In normal pregnant women, reactivation of Epstein-Barr virus (EBV) frequently occurs. Cellular immune response is apparently supperessed, but the antibodies cross the placenta and protect the infant against primary infection for many months. The Epstein¡VBarr virus (EBV) encoded latent membrane protein 1 (LMP1) which plays a crucial role in the long-term persistence of this virus within the cells of the immune system. Not only is this protein critical for the transformation of resting B cells by EBV, it also displays pleiotropic effects on various cellular proteins expressed in the host cell. These include upregulation of express- ion of B cell activation antigens, adhesion molecules, and various components of the antigen processing pathway. Here we test 45 human placenta samples for LMP1 of Epstein¡VBarr virus (EBV) by RT-PCR and immunohistochemistry. Eight of forty five (18%) are positive , and thirty seven of forty five (82%) are negative for LMP expression. It is noticed that all LMP protein expression were shown on the maternal side but not on the fetal side. The placenta may serve as a protection barrier for fetus against Epstein-Barr virus infection.

Le lymphome de Hodgkin est un cancer des ganglions lymphatiques caractérisé par la présence de cellules multinucléées Reed-Sternberg et de leur précurseur, les cellules mononucléées de Hodgkin. Ces cellules sont les cellules diagnostiques de ce cancer. Elles sont responsables de certains symptômes ressentis par les patients et aussi des cas de résistances aux agents thérapeutiques lors des traitements. Dans 10 à 20 % des cas de lymphome de Hodgkin, les patients ne répondent pas aux traitements et décèdent ou encore souffrnt de rechutes ou de complications qui se développent suite à l'exposition aux agents chimio- et radiothérapeutiques. Ces problèmes amènent un besoin de cibler de nouvelles avenues pour développer des traitements plus spécifiques permettant de diminuer les doses et d'augmenter le taux de réussite des traitements pour ce cancer. L'approche utilisée dans ce travail est l'étude du phénomène qui amène la multinucléation des cellules. En effet, les cellules mononucléées de Hodgkin subissent divers dommages qui amènent ces dernières à l'endomitose et puis à la multinucléation. II est connu que le virus Epstein-Barr joue un rôle dans le developpement de divers cancer, incluant le lymphome de Hodgkin, toutefois, le mécanisme par lequel ce virus provoque le développement du cancer est inconnu. L'oncoprotéine LMP1, exprimée par le virus Epstein-Barr, est le princical oncogène du virus, cette protéine, lorsqu'exprimée dans certaines lignées cellulaires hodgkiniennes amène la multinucléation des cellules. Ce phénomène est accompagné par la diminution de l'expression des protéines du complexe des shelterins TRF1, TRF2 et POT! dans la lignée cellulaire BJAB tTA LMP1. Ces protéines ont pour rôle de protéger l'intégrité des télomères et en présence de LMP1, ces dernières sont inhibées. Les problèmes pouvant résulter de la déprotection des télomères sont nombreux, notamment, il peut y avoir des jonctions d'extrémités non homologues qui amènent la fusion de chromosomes voisins ce qui provoque des complications au niveau de la mitose. Dans cet ouvrage, l'impact de la déprotection des télomères sur la multinucléation des cellules B est étudié avec un modèle d'induction de la protéine LMP1 et avec un système d'inhibition des protéines TRF2 et POT1.

Epstein-Barr Virus (EBV) is well known to manipulate the host ubiquitin machinery to facilitate its latent persistence and oncogenesis, exemplified by LMP1 signal transduction that activates multiple transcription factors, including NFκB, AP1, and IRF7/IRF4, which promote cell survival and outgrowth, and control immune response and inflammation. It is therefore vital to delineate the detailed mechanisms underlying LMP1 signal transduction for understanding EBV-mediated oncogenesis. p62 (also called SQSTM1, Sequestosome 1) is a ubiquitin sensor and a signal transducing adaptor that interacts with TRAF6 and facilitates the recruitment of ubiquitinated signal intermediators for the activation of NFκB and AP1 in diverse contexts. In turn, p62 is induced by NFκB. However, the interaction between p62 and EBV latency has never been studied. We have recently published interesting and important results, which imply a crucial role for p62 in EBV-mediated oxidative stress. In this study, we further show that p62 is upregulated in EBV latency, with the contribution of LMP1-mediated NFκB and AP1 activities. In turn, p62 participates in LMP1 signal transduction through its interaction with TRAF6, promoting TRAF6 ubiquitination. shRNA-mediated p62 depletion downregulates LMP1-TRAF6 interaction and TRAF6 ubiquitination, and significantly impairs AP1 activity; however, with no detectable effects on NFκB activity. These observations imply that TRAF6-p62 interaction differentiates LMP1 signaling to NFκB and AP1 activation. As a consequence, p62 depletion promotes etoposide-induced apoptosis. These findings identify p62 as a novel player in EBV LMP1 signaling to AP1 activation that is crucial for LMP1-mediated ROS production.

