京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第25515号 / 生博第531号 / 新制||生||70(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 荒木 崇, 教授 野田 岳志, 教授 谷口 雄一 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Einleitung: Die im Genom der Schweine integrierten porzinen endogenen Retroviren (PERV) gehören zu den potentiellen humanpathogenen Erregern, die eines der Risiken bei der Xenotransplantation darstellen. Für die Abschätzung des Infektionsrisikos von PERV sind drei Verfahrensweisen von Bedeutung: Erstens die Evaluierung der PERV-Freisetzung aus porzinen Zellen und Geweben; Zweitens die Etablierung eines In vivo-Infektionsmodells und Drittens ein retrospektives Screenen von Patienten. Methoden: Die PERV-Expression in Inselzellen von Schweinen der Deutschen Landrasse wurde in vitro und in vivo evaluiert. Anschließend wurde PERV auf nicht-humanen Primatenzellen passagiert. In einem zweiten Modellversuch wurde murinen Zellen in vitro und SCID-Mäusen in vivo zellfreies PERV appliziert. Schließlich wurden Seren von Patienten analysiert. Ergebnisse: Die untersuchten Inselzellen setzten keine Viruspartikel frei und konnten somit weder humane Zellen noch BALB/c-Mäuse infizieren. Die verwendeten Affenzellen produzierten infektiöses PERV mit geringer Replikation. Weder in den murinen Modellversuchen noch in den untersuchten Patienten wurde eine Übertragung von PERV beobachtet. Schlussfolgerung: Schweine der Deutschen Landrasse könnten als Ausgangsbasis für die Zucht sicherer Schweine für die Xenotransplantation dienen. Da keine Infektion verschiedener muriner Zellen mit PERV beobachtet wurde, muss angenommen werden, dass Publikationen anderer Arbeitsgruppen, die eine PERV-Infektion in SCID-Mäusen diagnostizierten, ein falsch-positives Ergebnis aufgrund von Mikrochimärismus oder aufgrund von Pseudotypisierungen mit murinen endogenen Retroviren wiedergeben. Unsere Befunde werden dadurch erhärtet, dass der Rezeptor für PERV-A auf murinen Zellen nicht exprimiert wird und diese auch in vitro nicht infiziert werden konnten. In Übereinstimmung mit den weltweit etwa 200 behandelten Patienten konnte auch in den beiden neuen Studien keine Übertragung von PERV festgestellt werden. / Objective: Porcine endogenous retroviruses (PERVs) are integrated in the porcine genome and are able to infect human cells in vitro. Therefore, PERVs are one of the possible pathogens which poses a risk for xenotransplantations. In this study three significant methods were used to evaluate the infectious risk of PERV: The release of PERV particles from porcine cells and tissues, the formation of an in vivo infection model and a retrospective screening of patients. Methods: Islet cells from german landrace pigs were co-cultivated with human cells in vitro and were transplanted in BALB/c mice in vivo. Serial passaging experiments were performed with nonhuman primate cells. Murine cells were incubated in vitro and SCID mice in vivo with PERV. Sera of patients who were treated ex vivo with porcine liver cells and who had received islet cells were investigated for antibodies against PERV. Results: No virus release were observed in german landrace islet cells, thus they were neither able to infect human cells nor BALB/c mice. The used nonhuman primate cells released low replicating PERVs. None of the murine cells could be infected by PERV and no provirus integration was observed in different SCID mice organs. PERV-specific antibodies were found in none of the investigated patients. Conclusion: German landrace pigs could be used as a source for breeding safe genetically modified pigs suitable for xenotransplantation. Since there were no detectable PERV infection of different murine cells and SCID mice, it have to be supposed, that previously reported PERV transmissions to SCID mice might be due to microchimerism or to pseudotyping of murine endogenous retroviruses. Our results were confirmed by the fact, that the receptor for PERV-A is not expressed on murine cells and that these cells could not be infected in vitro. The absence of a PERV transmission in the investigated patients, correspond to the results obtained from approximately twohundred treated patients worldwide.

