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1
BARAT, CORINNE. "Etude de l'initiation de la reserve transcription du genome du retrovirus humain, hiv-1". Paris 6, 1992. http://www.theses.fr/1992PA066024.
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Chez le retrovirus hiv-1, la replication de l'arn genomique viral est assuree par la reserve transcriptase virale rtp66-p51. Cette reserve transcription est initiee a partir d'un trna#l#y#s#,#3 cellulaire hybride a l'arn pres de son extremite 5. Dans une analyse de cette etape d'initiation, nous avons etudie les interactions entre ce trna amorce et les autres membres du complexe d'initiation de la reserve transcription, en particulier la reserve transcriptase rtp66-p51 et la proteine de nucleocapside ncp15. Nous avons montre une interaction specifique entre la reserve transcriptase et le trna#l#y#s#,#3 initiateur, et les sites d'interaction sur le trna et sur la proteine ont pu etre localises. L'importance de la structure en heterodimere de la reverse transcriptase pour son interaction avec le trna amorce et pour son activite polymerase a egalement ete mise en evidence. La proteine de nucleocapside, outre sa capacite a promouvoir la fixation du trna initiateur sur l'arn viral, semble participer a un complexe ternaire avec le trna#l#y#s#,#3 et la reserve transcriptase. Ce complexe pourrait etre implique dans les etapes de selection des trna encapsides, d'hybridation du trna#l#y#s#,#3 a l'arn viral, et d'initiation de la reverse transcription
2
Broders, Florence. "Analyse de la transcription des genes alpha globine dans les erythroblastes aviaires normaux et transformes par un retrovirus". Paris 7, 1988. http://www.theses.fr/1988PA077023.
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LAVIGNON, MARC. "Action d'oligonucleotides modifies sur la transcription inverse ou sur la traduction. Etude de leur effet sur la proliferation de retrovirus murins". Paris 7, 1991. http://www.theses.fr/1991PA077241.
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Nous montrons que des oligodesoxyribonucleotides d'anomerie alpha sont capables d'inhiber in vitro l'initiation de la transcription inverse qui est une etape clef du cycle replicatif des retrovirus. En effet, leur hybridation parallele sur une sequence complementaire d'acide nucleique ne leur permet pas de servir d'amorce a la transcriptase inverse. Lors de competitions entre une amorce d'anomerie beta et un oligonucleotide alpha, nous obtenons une inhibition de la synthese d'adn. Par ailleurs, la reconnaissance des duplex alpha-beta par la transcriptase inverse permet de pieger celle-ci et d'inhiber son activite polymerase et rnase h. Ex vivo, nous montrons qu'un oligonucleotide alpha, dirige contre le site de fixation de l'arnt (site pbs) qui sert d'amorce a la transcription inverse chez les retrovirus, est capable de diminuer la dissemination du retrovirus de friend et d'un virus recombinant de moloney. Cette inhibition est observee lors d'une infection de novo de cellules prealablement electropermeabilisees en presence de l'oligonucleotide. Nous emettons l'hypothese que cet oligonucleotide alpha agit lors des etapes tardives de la transcription inverse mettant en jeu le site pbs et non au moment de l'initiation de celle-ci. Enfin, nous montrons que la modification d'un oligodesoxyribonucleotide beta (en l'occurrence l'adjonction d'une molecule de 9-amino-ellipticine en 5), dirige contre le site d'initiation de la synthese de la proteine env, est necessaire pour obtenir un effet sur la proliferation du retrovirus de friend. Ce couplage n'augmente ni l'affinite de l'oligonucleotide pour sa cible ni son activite inhibitrice de la synthese proteique in vitro. Il protege l'oligonucleotide contre les degradations nucleasiques en 3 et facilite, peut etre, sa penetration cellulaire
4
Prats, Anne-Catherine. "Etude de l'expression genetique et de la constitution des particules virales infectieuses chez le retrovirus murin mulv". Toulouse 3, 1988. http://www.theses.fr/1988TOU30172.
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Yoshinaga, Noriyoshi. "A screening for DNA damage response molecules that affect HIV-1 infection". Kyoto University, 2019. http://hdl.handle.net/2433/243296.
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Laverdure, Sylvain. "Régulation de la transcription bidirectionnelle chez le Virus de l'Immunodéficience Humaine de type 1". Thesis, Montpellier 1, 2012. http://www.theses.fr/2012MON13514/document.
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Le génome des rétrovirus existe sous deux formes différentes : sous forme d'ARN simple brin, qui est traduit ou encapsidé, ou sous forme d'ADN double brin intégré dans le génome de la cellule hôte infectée. Cette dernière forme, l'ADN proviral, est indispensable à la production de tous les ARNm viraux nécessaires à la synthèse des protéines virales, qui en retour agissent sur la région promotrice située au niveau du LTR 5'. Cependant, l'ADN proviral possède un second LTR à son extrémité 3', capable de réguler une transcription antisens, orientée dans la direction opposée à celle contrôlée par le LTR 5'. L'ADN proviral a donc deux brins codants, ce qui offre au virus un plus grand potentiel de synthèse protéique. Dans le cas du Virus de l'immunodéficience Humaine de type 1 (VIH-1), la transcription antisens permet la production d'une protéine, appelée ASP (Antisense Protein). Dans ce manuscrit, nous démontrons que cette activité transcriptionnelle antisens s'exprime préférentiellement dans les cellules d'origine monocytaire, en particulier les cellules dendritiques ; une localisation membranaire de la protéine ASP a par ailleurs été mise en évidence dans ce type cellulaire. Nos résultats suggèrent également que la transcription antisens du VIH-1 est indépendante de la protéine Tat, et que par ailleurs les deux types de transcriptions ne sont pas exprimés simultanément au sein d'une même cellule. En outre, nos données soulignent que la séquence codante de la protéine ASP est très fortement conservée parmi les différents isolats viraux. Sur la base de l'ensemble de ces résultats, notre hypothèse est que la protéine ASP du VIH-1 possède des fonctions cruciales dans le cycle réplicatif des rétrovirus, indépendantes de la production viraleGenome of retroviruses exists in two different forms: as single-stranded RNA that is translated or packaged, or as double-stranded DNA integrated into the genome of the infected host cell. The latter form, the proviral DNA, is essential for the production of all viral mRNAs required for the synthesis of viral proteins, which in turn act on the promoter region located at the 5 '-LTR. However, the proviral DNA has a second LTR at its 3 '-end, capable of regulating antisense transcription oriented in the opposite direction to that controlled by the 5'-LTR. The proviral DNA has then two coding strands, which gives the virus a greater potential for protein synthesis. In the case of the Human Immunodeficiency Virus type 1 (HIV-1), antisense transcription allows the production of a protein called ASP (Antisense Protein). In this manuscript, we demonstrate that this antisense transcriptional activity is preferentially expressed in cells of the monocyte lineage, in particular dendritic cells; a membrane localization of the ASP protein was also observed in this cell type. Our results also suggest that the antisense transcription of HIV-1 is Tat-independent, and what's more that the two types of transcription are not expressed simultaneously within the same cell. In addition, our data highlight that the ASP protein coding sequence is highly conserved among different viral isolates. Based on these results, our hypothesis is that the ASP protein of HIV-1 has critical functions in the replicative cycle of retroviruses, distinct from viral production
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ROUSSET, RAPHAEL. "Caracterisation des interactions entre l'oncoproteine tax1 du retrovirus htlv-i et differents facteurs cellulaires impliques dans le controle de la transcription et de la proliferation". Lyon, École normale supérieure (sciences), 1997. http://www.theses.fr/1997ENSL0069.