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.

L’infection chronique est à l’origine de 15-20% des cancers dans le monde. Dans la plupart des cas, les infections sont éliminées par le système immunitaire, sans incidence importante sur les hôtes infectés. Toutefois, les oncovirus peuvent échapper au système immunitaire et induire une transformation cellulaire, ce qui constitue deux éléments clés de la cancérogenèse associée aux virus. L’EBV est un herpesvirus ubiquitaire à ADN double brin qui infecte plus de 90% de la population, avec un tropisme spécifique pour les cellules B. Après primo-infection, le virus persiste dans l’hôte pour toujours. L’EBV est responsable de la mononucléose infectieuse bénigne et est associé à plusieurs tumeurs malignes telles que le lymphome de Burkitt, le lymphome de Hodgkin et certaines formes de cancers gastriques. Les récepteurs Toll-like (TLRs) mammaires jouent un rôle important dans la défense de l’hôte lors de l’infection pathogène en régulant et reliant les réponses immunitaires innées et adaptatives. Dans cette étude, nous avons montré que l’EBV pouvait altérer la régulation et l’expression de TLR9, une des molécules effectrices majeures de la réponse immunitaire innée. L’infection par l’EBV des lymphocytes B primaires humains a entraîné l’inhibition de la fonctionnalité de TLR9. Nous avons observé que l’EBV exerçait sa fonction inhibitrice en diminuant les niveaux d’ARNm et de la protéine du récepteur TLR9. De plus, nous avons établi que LMP1, oncoprotéine majeure de l’EBV, inhibait fortement la transcription de TLR9. La sur-expression de LMP1 par transfection transitoire ou transduction des cellules B réduit l’activité du promoteur de TLR9, l’ARNm et les niveaux protéiques. Bloquer la voie de signalisation de NF-κB induite par la signalisation de LMP1 permet de récupérer l’activité du promoteur de TLR9 et l’expression de la protéine. L’ensemble de nos résultats mettent en évidence un nouveau mécanisme utilisé par l’EBV pour supprimer la réponse immunitaire de l’hôte en dérégulant la transcription de TLR9 via l’activation de NF-κB par LMP1 / Chronic infection causes about 15-20% of cancer worldwide. In most cases, infections are cleared by the immune system with no dramatic consequence for the infected hosts. However, oncoviruses can escape the immune system and induce cellular transformation, two key events in cancer mediated by viruses. EBV is a ubiquitous double-stranded DNA herpesvirus, which infects more than 90% of the population with a specific tropism to B-cells. Upon primo-infection the virus persists in the host for lifetime. EBV is responsible of the benign infectious mononucleosis and is associated to several malignancies such as the Burkitt lymphoma, Hodgkin’s lymphoma and some forms of gastric cancers. Mammalian Toll-like receptors (TLRs) play a key role in host defense during pathogen infection by regulating and linking the innate and adaptive immune responses. TLRs belong to a family of receptors that recognize pathogen-associated molecular patterns and are expressed on immune and non-immune cells, endowing them with the capacity to sense pathogen-derived products and to alert the immune system . In this study we show that EBV can alter the regulation and expression of TLR9, one of the key effector molecules of the innate immune response. EBV infection of human primary B cells resulted in the inhibition of TLR9 functionality. Stimulation of TLR9 on primary B cells led to the production of IL-6, TNFα and IgG, which was inhibited in cells infected with EBV. We observed that EBV exerts its inhibitory function by decreasing TLR9 mRNA and protein levels. This event was observed twelve hours post EBV infection of primary cells as well as in an immortalized B cell line, demonstrating the specific role of the virus to turn down TLR9 levels. In addition, we determined that the EBV oncoprotein LMP1 is a strong inhibitor of TLR9 transcription. Over expression of LMP1 by transient transfection or transduction of B cells, reduced TLR9 promoter activity, mRNA and protein levels. Blocking the NF-κB pathway induced by LMP1 signaling, recovered TLR9 promoter activity and protein expression. Moreover LMP1 mutants deficient in activating the NF-κB pathway inversely restored TLR9 transcription. Taken together, our study reveals a novel mechanism used by EBV to suppress the host immune response by deregulating the TLR9 transcript through LMP1-mediated NF-κB activation