Transmissible spongiform encephalopathies (TSEs) or prion diseases are a class of fatal brain disorders better known as Creutzfeldt-Jacob Disease (CJD) in humans, bovine spongiform encephalopathy (BSE) in cattle, scrapie in sheep, and chronic wasting disease (CWD) in deer and elk. The infectious agent responsible for these diseases is a misfolded prion protein capable of catalyzing a conformational change in normal cellular prion proteins (PrPC) into aberrant disease-causing structural isoforms (PrPSc). Although the etiological agent for TSEs has clearly been defined as PrPSc, there are important gaps in our understanding of how these proteins target and invade brain tissue. It remains to be established how ingested PrPSc ultimately reach the brain and also to understand why these tissues are particularly targeted, notwithstanding that several other tissues highly express prion proteins. Certain viruses, retroviruses in particular, efficiently hijack host proteins and can carry these proteins with them when they are released from a cell. Several lines of evidence have shown that prions and retroviruses can interact and associate at various stages of the retroviral replication cycle. Of special interest is that most retroviruses can cross the blood-brain barrier and could therefore deliver host-derived proteins to neuronal cells. In view of these observations, this thesis investigates whether retroviruses can act as vectors to capture prions from an infected cell and deliver them to a susceptible target cell. In this work, I have cloned human and mouse prion cDNAs from PBMCs and the murine cell line NIH 3T3. Either a FLAG epitope tag or the eGFP reporter protein cDNA was inserted into a region of the prion cDNA that is predicted to be amenable to such genetic insertions without affecting protein folding or expression. I then confirmed using both fluorescent and confocal microscopy and that the recombinant proteins had a similar cell distribution to the endogenous prion protein. Using Western blot analysis, I then showed that endogenous and overexpressed prion proteins can be detected in co-transfected cells producing HIV and murine leukemia virus (MLV) retroviral particles. Finally, I went on to show that prions are also present at high levels in HIV and MLV retroviral particles released from these cells. This work constitutes the first step in determining whether retroviruses can act as vectors for prion dissemination. Establishing a strong and clear association between retroviruses, pathogenic prions and prion disease would provide the rationale for preventive measures to be taken directly against retroviruses in order to protect humans and animals that have been newly exposed to PrPSc-infected products or those who are genetically predisposed to develop prion diseases. Anti-retroviral drugs could also be potentially used to delay disease progression and reduce prion transmission in human and animal tissues. The availability of such a treatment would constitute a significant advancement because there is currently no cure or treatment for prion diseases.

Endogenous retrovirus ERVWE1 is an integral part of the human genome. In the course of evolution, a protein encoded by the env gene of this retrovirus - Syncytin-1 - has gained unique function in human development. It mediates cell-to-cell fusion of placental cytotrophoblasts. Receptor that binds to Syncytin-1 is expressed in different cell types. Syncytin-1-mediated fusion is essential in placenta, but it can cause disruption of tissue integrity in other cell types. ERVWE1 expression is regulated by promoter DNA methylation, transcription factor GCM1 and efficient mRNA splicing. This thesis concerns the ERVWE1 expression and its regulation in non-placental tissues. It was found out that the moderate GCM1 overexpression was not sufficient to induce Syncytin-1 expression. Neither treatment with DNA demethylation agent 5-azacytidine nor with Syncytin-1 activator forskolin was able to manage Syncytin-1 expression. This thesis extends previous findings concerning high syncytin-1 expression in seminomas. In same tissues, there was found elevated TET1 expression on mRNA level in comparison with controls. The presence of the TET1 demethylation enzyme can influence ERVWE1 promoter DNA methylation. Previously unreported splicing variant of TET1 has been found during the construction of human TET1 expression...