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L'infection par le retrovirus htlv-i entraine divers syndromes proliferatifs et degeneratifs chez l'homme. La proteine tax1, produite par le virus, possede des activites transactivatrices et oncogeniques qui sont en grande partie responsables des pathologies associees a htlv-i. Les etudes que nous avons engagees ont permis de caracteriser plusieurs proteines qui interagissent avec tax1 dans la cellule. Des etudes effectuees in vivo et in vitro montrent que tax1 favorise la premiere etape de la mise en place du complexe d'initiation de la transcription, en recrutant directement tfiid. Par ailleurs, le systeme du simple hybride dans la levure a permis d'identifier 3 facteurs de transcription, creb, crem et atf1, capables de cooperer in vivo avec tax1 sur le motif tre1 du promoteur htlv-i. Dans un deuxieme temps, nous avons recherche d'autres partenaires cellulaires de tax1 par un criblage avec le systeme des deux hybrides dans la levure. Deux sous-unites du proteasome, hc9 et hsn3, ont ete ainsi isolees. Une etude fonctionnelle suggere que tax1 favorise l'association de p105 avec le proteasome et accelere ainsi le clivage de p105 en p50 dans la voie nf-b. Lors de ce criblage, nous avons egalement isole l'homologue humain de la proteine de souris int-6 impliquee dans la proliferation cellulaire. En interagissant avec hint-6, tax1 altere sa localisation nucleaire en points. Ce mecanisme pourrait modifier la fonction d'hint-6 et donc participer au processus de transformation cellulaire induit par tax1. Enfin, six nouvelles proteines contenant des domaines pdz ont ete isolees. Leur interaction avec tax1 est assuree par l'extremite c-terminale de la proteine virale, qui possede le motif consensus x-t/s-x-v-cooh implique dans l'interaction avec les domaines pdz. De part les fonctions des proteines de cette famille dans la cellule, il est possible que ces interactions soient a l'origine de certains desordres proliferatifs et degeneratifs qui sont associes a htlv-i.
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Hu, Lijuan. "Endogenous Retroviral RNA Expression in Humans". Doctoral thesis, Uppsala : Acta Universitatis Upsaliensis : Universitetsbiblioteket [distributör], 2007. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-8213.
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TOUNEKTI, NACEUR. "Etude structurale de l'extremite cinq prime terminale non codante de l'arn du retrovirus murin de moloney : application a l'etude de la dimerisation et du complexe d'initiation de la transcription inverse". Paris 11, 1993. http://www.theses.fr/1993PA112452.
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Dans le but d'inhiber la propagation retrovirale par des oligonucleotides de synthese nous avons mene une etude physico-chimique et structurale des phenomenes de dimerisation et de formation du complexe d'initiation de la transcription inverse. Nous avons montre que la dimerisation de l'arn du virus de moloney (mo-mulv) est un processus lent qui implique d'une part, des changements structuraux au sein de la sequence d'encapsidation psi (particulierement les sequences: deux cent dix-deux cent vingt) et (deux cent soixante dix-huit-trois cent dix) et d'autre part, des transitions conformationnelles au niveau des regions adjacentes a psi. Nous avons aussi montre que psi garde une structure intrinseque independante du reste de la molecule d'arn. Par ailleurs, la formation du complexe d'initiation de la transcription inverse, par fixation specifique de l'arn#t proline sur le site pbs, induit des changements structuraux au sein de la sequence (un-deux cent quinze) et impliquerait d'autres interactions entre l'amorce et l'arn viral. Enfin le ciblage du site pbs ou de sequences particulieres au sein de psi par des oligonucleotides de synthese a abouti a une inhibition significative de la replication retrovirale
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Mommert, Marine. "Modulation de l'expression des rétrovirus endogènes humains dans des contextes d'inflammation et d'immunosuppression". Thesis, Lyon, 2018. http://www.theses.fr/2018LYSEN044.
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Le sepsis est défini par l’apparition de dysfonctions d’organes, multiples et mortelles, causées par une réponse de l’hôte dérégulée suite à une infection. L’hétérogénéité de la maladie représente un défi clinique majeur au regard de la prise en charge thérapeutique, et à ce jour les marqueurs proposés ne suffisent pas à stratifier les patients. Les rétrovirus endogènes humains (HERV) pourraient être des marqueurs pertinents,compte tenu des propriétés immunosuppressives de leurs enveloppes et de leur expression dans des maladies inflammatoires et auto-immunes. Cette thèse a pour objectif de savoir dans quelle mesure les HERV sont exprimés et modulés, dans des conditions d’inflammation et d’immunosuppression. Pour cela,nous avons utilisé une puce à ADN haute densité permettant (i) l’analyse de la transcription de 363 689HERV et 1500 gènes, et (ii) une lecture fonctionnelle de l’activité des LTR. L’expression des HERV a été objectivée (i) dans un modèle ex-vivo de tolérance à l’endotoxine sur des cellules mononuclées du sang périphérique (PBMC) d’individus sains et (ii) sur sang total provenant d’individus sains et de patients en choc septique, stratifiés ou non en fonction du statut immunitaire. (1) De 5,6% à 6,9% des HERV sont exprimés dans le compartiment sanguin et environ 20% des LTR possèdent une fonction promotrice ou polyA, les deux fonctions étant mutuellement exclusives. (2) Le contenu du transcriptome HERV est modulé ex vivo dans le contexte de tolérance à l’endotoxine laissant apparaitre deux grands phénotypes transcriptionnels. L’expression de certains loci HERV est corrélée au statut immunitaire de patient septique.L’évaluation d’une signature moléculaire complexe sur une cohorte de validation, permet la séparation en deux groupes présentant des critères de sévérité distincts, suggérant les HERV/MaLR comme biomarqueurs de stratification. (3) L’analyse de la co-expression des gènes et des HERV a permis d’intégrer ceux-ci au sein de réseaux associées à la réponse de l’hôte et de proposer des hypothèses fonctionnellesSepsis is defined as a life-threatening organ dysfunction caused by a dysregulated host response to infection.The heterogeneity of the disease present a major clinical challenge with regard to the therapeutic coverage,and this day the proposed markers are not enough to stratify patients. The human endogenous retrovirus(HERV) could be relevant markers, considering the immunosuppressives properties of their envelopes andtheir expression in inflammatory and autoimmune disease. The aim of this thesis is to know to what extentthe HERVs are expressed and modulated, in inflammatory and immunocompromised contexts. For this, weused a high density DNA chip allowing (i) the transcription analysis of 363,689 HERV and 1500 genes,and (ii) a functional reading of LTRs activities. The HERVs expression was objectified (i) in endotoxintolerance ex vivo model in peripheral blood mononuclear cells (PBMCs) of healthy volunteers and (ii) inwhole blood of healthy volunteers and septic shock patients, stratified or not according to immunity state.(1) Of 5,6% at 6,9% of HERVs are expressed in the blood compartment and around 20% of LTRs have apromoter or polyA function, both functions being mutually exclusive. (2) The HERV transcriptome ismodulated in ex vivo endotoxin tolerance model letting appear two higher transcriptional phenotypes. Theexpression of some HERVs loci are correlated of the immunity state of the septic shock patients. Theevaluation of molecular signature in validation cohort, allowed to separate in two patients groupspresenting different severity criteria, suggesting HERV/MaLR as biomarkers of stratification. (3) The coexpressedanalysis of genes and HERVs allowed to integrate these within signaling pathways associated atthe host immune response and to provide functional hypothesis
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Simonneau, Lionel. "Etude de l'expression des cristallines et de leurs proprietes aggregatives dans les cultures de cellules epitheliales de cristallin de boeuf et de la neurotine embryonnaire de caille normale ou transformee par des retrovirus aviaires". Paris 7, 1988. http://www.theses.fr/1988PA077154.
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Bernaud, Julien. "Propriétés physiques de capsides virales étudiées à l'échelle du virus unique par microscopie à force atomique : exemples du rétrovirus VIH-1 et du parvovirus AAV". Thesis, Lyon, École normale supérieure, 2015. http://www.theses.fr/2015ENSL1028/document.
