Generation of Transgenic Mice to Evaluate Promoter Activity and Specificity of Two Human Endogenous Retrovirus Long Terminal Repeats = Untersuchungen zur Promotor-Aktivität und -Spezifität von zwei Long Terminal Repeats humaner endogener Retroviren in transgenen Mäusen

Generation of Transgenic Mice to Evaluate Promoter Activity and Specificity of two Human Endogenous Retrovirus Long Terminal Repeats Human Endogenous Retrovirus Long Terminal Repeats (HERV-LTRs) comprise 1.8% of the human genome (52.7 Mb). These sequences contain all the signal structures necessary for the regulation of gene transcription, such as promoters, enhancers and transcription factor binding sites. There is evidence that HERV-LTRs regulate gene expression in tissue-specific manner. This potential could be used to drive the expression of therapeutic genes, delivered by retroviral vector systems, in a safe and efficient manner. The HERV-H-H6 LTR and the HERV-L LTR were chosen for the generation of transgenic mice. Their promoter activity and specificity had prior been tested in a luciferase expression vector in vitro (Schoen et al., 2001). HERV-L was cloned into a luciferase expression vector and HERV-H-H6 was inserted into a enhanced green fluorescent protein (EGFP) expression vector. Transgenic mice were generated by DNAmicroinjection into pronuclei of zygotes. One pBL-HERV-L transgenic line and four pEGFP-HERV-H-H6 transgenic lines were established and analyzed. While the HERV-L promoter was not active in transgenic animals, pEGFP-HERV-H-H6 was expressed in gonads of mice of two transgenic lines. As only a single, non-expressing transgenic line was available, HERV-L promoter activity and specificity could not be evaluated. Additional transgenic lines have to be established. Expression level and pattern of the HERV-H-H6 promoter indicate specificity for gonad tissue. Whether the HERV-H-H6 promoter activity is linked to steroid production in cells remains to be clarified. Evaluating promoter activity in transgenic mice in two different expression vectors is not exclusively about the promoters, but also involves knowledge about the reporter genes. Advantages and limits of current applications of both luciferase and EGFP (with focus on the EGFP gene) are described in REVIEW OF THE LITERATURE. The conjunction of EGFP with the HERV-H-H6 promoter is to be seen critically, as all published methods for detection of EGFP in mice are described with EGFP linked to strong promoters. Problems like autofluorescence in fluorescence microscopy might be encountered when weaker promoters, such as HERV-LTRs, drive EGFP expression.Untersuchungen zur Promotor-Aktivität und –Spezifität von zwei Long Terminal Repeats humaner endogener Retroviren in transgenen Mäusen 1.8% des humanen Genoms bestehen aus Long Terminal Repeats Humaner Endogener Retroviren (HERV-LTRs). Solche Sequenzen enthalten alle Strukturen, die für die Regulierung von Transkription benötigt werden: Promotoren, Enhancer and Bindungsstellen für Transkriptionsfaktoren. Es gibt Hinweise, daß HERV-LTRs die Expression von Genen gewebespezifisch regulieren können. Eingebaut in retrovirale Genfähren, könnten HERV-LTRs therapeutische Gene sicher und effizient aktivieren. Zur Generierung transgener Mäuse wurden der HERV-H-H6 LTR und der HERV-L LTR ausgewählt. Deren Promoter Eigenschaften, wie Aktivität und Gewebespezifität, waren bereits in vitro untersucht worden (Schoen et al., 2001). Der HERV-L LTR wurde in einen Luciferase Expressionsvektor und der HERV-H-H6 LTR in einen Enhanced Green Fluorescent Protein (EGFP) Expressionsvektor kloniert. Transgene Mäuse enstanden durch DNA-Mikroinjektion in den Vorkern von Zygoten. Eine pBL-HERV-L transgene Linie und vier pEGFP-HERV-H-H6 transgene Linien wurden gezüchtet und auf Integration sowie Expression der Genkonstrukte untersucht. Während der HERV-L Promoter keine Aktivität zeigte, war Expression von pEGFP-HERV-H-H6 in Keimdrüsen von Mäusen aus zwei transgenen Linien nachweisbar. Da für das Genkonstrukt pBL-HERV-L nur eine einzige, nicht-exprimierende transgene Linie aufgebaut werden konnte, können keine Aussagen über die Aktivität und Gewebespezifität des HERV-L Promoters getroffen werden. Zu diesem Zwecke müssten weitere pBL-HERV-L transgene Linien untersucht werden. Das Expressionsmuster des pEGFP-HERV-H-H6 Genkonstruktes, weißt auf eine mögliche Gewebespezifität für Keimdrüsen hin. Eine eventuelle Verknüpfung der Aktivität des HERV-H-H6 LTRs mit der Produktion von Steroidhormonen müsste weitergehend geklärt werden.Da in dieser Arbeit zwei unterschiedliche Reportergen Systeme in der Maus angewandt wurden, sind im Literaturteil Vorteile und Einschränkungen von aktuellen Nachweisverfahren beider Reportergene, mit Schwerpunkt EGFP, in Mausgewebe zusammengefasst. Die Verbindung von EGFP mit dem HERV-H-H6 Promoter ist als kritisch zu beurteilen: Alle beschriebenen Nachweisverfahren für EGFP in der Maus gründen auf Mausmodellen, in denen das EGFP von einem starken Promoter kontrolliert wurde. Bei potenziell schwächeren Promotoren, wie HERV-LTRs, können Probleme auftreten, wie z.B. Autofluoreszenz bei der Fluoreszenzmikroskopie