Les rétrovirus endogènes (ERV) sont des éléments constitutifs de la plupart des génomes eucaryotes, et représentent chez l’humain environ 400000 loci. Les HERV sont divisés en familles distinctes, composées d’éléments apparentés mais structurellement hétérogènes. Leur activité peut être néfaste, neutre, mais aussi bénéfique. La majorité des HERV semble silencieuse dans les cellules somatiques. Cependant certains présentent une forte activité en contextes physiologiques. Par ailleurs, une expression significative de HERV est fréquemment observée dans des contextes pathologiques, tels que les cancers. La mise sous silence des éléments répétés est supposée se produire principalement par la méthylation de leur ADN. Nous nous sommes donc intéressés à l’implication de la méthylation des régions régulatrices des HERV, les LTR, dans le contrôle de leur expression. D’une part cette étude nous a permis de mettre en évidence une méthylation locus- et tissu- spécifique de LTR HERV en contexte physiologique, impliquant notamment des modalités particulières de méthylation contrôlant l’expression placentaire de HERV domestiqués. D’autre part ce travail nous a permis de déterminer que six loci HERV-W, incluant un locus domestiqué, sont réactivés de manière autonome dans des tumeurs testiculaires sous l’influence d’un changement de modalité de méthylation intra-famille. Ainsi la méthylation des HERV influence leur expression, mais sous des modalités variables selon les loci et les contextes concernés / Endogenous retroviruses are constitutive elements of most eukaryotic genomes. They represent about 400,000 loci in the human genome. HERVs are divided into distinct families on the basis of phylogenetic identities but are highly heterogeneous in structures. Their activity can be detrimental, neutral, or beneficial to the host. Majority of HERVs seems silent in somatic cells. Still, some are highly expressed in physiological contexts. Besides, a significant expression of HERVs is frequently observed in pathological contexts such as cancers. Silencing of repeated elements is supposed to occur mainly through DNA methylation. We were therefore interested by the implication of HERV regulatory region (LTR) methylation in the control of their expression. First, this study identified locus and tissues –specific HERV LTR methylation in physiological context, worth noting particular methylation modalities that control domesticated HERVs placental expression. Second, we could determine a change in intra-family LTR methylation modalities in testicular tumors leading to the autonomous reactivation of six HERV-W loci, among which a domesticated one. Thus methylation clearly influences HERVs expression, but under modalities varying upon the loci and the contexts

Le génome humain contient environ 200 000 séquences d'origine rétrovirale (HERV), intégrées au fil de l'évolution et organisées aujourd'hui en familles multicopies complexes globalement réprimées par un contrôle épigénétique. L'étude du transcriptome HERV au niveau locus est compliquée par les similarités phylogénétiques au sein d'une famille et par la profusion des sites d'intégration, deux propriétés inhérentes aux éléments transposables. Dans ce travail, nous avons utilisé une méthode de conception de sondes de détection de 25 mer afin d'adresser la question de l'expression individuelle des HERV. Une puce à ADN haute densité intégrant plus de 5 500 séquences HERV et permettant une lecture fonctionnelle de l'activité de leurs LTRs a été utilisée sur un panel de tissus sains et cancéreux. Cela a permis d'identifier 1 718 séquences HERV actives, dont 326 LTRs promotrices et 209 LTRs polyA. L’étude de l’environnement génomique a mis en évidence une fenêtre d’environ 8 kb en amont des LTRs promotrices, caractérisée par une sous-représentation en gènes cellulaires en orientation sens. Nous avons également montré que le transcriptome des rétrovirus endogènes humains suit des règles de tropisme d’expression, qu’il est sensible aux états de différenciation cellulaire et qu’il ne semble pas être corrélé à l’âge des familles. Une première tentative d’exploitation de ce répertoire HERV dans un contexte clinique a visé à rechercher de nouveaux marqueurs diagnostiques