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Les virus sont des parasites biologiques de taille nanométrique. Détournant la machinerie cellulaire de la cellule infectée, ils mettent en place une stratégie de réplication permettant la production de nouveaux virus. Un virus est constitué d’une capside protéique protégeant le génome viral, long polymère d’ADN ou ARN, et possède dans certains cas une enveloppe lipidique. Des travaux récents suggèrent que les propriétés physiques des virus sont importantes pour comprendre certaines étapes du cycle viral. Dans le but de relier le comportement biologique des virus à leurs propriétés physiques, nous avons utilisé une approche combinant l’imagerie AFM et des mesures mécaniques à l’échelle nanométrique, en lien avec la modélisation physique des capsides virales. Nous avons développé des outils d’analyse automatisée des images et courbes de forces obtenues pour quantifier les propriétés physiques de capsides virales et l’effet du microenvironnement. Nous avons étudié deux virus très différents : le rétrovirus VIH-1, responsable du SIDA et le vecteur AAV, utilisé en thérapie génique. Ce travail a permis la caractérisation des propriétés morphologiques et mécaniques de pseudo-particules virales et de cores du VIH-1, à l’échelle du virus unique et sur des populations de centaines de virus. En nous intéressant à l’effet de la nature de l’ARN encapsidé dans les particules virales in cellulo, nous avons montré un rôle structurant pour l’ARN viral du VIH-1 et en particulier son signal d’encapsidation psi. Enfin, nous avons initié l’étude de l’effet de la retro-transcription (conversion du génome viral ARN en ADN) au sein du core VIH-1 sur la stabilité de celui-ci. L’étude du parvovirus AAV existant sous forme de plusieurs variants naturels (sérotypes) nous a permis de comparer les propriétés physiques des capsides à l’équilibre thermodynamique et hors d’équilibre. En faisant varier le microenvironnement (température et pH), nous avons sondé son influence sur la stabilité des capsides AAV. Nous avons pu montrer en particulier que la capside AAV8 est plus rigide que AAV9 alors que sa stabilité thermique est réduite, en relation avec des propriétés biologiques différentes pour ces deux sérotypes. En outre, la rigidité des capsides AAV8 diminue dans un environnement acide imitant l’endosome tardif, et ceci se traduit par une plus grande stabilité thermique. Enfin, nous avons quantifié l’effet de la longueur et de la nature du génome sur la stabilité des capsides AAVViruses are nanometer size biological parasite, which highjack the cellular machinery of the infected cells to replicate and thereby produce new viruses. A virus consists of a protein capsid, protecting the viral genome, a long polymer of DNA or RNA, and in some cases is surrounded by a lipid envelope. Recent work suggests that the physical properties of viruses are important in order to understand the viral cycle. In order to link the biological behavior of the virus to their physical properties, we used an approach combining AFM imaging and mechanical measurements at the nanometer scale, in connection with the physical modeling of viral capsids. We have developed automated image and force curves analysis tools to quantify the physical properties of viral capsids and the effect of the microenvironment. We have focused on two very different viruses: the HIV-1 retrovirus, responsible for AIDS and the AAV vector used in gene therapy. This work has led to the characterization of the morphological and mechanical properties of virus-like particles and cores of HIV-1 at the single virus level and on populations of hundreds of viruses. Focusing on the effect of the nature of the RNA encapsidated inside the viral particles in cellulo, we have highlighted the structural control of the viral RNA, and more precisely the psi packaging signal, on both HIV-1 VLPs and cores. Finally, we have initiated the study of the effect of reverse transcription (conversion of viral genomic RNA into DNA) within the cores HIV-1 on its stability. The study of parvovirus AAV existing form of several natural variants (serotypes) allowed us to compare the capsid physical properties at thermodynamic equilibrium and out of equilibrium. By varying the microenvironment (temperature and pH), we probed its influence on the stability of the AAV capsid. We have shown in particular that the AAV8 virus is stiffer than AAV9 while thermal stability is reduced, in relation to different biological properties for these two serotypes. In addition, the rigidity of AAV8 capsids decreases in an acidic environment mimicking the late endosome transport, and this results in a higher thermal stability. Finally, we quantified the effect of the length and nature of the confined genome on the thermal stability of AAV capsids
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Sullivan, Timothy A. "Studies of entry, reverse transcription, and regulation of splicing in retroviruses". View the abstract Download the full-text PDF version, 2008. http://etd.utmem.edu/ABSTRACTS/2008-054-Sullivan-index.htm.
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Thesis (M.S.)--University of Tennessee Health Science Center, 2008.Title from title page screen (viewed on February 24, 2009). Research advisor: Lorraine M. Albritton Ph.D. Document formatted into pages (vii, 81p. : ill.). Vita. Abstract. Includes bibliographical references (p. 65-74).
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Voronin, Yegor A. "Investigation of initiation of reverse transcription in retroviruses using vectors with two primer-binding sites". Morgantown, W. Va. : [West Virginia University Libraries], 2003. http://etd.wvu.edu/templates/showETD.cfm?recnum=3136.
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Tolentino, Felipe Thadeu 1983. "Produção e avaliação de vetores retrovirais visando à diferenciação de neurônios olfativos in vitro pela superexpressão de fatores de transcrição definidos". [s.n.], 2014. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316718.
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Orientador: Fabio PapesDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O Sistema Sensorial Olfativo de mamíferos é composto por vários subsistemas na cavidade nasal. Dentre estes, destacam-se o sistema olfativo principal e o sistema olfativo acessório ou vomeronasal. O primeiro realiza a detecção geral de odores e parece participar também da detecção de algumas substâncias que levam a respostas comportamentais instintivas (feromônios), enquanto o último é especializado na detecção desta classe de semioquímicos. A detecção dos estímulos sensoriais olfativos resulta em informações importantes que dependem de vias complexas para sua interpretação e para a geração de respostas apropriadas por parte do sistema nervoso central. Existem vários pontos ainda desconhecidos sobre o funcionamento do sistema olfativo, tanto no que diz respeito aos mecanismos moleculares subjacentes à escolha dos receptores a serem expressos por um dado neurônio sensorial ¿ sendo que cada neurônio olfativo expressa apenas um receptor dentro de uma grande família multi-gênica ¿ quanto em relação ao processamento da informação sensorial em centros cerebrais superiores. Neurônios sensoriais olfativos cultivados eficientemente in vitro seriam extremamente úteis, pois poderiam ser utilizados como ferramenta para o estudo destes problemas, como a investigação da atividade das células sensoriais olfativas, possibilitando, por exemplo, uma melhor compreensão dos mecanismos genéticos e moleculares por trás da expressão dos receptores olfativos e de suas propriedades de detecção. Neste trabalho foram desenvolvidas ferramentas baseadas em vetores retrovirais com o objetivo de induzir a diferenciação celular de neurônios olfativos in vitro, utilizando uma combinação de fatores de transcrição, por meio de transdução viral em células-alvo (fibroblastos murinos). Os retrovírus produzidos foram testados e algumas combinações de fatores de transcrição foram preliminarmente testadas, sendo capazes de induzir mudanças moleculares em fibroblastos acompanhadas da expressão de marcadores de neurônios sensoriais olfativos
Abstract: The mammalian Olfactory System enables the vast majority of animal species to identify the presence and quality of food, predators, competitors, conspecifics and potential mates in the environment. Olfactory stimuli detected by sensory neurons are interpreted by brain processing pathways to generate appropriate behavioral and endocrine responses. Despite its central importance in mammalian physiology, several aspects about the biology of this sensory system remain uncharacterized. For example, it is known that each olfactory sensory neuron (OSN) in the nasal cavity expresses only one gene out of a large multi-gene family coding for receptors involved in odorant and pheromone detection. However, the molecular mechanisms behind this process of olfactory receptor gene choice are not fully understood. The study of this and many other aspects of olfaction has been made difficult by the lack of appropriate in vitro cellular models. An efficient way to obtain cultured OSNs would thus be extremely useful, enabling researchers to investigate the sensory neuron¿s activity in a controllable environment, avoiding obstacles imposed by the cellular heterogeneity found in sensory organs in vivo. In this study, we aimed at obtaining OSNs directly differentiated from mouse embryonic fibroblasts (MEF) using the forced expression of specific transcription factors via retroviral vectors. We therefore developed tools based on retroviral vectors with the objective of differentiating olfactory sensory neurons in vitro, using viral transduction in target cells (murine fibroblasts) with combinations of select transcription factors. Retroviruses were tested and some combinations of transcription factors were tested on a preliminary basis, which were capable of inducing molecular alterations on fibroblasts followed by the expression of olfactory sensory neuron markers
Mestrado
Genetica Animal e Evolução
Mestre em Genética e Biologia Molecular
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Leung, Danny Chi Yeu. "Transcriptional silencing of endogenous retroviruses : interplay between histone H3K9 methylation and DNA methylation". Thesis, University of British Columbia, 2011. http://hdl.handle.net/2429/38966.