L'objectif de notre travail est de mieux comprendre le rôle de LMP1 et de ses variants délétés de 30 ou 69pb en C-terminal dans le développement du lymphome de Hodgkin (LH) chez les patients VIH+. Le séquençage de LMP1 dans des échantillons de sang total et de cellules oropharyngées de patients de la cohorte Lymphovir (LH/VIH+) et de deux populations contrôles (VIH+ et LH) a permis d'étudier la fréquence des variants de LMP1 in vivo. Ce travail a révélé que le variant LMP1-del30 semble plus fréquent chez les patients avec une immunosuppression modérée, durant laquelle l'incidence des LH est élevée. Par ailleurs, nous avons établi un modèle cellulaire dérivé de LH, exprimant LMP1 de façon inductible, montrant des différences d'expression des cytokines et de progression du cycle cellulaire en fonction des variants de LMP1. Notre travail supporte l'hypothèse d'une plus grande oncogénicité du variant LMP1-del30 et suggère qu'il pourrait être un facteur de risque de développer un LH. / The aim of this work was to improve our understanding of the role of LMP1 and its 30bp and 69bp deletion variants in the development of Hodgkin's lymphoma (HL) among HIV+ people. Blood and saliva samples were collected from HL/HIV+ patients recruited in the Lymphovir cohort, as well as in two control populations (HIV+ and HL). Next-Generation Sequencing allowed us to determine the frequency of LMP1 variants in these samples. This work showed that the del30-LMP1 variant seems to be more frequent in patients with a moderate immunosuppression, associated with a higher HL incidence. Besides, we established three HL-derived cell lines expressing WT-LMP1 or its variants and showed differences of cytokine expression and progression of the cell cycle depending on the variant. Our work supports the hypothesis of a greater oncogenicity of del30-LMP1 variant and suggests that it could be a risk factor for the development of HL.

<p>Epstein-Barr virus (EBV) is a ubiquitous human pathogen that establishes a lifelong latent infection in over ninety percent of all adult humans worldwide. While typically benign, EBV has been causally associated with a number of human malignancies in the settings of immune suppression, genetic, and/or environmental factors. While a highly successful pathogen based on prevalence, the ability of the virus to immortalize human B cells (a stage of infection thought to be critical for the establishment of latency) is quite poor. We hypothesize that the interactions between the virus and the human host early after infection are ultimately important for the outcome of viral latency establishment. To answer this question we broadly profiled primary human B cells at both early and late times after EBV infection to assay both host mRNA expression and the host-driven response to apoptotic stimuli. We found that EBV infection induces host gene expression signatures early after infection that are functionally distinct from the gene expression program late after infection. These studies also led to the novel discovery that viral gene expression is controlled differently early after infection, including the delayed expression of a viral protein that is critical for the establishment of latency. Furthermore, we have also shown that EBV can use a single viral protein to alter and repress host apoptotic sensitivity in the face of an anti-viral apoptotic response.</p> / Dissertation

Nasopharyngeal carcinomas (NPC) is highly induced by Epstein-Barr virus (EBV). EBV infection encoded latent membrane protein 1 (LMP1) is expressed in latent stage II and III of EBV infection nasopharyngeal cells. LMP1 was reported to be associated with increased tumorigenesis, through the activation of nuclear factor-£eB (NF-£eB). In this study, LMP1 overexpressing NPC cell line (TW04) was used for assays of cell proliferation, apoptosis and migration under drug (Sodium Salicylate, Cisplatin and 5-Fluorouracil) treatment. Sodium Salicylate is one of non-steroidal anti-inflammatory (NSAID) drug, which inhibits the downstream of NF-£eB pathway, e.g., COX-1. Cisplatin and 5-Fluorouracil are traditional chemotherapy durgs used in many cancers. Our result shows that overexpression of LMP1 affects cell proliferation, apoptosis , invasion and migration ability. It indicates that LMP1-overexpression is an important marker for NPC therapy.