du cancer de la prostate à partir de prélèvements urinaires, par la réalisation d’une étude pilote sur 45 patients / The human genome contains around 200,000 endogenous retroviral sequences (HERV) integrated during the evolution and which are nowadays organized into complex multicopy families, globally repressed by epigenetic control. The study of the HERV transcriptome at the locus level is complicated by phylogenetic similarities within one family and by the profusion of integration sites, two inherent characteristics of transposable elements. In this work, we used a method aiming to optimally characterize individual loci associated with 25 mer probes. A custom microarray dedicated to more than 5,500 HERV sequences and allowing a functional interpretation of the LTRs expression was used on a panel of normal and tumor tissues. We therefore identified 1,718 active HERV sequences, including 326 promoter LTRs and 209 polyA LTRs. The study of the genomic environment has highlighted an approximately 8 kb zone upstream of promoter LTRs characterized by a drastic reduction in sense cellular genes. We also showed that the HERV transcriptome follows tropism rules, is sensitive to the state of cell differentiation and, unexpectedly, seems not to correlate with the age of the families. In a first attempt to use the HERV repertoire in clinical, we sought to identify new markers of prostate cancer from urine samples. This goal was pursued by conducting a pilot study on 45 patients

Ao longo dos processos de imortalização e transformação maligna, as células adquirem inúmeras alterações genéticas, que são causadas por fatores endógenos e exógenos como agentes biológicos e a geração de espécies reativas de oxigênio. Neste trabalho, uma linhagem celular espontaneamente transformada foi clonada a partir de explantes de embriões de camundongos C57bl/6. Esta linhagem mostrou-se produtora de pigmento escuro; a análise citoquímica e ultraestrutural permitiu caracterizar a linhagem como tendo origem melanocítica. A linhagem, denominada Mgal3, mostrou-se tumorigênica quando implantada no tecido subcutâneo de animais singenéicos, apresentando capacidade de disseminação linfática, dando origem a metástases em linfonodos, o que permitiu caracteriza-la como uma linhagem de melanoma murino. O processo de transformação deste melanoma caracterizou-se pela expressão de genes retrovirais endógenos, com expressão do antígeno associado a melanoma (MAA), reconhecido pelo anticorpo monoclonal MM2-9B6; ausência de mutações nos exons 5 a 8 do gene supressor de tumor TP53; e, silenciamento do gene CDKN2a, que codifica duas proteínas que atuam em redes de supressão de tumores, p16INK4a e p19ARF. A perda de expressão de pelo menos um destes produtos gênicos parece associada a mecanismos epigenéticos, uma vez que o tratamento de Mgal3 com o inibidor de DNA metiltransferase 5-Aza-2-deoxicitidina, restaurou a transcrição de pelo menos um dos transcritos do gene CDKN2a. Da mesma forma, observamos que o gene LGALS3, que codifica a lectina animal galectina-3 também é silenciado nesta linhagem, mostrando que esta molécula não está associada à manutenção desta célula transformada em condições de cultivo. / A novel murine melanoma cell line named Mgal3 was generated from embryo explants. This cell line gave rise to metastatic tumors when injected subcutaneously in C57bl/6 mice. Tumor histogenesis was determined at the cytochemical (Fontana Masson staining), immunohistochemical (staining with anti-HMB45 and anti-S100) and ultrastructural levels. Mgal3 produces high amounts of retroviral C particles and was recognized by the mAb MM2-9B6, which reacts with a melanoma associated antigen derived from the envelope of the ecotropic retrovirus MelArv. No mutations were found in TP53 exons 5-8, however loss of CDKN2a expression was observed. Treatment of Mgal3 with the demethylating agent azadeoxycytidine indicated that at least one of the genes encoded at the CDKN2a locus was silenced by promoter hypermethylation. Furthermore, this cell line did not express the animal lectin, galectin-3. The galectin-3 gene promoter seemed to be hypermethylated, since treatment of Mgal3 with azadeoxycytidine led to the de novo expression of the lectin.