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Endogenous retroviruses (ERVs) are found in genomes of all higher eukaryotes. As retrotransposition is deleterious, pathways have evolved to repress these retroelements. While DNA methylation transcriptionally represses ERVs in differentiated cells, this epigenetic mark is dispensable for maintaining proviral silencing during early stages of mouse embryogenesis and in embryonic stem cells (mESCs). Studies in diverse species have found histone H3K9 methylation and DNA methylation to function together to repress retrotransposons. However, until recently, little was known about the role of this histone modification in proviral silencing in mESCs. Interestingly, our analysis of mESCs lacking G9a, an H3K9-specific lysine methyltransferase (KMTase) revealed that although ERVs lost H3K9 di-methylation (me2) and DNA methylation, they remained silent. Strikingly, the levels of H3K9 tri-methylation (me3) remained unaltered, suggesting that this mark may instead be responsible for maintaining these parasitic elements transcriptionally inactive. The first stage of my research focused on identifying the enzyme depositing H3K9me3 at ERVs and on determining its role in proviral silencing. I discovered that Setdb1, another H3K9-specific KMTase, was indeed depositing H3K9me3 at a subset of ERVs and was required for maintaining transcriptional repression. Interestingly, this silencing pathway operated independently of DNA methylation. Through collaboration, we also discovered that this pathway played a diminished role in differentiated cells. Taken together, these findings indicate the existence of a DNA methylation-independent proviral silencing pathway in mESCs. The second stage of my research focused on the establishment of transcriptional repression of newly integrated proviruses. By employing an exogenous retroviral construct, I discovered a dramatic silencing defect in mESCs lacking G9a, which phenocopied cells depleted of the de novo DNA methyltransferases. Furthermore, efficient DNA methylation of proviruses required G9a-mediated H3K9me2. These findings reveal that histone modifications and DNA methylation function in concert to defend the genome against invading retroviral elements in mESCs. Taken together with discoveries regarding the mechanism of DNA demethylation in early embryos, I propose that cells undergoing DNA methylation reprogramming predominantly employ histone modification-based pathways to maintain these parasitic elements in a silent state; however, the establishment of transcriptional repression for newly integrated elements also requires de novo DNA methylation.
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Azevedo, Filipe Costa. "A transcriptase reversa como alvo terapêutico em doenças retrovirais". Master's thesis, [s.n.], 2013. http://hdl.handle.net/10284/4082.
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Projeto de Pós-Graduação/Dissertação apresentado à Universidade Fernando Pessoa como parte dos requisitos para obtenção do grau de Mestre em Ciências FarmacêuticasO impacto imediato da descoberta da enzima transcriptase reversa veio alterar até então o dogma central da biologia molecular, ou seja, que a transferência da informação genética é unidirecional: ADN-> ARN-> Proteína (Gilboa et al., 1979). As transcriptases reversas retrovirais são máquinas moleculares complexas com partes móveis e atividades múltiplas (Flint et al., 2009). Esta enzima também permitiu compreender a persistência de infeções retrovirais, bem como aspetos patogénicos do vírus da imunodeficiência adquirida (VIH) (Flint et al., 2009). Os retrovírus possuem um genoma composto por duas cadeias simples de ARN e replicam o ARN viral por transcrição reversa pela ação da enzima transcriptase reversa (Tenório et al., 2008). A família dos retrovírus tem vindo a ser um dos principais alvos de estudo de cientistas nas últimas décadas por ser causadora de doenças graves em humanos, como a síndrome da imunodeficiência adquirida (SIDA) (Tenório et al., 2008). O vírus responsável é o Vírus da Imunodeficiência Humana (VIH) e trata-se de um retrovírus que modifica a composição genética das células que infeta, destruindo-as. São conhecidos dois tipos de vírus: VIH-1 e VIH-2 (Araújo, 2005). O avanço mais significativo, em termos de gestão da infeção VIH-1, pode ser atribuído ao tratamento dos pacientes através da utilização de fármacos antivirais, os quais podem suprimir a replicação do VIH-1 a níveis indetetáveis (Arts e Hazuda, 2012). Até à data, estão disponíveis cerca de 36 medicamentos para o tratamento de infeções por VIH, todos eles aprovados pela Food and Drug Administration (FDA) (U.S. Department of Health & Human Services, 2013). Entre eles, destacam-se duas classes de fármacos que inibem especificamente a enzima transcriptase reversa - inibidores da transcriptase reversa análogos de nucleosídeos (ITRAN) e inibidores da transcriptase reversa não análogos de nucleosídeos (ITRNAN) (Collier et al., 1996; D’Aquila et al., 1996; Stas- zewski et al., 1996). Uma das grandes ameaças a todas as terapias antivirais que existem atualmente, será sempre o aparecimento de estirpes virais resistentes à ação dos fármacos existentes (Sleiman et al., 2012). Por isso, é importante a presença constante de novos conhecimentos sobre toda a biologia da replicação viral, de forma a se obterem novas terapias em alternativa aos fármacos clássicos já existentes (Buckheit et al., 2010). The immediate impact of the discovery of the enzyme reverse transcriptase amends by then the central dogma of molecular biology , in other words, the transfer of genetic information is unidirectional : DNA - > RNA - > Protein (Gilboa et al., 1979) . The retroviral reverse transcriptases are complex molecular machines with moving parts and multiple activities (Flint et al., 2009). This enzyme also allowed us to understand the persistence of retroviral infections and pathogenic aspects of human immunodeficiency virus (HIV) (Flint et al., 2009). Retroviruses have a genome consisting of two single strands of RNA and replicate the viral RNA by reverse transcription by the action of the enzyme reverse transcriptase (Tenório et al., 2008). The family of retroviruses has been a main target of study in recent decades to be a cause of serious diseases in humans, such as acquired immunodeficiency syndrome (AIDS) (Tenório et al., 2008). The virus responsible is the human immunodeficiency virus (HIV), and it is a retrovirus that modifies the genetic composition of the infecting cells, destroying them. There are known two kinds of viruses: HIV-1 and HIV-2 (Araújo, 2005). The most significant, in terms of management of HIV-1 infection can be attributed to treatment of patients through the use of antiviral drugs which can suppress the replication of HIV-1 to undetectable levels (Hazuda and Arts , 2012). To date are available approximately 36 drugs for the treatment of HIV infections, all approved by the Food and Drug Administration (FDA) (U.S. Department of Health & Human Services, 2013). Among them, we highlight two classes of drugs that specifically inhibit the reverse transcriptase - nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) (Collier et al., 1996; D ' Aquila et al., 1996; Stas- zewski et al., 1996). One of the major threats to all antiviral therapies that will always be the emergence of viral strains resistant to the action of available drugs (Sleiman et al., 2012). Therefore, it is important to the continuing presence of new knowledge about the biology of the whole viral replication in order to obtain new therapies alternative to existing classical drugs (Buckheit et al., 2010).
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Soufuku, Kozue. "Transcription Profiling Demonstrates Epigenetic Control of Non-retroviral RNA Virus-Derived Elements in the Human Genome". Kyoto University, 2016. http://hdl.handle.net/2433/215439.
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Thompson, Peter Jeffrey. "Transcriptional silencing of endogenous retroviruses by the novel lysine methyltransferase co-repressor hnRNP K". Thesis, University of British Columbia, 2015. http://hdl.handle.net/2429/55760.
