pathways. Here, we review LMP1's molecular and biological functions, highlighting the interface between LMP1 and the cellular signal transduction network as an important factor of virus-host interaction and a potential therapeutic target.