Endogenous retroviruses (of the HERV-W family) represent about 8% (1%) of our genome. Their endogenous and exogenous forms (MSRV, Multiple Sclerosis-associated RetroVirus) can alter the regulation of the immune system and be involved in inam- matory and autoimmune pathologies (Multiple Sclerosis). The MSRV envelope protein (ENV) stimulates T lymphocytes by acting as a superantigen. It also interacts with mono- cytes and dendritic cells via membrane receptors, thereby provoking inammatory cytokine production. Our studies are based on the characterisation of the immunological cascade leading from the interaction of the viral envelope with its receptor to the pathological inammatory reaction. The work presented in this thesis combines an in vitro cellular and molecular approach with an in vivo validation using an animal model (mouse). The chosen animal model is Experimental Autoimmune Encephalomyelitis (EAE) in which the complete Freund's adjuvant can be replaced by ENV. Its effects on the murine organism are evaluated on several levels: analysis of behaviour (clinical score) and brain (IRM), cellular and molecular analysis of the immune system. Furthermore, we want to generate a transgenic mouse model expressing different ENVs (MSRV/HERV) under the control of different promoters since MSRV/HERV are found only in great apes. This model, of which the rst steps of elaboration are part of this work, will allow us to study the behaviour of the ENV over-expressing animals as well as their brain and the effects on the immune system.

<p>Human endogenous retroviruses (HERVs) represent approximately 7% of the human genome. This investigation was focused on one particular HERV, ERV3, with the main purpose of characterising its gene expression patterns and genomic distribution of ERV3-like sequences. Furthermore, this careful expression study should provide insights into the biological role of HERVs. The impact of HERVs in health and disease is not yet clarified. ERV3 is expressed as three envelope (<i>env</i>) transcripts, of which two also contain a cellular gene, <i>H-plk</i> (human proviral linked <i>Krüppel</i>). ERV3 <i>env</i> expression was mainly investigated at the RNA level. The gene expression of two other HERVs, HERV-K and HERV-E was analysed and compared with ERV3 activity.</p><p>Real-time PCRs were developed and in combination with in situ hybridisation, it was found that ERV3 is expressed in a tissue- and cell-specific way. High levels of ERV3 mRNA (up to six times over Histone3.3) were demonstrated in placenta, sebaceous glands, foetal and adult adrenal glands, brown adipose tissue, corpus luteum, pituitary gland, thymus and testis. In monocytic cells including both normal monocytes and malignant U-937 cells, elevated mRNA levels were observed after retinoic acid (RA)-induced differentiation. ERV3-encoded Env protein was detected in selected cases, one following RA-treatment. In addition, several new ERV3-like sequences were discovered in the human genome. </p><p>ERV3 was found to have conserved open reading frames in contrast to other ERV3-like sequences in the human genome. This suggests that ERV3 may be involved in important cellular processes such as differentiation, cell fusion, immunomodulation and protection against infectious retroviruses. The developed techniques and obtained results will allow further studies of HERV expression to better correlate HERV activity to both normal development and disease. </p>

<p>In the field of transplantation, the increasing deficit of human donors have lead to an interest in animals as an alternative source of organs and tissues. </p><p>Different <i>in vitro </i>systems and rodent models of xenotransplantation were used to examine the most significant barriers that have to be overcome, before isolated islets of Langerhans from pigs can be used as a cure for insulin-dependent diabetes mellitus in humans.</p><p>In clinical transplantation, islets are infused into the liver through the portal vein. During this procedure the islets are susceptible to harmful innate reactions triggered in blood. Adenoviral vectors generating transgenic expression of human complement regulatory proteins were evaluated in pig islets and shown to confer protection against acute complement-mediated damage. </p><p>Transplanted islets escaping this immediate destruction will be targets of a cellular immune response. Using a new mouse model of islet xenograft rejection, it was demonstrated that macrophages, effector cells in the rejection, were part of an MHC-restricted xenospecific immune response mediated by T cells. In a strain of knockout mice it was further shown that this process can proceed in the absence of an important signalling system, mediated by Toll-like receptors, between cells in innate and adaptive immunity. These findings illustrate some of the mechanistic differences compared to cellular islet allograft rejection which partly explain why immunosuppressive drugs used in clinical allotransplantation is not sufficient for preventing xenograft rejection. </p><p>Porcine endogenous retroviruses (PERV) remain a safety concern in xenotransplantation. Characterization of PERV in pig islets indicated that virus expression is low <i>in vitro </i>but increases during the immediate time period following transplantation. This suggests that antiviral therapies administered at the time of transplantation could be used for preventing the risk of PERV transmission after xenotransplantation.</p>