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Histone lysine methylation is essential for mammalian development and maintenance of somatic cell identity, as evidenced by a group of Mendelian diseases and cancers linked with mutations in lysine methyltransferases (KMTs). The transcriptional silencing of a class of retrotransposons known as endogenous retroviruses (ERVs) in murine embryonic stem cells (mESCs) provides a unique model system in which to investigate epigenetic regulation by the H3K9 family of KMTs and characterize novel molecular mechanisms of relevance to human biology and disease. In mESCs, class I and II ERVs are silenced by the SETDB1/KAP1 complex, which deposits histone H3K9 trimethylation (H3K9me3). In contrast, class III MERVL ERVs are silenced by the G9a/ GLP complex, which deposits H3K9me2. The molecular mechanisms governing the recruitment of these KMTs to their genomic ERV targets remain poorly understood. The goal of this work was to identify and characterize novel factors that regulate the functions of these KMTs in ERV silencing. In the first part of my thesis work, I identified the RNA-binding protein and transcription factor hnRNP K as a novel co-repressor for the SETDB1/KAP1 complex. HnRNP K coordinates recruitment of the KMT SETDB1 by KAP1 to its ERV targets. This function of hnRNP K involves a previously uncharacterized influence on the levels of chromatin protein SUMOylation. In the second part of my thesis work, I demonstrated that MERVL elements are also repressed by hnRNP K and can remain inactive in the absence of H3K9me2, likely due to the lack of transcriptional activators. HnRNP K forms a novel RNA-dependent complex with G9a/GLP, is required for global H3K9me2 and provides a repressive barrier to MERVL expression in the presence and absence of H3K9me2. Taken together my work has provided significant insights into the epigenetic repression of ERV transcription by KMTs and demonstrates that hnRNP K is a novel co-repressor for two different KMT complexes. As recent studies have linked mutations in HNRNPK to the novel Mendelian disorder Au-Kline syndrome and cancer, these insights should also guide future studies on the role of hnRNP K in regulation of KMT-mediated signaling pathways in human disease.Medicine, Faculty of
Medical Genetics, Department of
Graduate
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Palmer, Matthew T. "The influence of retroviral codon usage on the acquisition of the tRNA used to prime reverse transcription". Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2006. https://www.mhsl.uab.edu/dt/2007r/palmer.pdf.
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Whiting, Sam H. "Studies into the characteristics and mechanism of strand displacement synthesis by retroviral reverse transcriptase /". Thesis, Connect to this title online; UW restricted, 1997. http://hdl.handle.net/1773/11494.
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Tang, Hengli. "Identification and characterization of a cellular protein involved in the post-transcriptional regulation of retroviruses /". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 1998. http://wwwlib.umi.com/cr/ucsd/fullcit?p9904823.
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Anderson, Jeffrey A. "Retroviral recombination during reverse transcription an analysis of the mechanism, frequency, and effect of the viral packaging signal [psi] /". Morgantown, W. Va. : [West Virginia University Libraries], 2001. http://etd.wvu.edu/templates/showETD.cfm?recnum=1822.
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Thesis (Ph. D.)--West Virginia University, 2001.Title from document title page. Document formatted into pages; contains viii, 174 p. : ill. Vita. Includes abstract. Includes bibliographical references.
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Sharma, Amit. "Functional control of HIV-1 post-transcriptional gene expression by host cell factors". The Ohio State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=osu1330658547.
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Dekoninck, Ann. "Etude de la régulation transcriptionnelle du virus de la leucémie bovine: rôle de la chromatine et des facteurs de transcription PU.1 et Sp1/Sp3". Doctoral thesis, Universite Libre de Bruxelles, 2005. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/211015.
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Crowe, Brandon L. "Structural Features of Proteins Involved in Pfu RNase P or Recruitment of Viral Genomes to Human Chromatin". The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu1471621708.
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Bolinger, Cheryl Giles. "Study of translation control by a RNA helicase A-responsive post-transcriptional control element in Retroviridae". The Ohio State University, 2008. http://rave.ohiolink.edu/etdc/view?acc_num=osu1226513076.
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Comandur, Roopa. "Structure of Retroviral 5′-Untranslated Regions and Interactions with Host and Viral Proteins". The Ohio State University, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=osu148060178765983.
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Colin, Laurence. "Molecular control of gene expression in the HIV-1 and BLV retroviruses". Doctoral thesis, Universite Libre de Bruxelles, 2011. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209944.
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Après intégration dans le génome cellulaire de l’hôte, l’expression des rétrovirus dépend d’éléments agissant en cis localisés dans la longue répétition terminale 5’ (LTR5’) et la région leader, de facteurs de transcription cellulaires et viraux agissant en trans ainsi que de l’organisation chromatinienne du provirus intégré. Notre laboratoire a précédemment identifié dans le génome du rétrovirus HIV-1 (Human Immunodeficiency Virus type 1) une région intragénique importante (nt 4079-6026, où nt +1 est le début de U3 dans le LTR5’) composée du fragment 5103, du site hypersensible aux nucléases SH7 et du fragment 5105. Lors de ce travail, nous avons caractérisé physiquement et fonctionnellement différents sites de liaison pour des facteurs de transcription cellulaires localisés dans la région intragénique du virus HIV-1, dont trois sites de liaison pour le facteur inductible AP-1, dans des expériences de retard de migration sur gel et de transfection transitoire. Nous avons montré l’importance de ces trois sites AP-1 pour la réplication virale au niveau transcriptionnel dans des expériences d’infection et d’immunoprécipitation de la chromatine. De plus, nous avons caractérisé l’activité transcriptionnelle associée à la région intragénique du virus HIV-1. D’autre part, la structure nucléosomale du provirus intégré et les modifications épigénétiques associées jouent un rôle crucial pour l’expression des rétrovirus. La répression transcriptionnelle du rétrovirus oncogène BLV (Bovine Leukemia Virus) lui permet d’échapper au système immunitaire de son hôte bovin et favorise ainsi l’apparition de tumeurs. Dans ce contexte, nous avons montré que la méthylation de l’ADN au niveau du promoteur viral permet le maintient de la latence transcriptionnelle. En effet, la méthylation des dinucleotides CpGs localisés dans le LTR5‘ empêche le recrutement in vivo des facteurs de transcription activateurs CREB/CREM/ATF. Nous avons également montré que l’activation transcriptionnelle de l’expression du BLV par la combinaison PMA/ionomycine s’accompagne d’un remodelage chromatinien rapide mais transitoire au niveau du promoteur viral par des expériences de marquage indirect des extrémités et d’immunoprécipitation de chromatine. Nous avons ensuite démontré l’importance du site de liaison pour le facteur de transcription PU.1 et de la E-box 4 qui lie USF-1/-2, tous deux localisés dans la région dont l’accessibilité aux nucléases s’accroît après traitement des cellules, pour l’activation transcriptionnelle de l’expression virale par cette combinaison d’inducteurs. En conclusion, notre travail devrait permettre une meilleure compréhension des mécanismes transcriptionnels et épigénétiques régulant l’expression des rétrovirus HIV-1 et BLV.Doctorat en Sciences
info:eu-repo/semantics/nonPublished
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Westberg, Christopher Bryant. "Identification and characterization of three RNA helicase A binding proteins and their roles in the post-transcriptional regulation of simple retroviruses /". Diss., Connect to a 24 p. preview or request complete full text in PDF format. Access restricted to UC campuses, 2002. http://wwwlib.umi.com/cr/ucsd/fullcit?p3044786.
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Kelleher, Colleen Diane. "Characterization of polymerase and RNase H activities of Moloney murine leukemia virus reverse transcriptase in relation to models for retroviral plus-strand synthesis /". Thesis, Connect to this title online; UW restricted, 1999. http://hdl.handle.net/1773/11519.
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Gimenez, Juliette. "Implication de la méthylation dans le contrôle de l'expression de rétrovirus endogènes humains en contextes physiologiques et pathologiques". Thesis, Lyon 1, 2009. http://www.theses.fr/2009LYO10222.
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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 concernésEndogenous 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
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Chen, Wei-Kang. "Analysis of neural gene expression: glutamine synthetase and nitric oxide synthas 1". The Ohio State University, 2003. http://rave.ohiolink.edu/etdc/view?acc_num=osu1061581254.
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Dupressoir, Anne. "Etude, chez la souris, de la regulation de la transcription d'elements genetiques mobiles de type retroviral -les sequences iap- dans les processus de developpement normal et de transformation tumorale". Paris 7, 1995. http://www.theses.fr/1995PA077190.