Orientador: Deilson Elgui Oliveira / Resumo: Presente em aproximadamente 95% da população mundial o Vírus de Esptein-Barr (Epstein-Barr Virus - EBV) está relacionado com 1,5% dos casos de câncer no mundo com destaque para o carcinoma de nasofaringe (Nasopharyngeal Carcinoma – NPC) o qual segundo a Organização Mundial da Saúde, 90% dos carcinomas indiferenciados estão associados ao EBV. Expresso durante o ciclo de latência III e II a proteína latente de membrana 1 (Latent Membrane Protein 1, LMP1), um dos principais produtos oncogênicos do EBV mimetiza o receptor CD40 e mantém-se constitutivamente ativa na célula. Com papel importantíssimo no desenvolvimento do carcinoma de nasofaringe, LMP1 atua na imortalização e proliferação de linfócitos B por meio da deflagração de vias de sinalização intracelular como NFkB e PI3K/AKT. Atualmente são conhecidas sete variantes do gene BNLF1 que codifica LMP1 e apresentam diferenças nas sequências nucleotídicas e aminoacídicas, ambas consideradas oncogênicas por conservarem os domínios CTARs. Essas diferenças encontradas nas variantes de LMP1 podem estar relacionadas à agressividade dos tumores. Entre as variantes de LMP1 foram avaliadas no presente estudo as variantes B95.8 e M81, ambas associadas ao desenvolvimento de NPC. As propriedades transformantes dessas variantes de LMP1 foram analisadas frente ao programa biológico transição epitelial- mesenquimal (Epithelial-Mesenckymal Transition – EMT) que está diretamente relacionado com a progressão dos carcinomas. A avaliação das v... (Resumo completo, clicar acesso eletrônico abaixo) / Mestre

Submitted by CLEITON SILVA MARQUES null (biocleitond2@yahoo.com.br) on 2017-06-14T16:42:05Z No. of bitstreams: 1 MS 201513939-9 Dissertação Cleiton Silva Marques.pdf: 1951878 bytes, checksum: 324f65b41f0660609ec022c77b81f85e (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-06-19T13:53:05Z (GMT) No. of bitstreams: 1 marques_cs_me_bot.pdf: 1951878 bytes, checksum: 324f65b41f0660609ec022c77b81f85e (MD5) / Made available in DSpace on 2017-06-19T13:53:05Z (GMT). No. of bitstreams: 1 marques_cs_me_bot.pdf: 1951878 bytes, checksum: 324f65b41f0660609ec022c77b81f85e (MD5) Previous issue date: 2017-05-15 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Presente em aproximadamente 95% da população mundial o Vírus de Esptein-Barr (Epstein-Barr Virus - EBV) está relacionado com 1,5% dos casos de câncer no mundo com destaque para o carcinoma de nasofaringe (Nasopharyngeal Carcinoma – NPC) o qual segundo a Organização Mundial da Saúde, 90% dos carcinomas indiferenciados estão associados ao EBV. Expresso durante o ciclo de latência III e II a proteína latente de membrana 1 (Latent Membrane Protein 1, LMP1), um dos principais produtos oncogênicos do EBV mimetiza o receptor CD40 e mantém-se constitutivamente ativa na célula. Com papel importantíssimo no desenvolvimento do carcinoma de nasofaringe, LMP1 atua na imortalização e proliferação de linfócitos B por meio da deflagração de vias de sinalização intracelular como NFkB e PI3K/AKT. Atualmente são conhecidas sete variantes do gene BNLF1 que codifica LMP1 e apresentam diferenças nas sequências nucleotídicas e aminoacídicas, ambas consideradas oncogênicas por conservarem os domínios CTARs. Essas diferenças encontradas nas variantes de LMP1 podem estar relacionadas à agressividade dos tumores. Entre as variantes de LMP1 foram avaliadas no presente estudo as variantes B95.8 e M81, ambas associadas ao desenvolvimento de NPC. As propriedades transformantes dessas variantes de LMP1 foram analisadas frente ao programa biológico transição epitelial- mesenquimal (Epithelial-Mesenckymal Transition – EMT) que está diretamente relacionado com a progressão dos carcinomas. A avaliação das variantes de LMP1 foi feito por meio de transfecção transiente de células HEK293 e NP69. Células expressando as variantes de LMP1 apresentaram maior capacidade de migração e invasão em relação ao grupo controle. Ainda, células expressando as variantes de LMP1 demonstraram alteração para alguns genes relacionados ao processo de EMT. No entanto, as variantes de LMP1 não apresentam diferenças significativas na regulação do programa de EMT. / FAPESP: 2015/13939-9