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Afin d'etudier la regulation de l'expression des sequences iap au cours du developpement normal de la souris, nous avons genere des souris transgeniques (ltrlacz) portant le gene nls-lacz sous controle de la region regulatrice (ltr) de deux sequences iap differentes. Dans toutes les lignees obtenues, l'expression du transgene est restreinte aux cellules spermatogeniques souches ; ce resultat est compatible avec un role de ces transposons dans l'evolution de l'espece murine. Une correlation entre l'expression du transgene et son hypomethylation a egalement ete observee. Nous avons alors cherche a transformer les cellules germinales males en utilisant, cette fois, l'oncogene viral t de sv40 comme gene rapporteur. De maniere inattendue, ces souris ne developpent pas de tumeurs testiculaires mais des tumeurs cerebrales, pancreatiques et des pathologies du thymus. D'autres lignees transgeniques seront necessaires pour interpreter ces resultats. Nous avons egalement etudie la regulation de l'expression du transgene ltrlacz et des iap endogenes dans differentes tumeurs induites chez les souris transgeniques ltrlacz (par croisement avec des oncosouris): ces resultats encore preliminaires ne montrent pas d'induction du ltr iap transgenique au cours du processus tumoral ; ceci suggere que l'activation de la transcription des iap endogenes observee dans certaines tumeurs ne resulte pas de l'activation specifique des sequences cis-regulatrices de ces elements. Enfin, au cours d'une etude systematique de la regulation de l'expression des sequences iap endogenes au cours du vieillissement, nous avons mis en evidence l'induction de deux transcrits iap de taille anormale dans le foie des souris agees, et ce quelque soit la souche consideree. Nous avons fait l'hypothese que ces transcrits devaient correspondre a un effet de position associe a une sequence iap inseree dans un domaine soumis a regulation au cours du vieillissement (ou are pour age responsive element). La caracterisation de la region are est actuellement en cours
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Yilmaz, Alper. "Translational control of mRNAs transcribed from HIV-1 provirus and HIV-1 based lentiviral vectors". Columbus, Ohio : Ohio State University, 2007. http://rave.ohiolink.edu/etdc/view?acc%5Fnum=osu1189785802.
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Kim, Sang-Woo. "Walleye retroviral cyclins phosphorylate pRb tumor suppressor and the walleye dermal sarcoma retrovirus cyclin and G2/M cyclins repress transcription of p14[superscript]ARF tumor suppressor through interaction with TBX2, possibly contributing to tumorigenesis /". 2004. http://wwwlib.umi.com/dissertations/fullcit/3154241.
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Dobšová, Martina. "Lidský endogenní retrovirus ERVWE1: transkripční aktivace a změny methylace DNA v promotorové oblasti". Master's thesis, 2014. http://www.nusl.cz/ntk/nusl-332228.
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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...
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Larocque, Émilie. "Caractérisation des transcrits antisens chez les rétrovirus HTLV et étude comparative des fonctions des protéines traduites à partir de ces transcrits antisens". Thèse, 2015. http://hdl.handle.net/1866/13038.
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Le premier membre de la famille des rétrovirus humains HTLV (Virus T-lymphotropique Humain), HTLV-1, a été découvert en 1980 et l’on estime aujourd’hui à plus de 10 millions le nombre d’individus infectés à travers le monde. Après une période de latence d’environ 40 ans, 5% des individus infectés développent des leucémies, des lymphomes adultes de lymphocytes T (ATLL) ou encore une myélopathie associée à HTLV-1/ paraparésie spastique tropicale (HAM/TSP). L’apparition de la maladie serait en grande partie orchestrée par deux protéines virales, soit Tax et HTLV-1 bZIP factor (HBZ). L’expression du génome viral se fait à partir d’un transcrit sens de pleine longueur suite à un épissage alternatif, à l’exception du gène HBZ. HBZ est produite à partir d’un transcrit antisens initié dans la séquence terminale longue répétée (LTR)’3. Elle a été décrite comme étant capable de réguler négativement la transcription virale dépendante de Tax en se dimérisant avec des facteurs de transcription cellulaires tels que CREB-2 et certains membres de la famille Jun. HBZ a aussi un pouvoir prolifératif et bien que nous ne sachions toujours pas le mécanisme moléculaire menant à l’oncogenèse par HBZ, nous savons qu’elle module une multitude de voies de transduction de signaux, dont AP-1. Nous avons récemment mis en évidence un transcrit antisens nommé Antisense Protein of HTLV-2 (APH-2) chez HTLV-2 qui n’est associé qu’à une myélopathie apparentée au HAM/TSP. Ce n’est qu’en 2005 que HTLV-3 et HTLV-4 se sont rajoutés au groupe HTLV. Cependant, aucune corrélation avec le développement d’une quelconque maladie n’a été montrée jusqu’à ce jour. Le premier volet de ce projet de doctorat avait pour objectif de détecter et caractériser les transcrits antisens produits par HTLV-3 et HTLV-4 et d’étudier les protéines traduites à partir de ces transcrits pour ainsi évaluer leurs similitudes et/ou différences avec HBZ et APH-2. Nos études de localisation cellulaire réalisées par microscopie confocale ont montré que APH-3 et APH-4 sont des protéines nucléaires, se retrouvant sous la forme de granules et, dans le cas d’APH-3, partiellement cytoplasmique. Ces granules co-localisent en partie avec HBZ. Les analyses à l’aide d’un gène rapporteur luciférase contenant le LTR 5’ de HTLV-1 ont montré que APH-3 et APH-4 peuvent aussi inhiber la transactivation du LTR 5’ par Tax. Aussi, des études faisant appel au gène rapporteur précédé d’un promoteur de collagénase (site AP-1), ont montré que ces deux protéines, contrairement à HBZ, activent la transcription dépendante de tous les membres des facteurs de transcription de la famille Jun. De plus, les mutants ont montré que le motif fermeture éclair (LZ) atypique de ces protéines est impliqué dans cette régulation. En effet, APH-3 et APH-4 modulent la voie Jun-dépendante en se dimérisant via leur LZ atypique avec la famille Jun et semblent activer la voie par un mécanisme ne faisant pas par d’un domaine activateur autonome. Dans un deuxième volet, nous avions comme objectif d’approfondir nos connaissances sur la localisation nucléolaire de HBZ. Lors de nos analyses, nous avons identifié deux nouveaux partenaires d’interaction, B23 et la nucléoline, qui semblent être associés à sa localisation nucléolaire. En effet, ces interactions sont plus fortes suivant une délétion des domaines AD et bZIP de HBZ qui dans ce cas est localisée strictement au nucléole. De plus, bien que APH-3 et APH-4 puissent se localiser aux nucléoles, HBZ est la seule protéine traduite à partir d’un transcrit antisens pouvant interagir avec B23. Finalement, ces travaux ont clairement mis en évidence que HTLV-3 et HTLV-4 permettent la production de transcrits antisens comme chez d’autres rétrovirus. Les protéines traduites à partir de ces transcrits antisens jouent d’importants rôles dans la réplication rétrovirale mais semblent avoir des fonctions différentes de celles de HBZ au niveau de la régulation de la transcription de la voie Jun. HBZ semble aussi jouer un rôle unique dans le nucléole en ciblant les protéines nucléolaires de la cellule. Ces études démontrent que les protéines produites à partir de transcrits antisens chez les rétrovirus HTLV partagent plusieurs ressemblances, mais démontrent aussi des différences. Ainsi, les APH pourraient, en tant qu’outil comparatif, aider à mieux cibler les mécanismes moléculaires importants utilisés par HBZ pour induire la pathogénèse associée à une infection par HTLV.The first human T-cell lymphotropic virus (HTLV) family member was discovered in 1980 and it is estimated that approximately 10 million people are infected with HTLV-1 worldwide. After about 40 years, 5% of infected individuals will develop an adult T-cell leukemia/lymphoma (ATLL) while another 4% will develop HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It is believed that two viral proteins, Tax and HBZ, together orchestrate the oncogenic process. The viral proteins are expressed from an alternatively spliced sense transcript except for the HBZ gene. HBZ is translated from an antisense transcript initiated in the long terminal repeat (LTR)’3. This viral protein is capable of inhibiting Tax transactivation of the LTR5’ by dimerizing with cellular transcription factors such as CREB-2 and c-Jun. HBZ also has proliferating capacities and while the molecular mechanisms leading to the disease still need to be elucidated, it is well known that HBZ can modulate a multitude of signal transduction pathways like AP-1. We have recently discovered an antisense transcript termed Antisense Protein of HTLV-2 (APH-2) produced in HTLV-2. HTLV-2 is only associated to myelopathies resembling HAM/TSP. HTLV-3 and HTLV-4 were discovered in 2005 and have not been associated with any type of disease thus far. The first goal of this PhD project was hence to detect and characterize the antisense transcripts produced in HTLV-3 and HTLV-4, to study the functions of these translated proteins and to evaluate their similarities and/or differences shared with HBZ and APH-2. Our localization studies using confocal microscopy demonstrated that APH-3 and APH-4 are found in the nucleus as speckles, and for APH-3, also partially cytoplasmic. These two proteins can also partially colocalize with HBZ. Using a luciferase reporter plasmid bearing the HTLV-1 LTR5’, we demonstrated that APH-3 and APH-4 could inhibit Tax transactivation of the LTR5’. We also used a luciferase reporter plasmid bearing the collagenase promoter, which bears an AP-1 site, and demonstrated that both viral proteins could activate transcription in the presence of any of the Jun family of transcription factors. We generated several mutants and the atypical leucine zipper (LZ) found in APH-3 and APH-4 is crucial for this regulation. In fact, APH-3 and APH-4 using their atypical LZ dimerize with Jun family members and activate this pathway using a mechanism other than an autonomous activation domain. Our next goal was to investigate the significance of the HBZ nucleolar localization. During this project, we identified two new interacting partners, B23 and nucleolin, which seem to be associated with its nucleolar localization. In fact, these interactions are stronger when HBZ is deleted of its AD and bZIP domains and hence when HBZ demonstrates a stronger nucleolar distribution. Moreover, while APH-3 and APH-4 are also found in the nucleolus, HBZ is the only antisense protein able to interact with B23. Finally, this work clearly demonstrates that HTLV-3 and HTLV-4 can produce an antisense transcript alike other retroviruses. The encoded proteins play an important role in retroviral replication and seem to regulate Jun-dependant transcription differently than HBZ. HBZ also seems to have a unique role in the nucleoli by targeting specific cellular nucleolar proteins. Similarities but also differences are shared between the antisense proteins. Thus, the APH proteins represent a good comparative tool in order to better understand the molecular mechanisms involved in HTLV induced diseases.
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Slavková, Martina. "Reportérový expresní systém pro studium umlčování integrovaného proviru v transkribované oblasti genu". Master's thesis, 2015. http://www.nusl.cz/ntk/nusl-343810.
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Retroviral vectors are used as mighty tools for an introduction of recombinant genes into the recipient genome in gene therapy trials. In the vector design, great emphasis is put on safety and efficiency. In spite of a great progress in retroviral vector design with the purpose to stabilize its expression, e.g. introduction of protective elements into the viral regulatory sequences, the current approaches are still not sufficiently effective and the majority of proviruses is transcriptionally silenced. The understanding of the silencing mechanism is of special importance to the optimization of the vector design and handling. In this master thesis, I have designed and constructed an expression system for study of the mechanism involved in the silencing of retroviruses integrated inside gene bodies. This artificial system will be utilized for testing of hypothesis that retroviruses integrated into gene bodies are silenced by DNMT-dependent mechanism and this process is triggered by transcriptional read-through of the provirus from nearby host promoter. I have obtained preliminary results suggesting the validity of the suggested hypothesis; however the verification of general validity of this hypothesis for various retroviruses and elements will be a matter of further studies in our laboratory. Powered by...
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Landry, Josette-Renee. "Transcriptional regulation of human genes by endogenous retroviral elements". Thesis, 2003. http://hdl.handle.net/2429/14958.
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Human endogenous retroviruses (HERVs) and other long terminal repeat (LTR)-
containing elements comprise a significant portion (8%) of the human genome and are likely
vestiges of retroviral infections during primate evolution. Although the vast majority of
HERVs are now unable to code for retroviral proteins, an unknown number have retained
functional transcriptional elements within their LTRs and some of these regulatory sequences
have been shown to participate in the transcription of nearby genes. The overall objective of
my thesis was to further understand the role of HERVs in human gene regulation by
investigating LTRs that provide alternative promoters to cellular genes. When I began my
study, three putative endogenous retroviral promoters were identified by screening sequence
databases for chimeric (viral-cellular) transcripts. These searches revealed fusion transcripts
containing the LTRs of three HERV-E elements linked to the endothelin B receptor
(EDNRB), the apolipoprotein C-l (APOC1) and the Opitz syndrome gene, midline 1. To
confirm the authenticity of the chimeric transcript and to establish that the mRNAs were
transcribed from the retroviral LTRs, we performed 5'RACE and determined the genomic
organization for each gene. Our results indicated that the chimeric transcripts were
alternatively promoted by the retroviral elements, as they initiated within HERV-E LTRs but
spliced into the downstream coding sequence of the cellular genes. To determine the
expression pattern and the relative contribution of the retroviral promoter, we quantified the
percentage of transcripts which were chimeric in various tissues using real-time PCR. While
chimeric APOC1 transcripts could be detected in several tissues tested, the retroviral
promoter of EDNRB and MIDI appeared to be placenta-specific. Transient transfection
studies supported a role for the EDNRB and MIDI LTRs as strong promoters in placenta and suggested a function for the LTRs as enhancers. Further deletion and hybrid constructs
delineated regions within both LTRs necessary for strong promoter activity. Finally, to
further characterize the APOC1, EDNRB and MIDI genes, the non-retro viral (native)
promoters of these three genes were also analysed. These findings provide further evidence
that some endogenous retroviruses have evolved a biological function as transcriptional
regulatory elements by contributing alternative promoters to human genes.
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Nelson, David Troy. "Transcriptional regulatory elements in the long terminal repeats of the human endrogenous retrovirus, HERV-H". Thesis, 1997. http://hdl.handle.net/2429/6548.
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HERV-H sequences comprise a large family of human endogenous retroviruslike
elements. Previous DNA sequence comparisons of HERV-H long terminal repeats
(LTRs) have led to their classification into three subtypes, Type I, la and II. Type la
appears to have been generated by recombination between Type I and II LTRs. These
subtypes differ in evolutionary age and transcriptional activity with Type la LTRs being
younger in evolutionary terms and possessing stronger promoter function than the
other two subtypes. In this study, possible mechanics responsible for the functional
difference between LTRs have been explored. Type I and II LTRs each contain
different sets of repeated segments in their U3 regions which are disrupted in Type la
LTRs. Using reporter gene assays, both types of repeated segments were shown to
suppress activity of the human R-globin gene promoter when cloned at a distant site.
Both sets of repeats also repress promoter activity of a Type la LTR when directly
inserted within its U3 region. In further support of these findings, removal of the
strongly supressing Type II repeat set from a Type II LTR increased promoter activity in
one test cell line. However, this result was not observed in all cell lines or with both
LTR types, emphasizing the complexity and cell-type dependence of HERV-H
promoter regulation. In addition, using deletion constructs, two positive regulatory
segments have been localized within the Type la LTR, both of which contain a
potential binding site for the transcription factor Sp1. Gel mobility shift assays
demonstrated that fragments containing these sites do bind Sp1. Although Type I
LTRs are generally similar to Type la LTRs in the regions surrounding the Sp1 sites,
there are sequence differences within the sites. Gel shift analysis revealed no, or
much reduced, Sp1 binding of Type I LTR fragments containing these sites. Thus, it
appears that the loss of repeated suppresser elements and the acquisition of Sp1
binding sites have both contributed to the relatively strong transcriptional activity of the
Type la LTRs.
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Levy, David E. "Expression of Endogenous Retroviruses in Inbred Mice : Coordinate Regulation and Structure of Multiple Transcription Units". Thesis, 1985. https://thesis.library.caltech.edu/11366/1/Levy_DE_1985.pdf.
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The control of expression of the murine antigen Gix and of other products of endogenous retroviruses, in strain 129 mice and in its congeneic partner strain 129 Gix-, is an example of the coordinate expression of a dispersed family of independent transcription units. In order to provide a molecular description of the Gix phenotype, evidence is presented, from DNA and RNA hybridization analyses using heterologous viral probes, indicating that this phenotype is specified by a distinct regulatory gene, defined genetically, that acts in trans to control the levels of accumulation of specific mRNA species. The steady state levels of several, structurally distinct polyadenylated RNA species are reduced in Gix- mice, and a major reduction in transcription of these sequences accompanies this drop in abundance. Tissue specific patterns of accumulation of different sized RNA species were detected in numerous organs of the mouse, and the majority of these distinct transcripts were collectively regulated.
The isolation and characterization of cDNA copies of these endogenous retroviral transcripts demonstrated that they were derived from multiple, distinct transcription units. Differences among these RNA species were detected by S1 nuclease protection analyses , which confirmed the tissue specific patterns of RNA accumulation. The nucleotide sequences of endogenous virus cDNA clones fully documented the expression of distinct genes, the nature of the sequence heterogeneity, and the relationship of these normal cellular constituents to exogenous, infectious virus. The polymorphism was found to result from both single nucleotide changes and from deletions of different lengths of coding and non-coding information. Comparison of these sequences with exogenous virus demonstrated that the endogenous transcripts are closely related to the recombinant sequences of eukemogenic, mink cell focus forming viruses.
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Chang, Nien-Tzu y 張念慈. "Study on the Transcriptional Regulation of Human Endogenous Retroviruses and their Pathogenic Potentials". Thesis, 2008. http://ndltd.ncl.edu.tw/handle/55626230751393070845.
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博士國防醫學院
生命科學研究所
96
Endogenous retroviruses have a wide distribution within vertebrates; they are present in multiple copies dispersed throughout the genomes of host species. Human endogenous retroviruses (HERVs) constitute about 8% of the human genome. The prevalence and maintenance of HERVs elements suggest that they may play a role in the biology of the host species. HERVs are related to retroviruses with their characteristic LTR-gag-pol-env-LTR structure, in which LTRs possess the enhancer and the promoter functions for RNA transcription, viral integration and polyadenylation. Transcriptional activation of HERVs is supposed to be potentially pathogenic for producing retroviral proteins and RNA copies to form virus particles, proceeding to retro-transposition and insertion mutagenesis, altering the expression of neighboring or distant cellular genes from the insertion sites and eventually leading to neoplastic transformation or genetic disease. In most cases, HERVs are either structurally defective or inactive due possibly to stringent negative control mechanisms. Since RNA transcripts of HERV-I in various human cancer cells were hardly detectable by Northern blots in our preliminary studies, we isolated the LTR of RTVL-Ia and constructed site-specific mutations for analysis of the promoter and enhancer functions by using chloramphenicol acetyl transferase (CAT) reporter assay. Our results showed that 5’-LTR of HERV-I possess bi-directional promoter activity and unidirectional enhancer activity. The ATAAAAA element, a TATA-like box, in 5’-LTR mainly exerts a promoter function while the CCAAT element exhibits a partial enhancer function, and these two elements in the LTR apparently provide maximum transcriptional activity. Therefore, the poor transcriptional activity of HERV-I LTR may be due to the AGTAAA segment at the presumed polyadenylation site which plays a negative regulatory role in controlling gene expression. P53 is a tumor suppressor protein, and its mutations are the most frequently reported genetic alterations in human cancers. P53 has been shown to repress or activate the transcription activity of several cellular and viral promoters, although its effect on HERVs is not known. We have found that wild type (wt) p53 can efficiently repress the transcriptional activity of HERV-I LTR presumably through the interaction of wtp53 with the TATA-binding proteins or CAAT-binding proteins. In addition, mutantp53(V143A) can more or less stimulate the transcriptional activity of HERV-I LTR. These results imply that, following p53 mutation, LTRs are likely to be activated and might aberrantly regulate their neighboring cellular genes during the tumor progression processes. To study the possible involvement of p53 in chromosomal HERV expression, we further examined the RNA transcripts of HERV-E, HERV-H, HERV-I, HERV-K and HERV-W multi-gene families in p53-null Saos-2 cells transfected with wtp53 or “hot-spot” site-specific p53 mutants. Quantitative real-time PCR analysis showed that limited RNA expression of these HERVs in Saos-2 cells were not affected by wtp53 but could be elevated by mtp53(D281G), through transient or stable transfection. 5-Azacytidine and trichostatin A, known to activate endogenous retroviruses in other animals, did not induce HERV expression in the parental Saos-2 cells or wtp53 transfectants but could additively increase HERV genes expression especially in cells stably transfected with mtp53(D281G). Our results suggest that the stringent controlled chromosomal HERV genes may be compromised by p53 mutation, especially at codon 281, in combination with epigenetic chromatin alterations, leading to the activation of potentially pathogenic retrotransposable gene functions.
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Chang, Che-Hao y 張哲豪. "Functional Characterization of a p53-Interacting Protein via Retroviral and Adenoviral Overexpression and Post-Transcriptional Gene Silencing". Thesis, 2004. http://ndltd.ncl.edu.tw/handle/70447961829712478657.
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碩士國防醫學院
生物化學研究所
92
Abstract RNAi technology has been developed and recognized as one of the most powerful tools in the field of functional genomics. Many investigators use it to solve biological problems in a variety of aspects. We took advantage of the inhibitory activity of RNAi to develop gene therapy based approach on the potential treatment of SARS virus infection, and to understand the molecular mechanism of a newly identified p53-interacting protein, pip. In the study of inhibiting growth of SARS virus, we designed a set of hairpin RNAs specifically targeting to each of the SARS replicases which is essential for viral propagation, and cloned into adenoviral expression system to produce silencing RNAs under the contron of RNA polymerase III. Two adenoviral short hairpin RNA viruses, Ad-shSARS3350 and Ad-shSARS18833, were thus constructed, and our in vitro results clearly demonstrated that the expression of SARS viral antigen was completely diminished and the transcripts of SARS replicase were entirely knocked down to an undetectable level. The second part of my thesis was to decipher the cellular function of pip protein. Initially, we created a pip knock down and a pip-overexpressing C2C12 stable cell lines. In couple with the parental C2C12, three different pip expression lines, C2C12/shipip C2C12, C2C12/pip, were employed to perform my research. Besides, I also determined optimal dosages to stress C2C12 for mimicking cellular damaged condition and for p53 induction. From extensively cell cycle analysis on the cell line created in response to various damaging treatments, I found that pip was functioned to potentiate stress-induced apoptosis and to compromise G1 and G2 arrest. In my study, it also revealed that pip significantly reduced the expression level and kinetics of p53 after damaging treatments. Moreover, we provided profound evidence to demonstrate that the function of pip was affected by the activation status of p53, and consequently the cellular functions of p53, growth arrest or apoptosis, was significantly affected by the presence of pip.
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Lo, Yuen Man Mandy. "Gene Localization and Transcriptional Dynamics in the Optimization of Transgene Expression". Thesis, 2013. http://hdl.handle.net/1807/35885.
Texto completoResumen
Gene transfer techniques such as retroviral transduction have many applications such as cell marking, cell reprogramming, and therapeutics. Transgene expression, however, is often variable and maintaining long-term expression is problematic in progenitor cell types. To better control transgene expression, research has focused on the optimized use of cis-regulatory elements, such as promoters, enhancers and insulators. In addition to controlling gene expression, these regulatory elements modulate the nuclear organization of the transgene. The integration site also exerts significant effects on steady state and temporal transgene expression via the neighbouring chromatin environment. The first part of this thesis describes the co-operation of modified β-globin intronic elements in providing high-level expression and favorable nuclear localization. I demonstrate that these elements are compatible with efficient lentivirus transduction for globin gene therapy purposes. In the second chapter, I examine high-expressing EGFP retroviral transgenes and show that such steady state expression may exhibit rapid transcriptional fluctuations, which is modulated by different transcriptional dynamics at different integration sites. Finally, in the last chapter, I evaluate the use of a 3’D4Z4 insulator element in maintaining long-term EGFP transgene expression in ES cells, and discover integration-site specific temporal dynamics in retroviral vector expression. Overall, my results demonstrate that using multiple regulatory elements and insulating these elements from different types of genomic loci optimize transgene expression and dynamics in progenitor cells.
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Auxt, Miroslav. "Úloha de novo DNA methyltransferáz v transkripčním umlčování retrovirů a retrovirových vektorů odvozených od ptačího sarkomového a leukozového viru". Master's thesis, 2010. http://www.nusl.cz/ntk/nusl-295802.
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