Human endogenous retrovirus H protects the genome of human embryonic stem cells from mutagenic retroelements activity

Human pluripotent stem cells (hPSCs), which include human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs), infinitely self-renew, and can differentiate into any cell type on the human body [1–3]. hESCs are derived from early human embryos and became widely used to study the molecular pathways specific to human embryogenesis [1, 4–8]. Considering the ethical challenge in using embryo-derived cells and the possible immune rejection, hiPSCs are currently more common for regenerative therapies [3, 9–11]. hiPSCs are reprogrammed from a somatic cell line of a patient, genetically modified, and then differentiated to the desired lineage to transplant them back to the patient. hiPSCs are the future of personalized medicine, but not every hiPSC line can differentiate to every given cell type, as a result of cell heterogeneity. To reduce this heterogeneity, a naïve cell state might be a solution [3]. Whereas cultured hPSCs reside in a primed state, the cells of pre-implantation embryos resemble naïve pluripotency [12–16]. By adjusting culture conditions, it is possible to support hPSCs in a naïve state, similar in gene expression signature to early embryos [4, 5, 17–19]. The similarity is reflected as well in transcripts of some of the L1, Alu, and SVA retroelements (REs) [5]. These REs are phylogenetically young and still active human transposons, which might be detrimental for the integrity of the genome [20–27]. Our research group had previously derived the different types of naïve cells, resembling the later stages of pre-implantation development and highly expressing human endogenous retrovirus H (HERVH) [6]. HERVH is a phylogenetically older endogenous retrovirus, which was transposing following New- and Old-World monkey separation [28–30]. Now, HERVH can’t mobilize, but its transcripts were shown to support pluripotency in later stages of human embryogenesis, reprogramming, and in cultured primed hPSCs [6, 7, 31, 32]. Here I show that HERVH controls the transposition of young REs. In HERVH-depleted hESCs, L1 transposition increases, which is measured by two transposition assays. The active L1 elements drive the transposition of non-autonomous REs, resulting in the accumulation of de novo Alus and SVAs integrations, shown by whole-genome sequencing of cells undergoing stable HERVH knock-down. A subgroup of HERVH has the potential to control L1 transposition. These HERVHlin loci contain lin motif, two tandem LIN28A binding sites [33]. HERVHlin is supposedly evolutionary younger than the other HERVH. There are around 100 of HERVHlin sequences in the human, chimp, and gorilla genomes, while less exist in orangutans, and none in other primates. Based on the analysis of the previously published CLIP-seq data [33] and performed RIP-qPCRs, the lin motif allows LIN28A to bind HERVHlin more efficiently than other HERVH transcripts. LIN28A is known to inhibit the maturation of let-7 microRNA [34–37], which in turn controls the transposition of L1 [38]. HERVHlin sponging LIN28A to allow let-7-mediated inhibition of L1 might be the molecular mechanism of HERVH-controlled transposition of young REs. The supporting experiment shows that a let-7 independent L1-ORFeus reporter does not change the transposition activity in HERVH-depleted cells. HERVHlin embedded itself in a previously conservative pluripotency-specific LIN28A-let-7 pathway to protect the genome of hESCs from the mutagenic activity of REs. This is an example of a new evolutionary event where the selfish transposon HERVH evolved to compete with other transposable elements, which could be harmful to the host

Interaction between the ovine Bst-2 paralogs and sheep Betaretroviruses

There is a delicate evolutionary balance between viruses and their hosts. The host has evolved the intrinsic, innate and adaptive immunity to fight viral infections. However, viruses have acquired several counteracting measures to evade host defences. Ovine Betaretroviruses, including the exogenous and pathogenic Jaagsiekte sheep retrovirus (JSRV) and the highly related endogenous enJSRVs are a unique model system to investigate virus-host interaction over long evolutionary periods. Sheep have co-opted some defective enJSRV loci to (i) counteract infection by exogenous viruses and likely (ii) to cope with the continuous retroviral invasion of their genome. In addition, various genes of the innate and intrinsic immunity of the host have evolved to block viral replication. The work presented in this thesis focuses on the ovine bone marrow stromal cell antigen 2 (Bst-2)/ tetherin, a recently identified cellular restriction factor with a broad antiviral activity, and its interaction with sheep Betaretroviruses. In sheep, the BST-2 gene is duplicated into two paralogs termed oBST-2A and -2B. Studies presented in this thesis show that oBST-2B possesses several biological properties distinct from the paralog oBST-2A and from all the other BST-2 orthologs. oBST-2A prevents the release of JSRV/enJSRV viral particles by ‘tethering’ them at the cell membrane similarly to what observed by human BST-2. On the other hand, oBST-2B, does not reach the cell membrane but remains within the Golgi stacks and the trans-Golgi network. Several lines of evidence obtained in this thesis suggest that oBST-2B reduces significantly Env incorporation into viral particles. Therefore, oBST-2B possesses a unique antiviral activity that complements the classical tethering restriction provided by oBST-2A

Charakterisierung der solitären retroviralen Promotoren DQ-LTR3 und DQ-LTR13 im Kontext der Pathogenese des Typ 1 Diabetes mellitus

Der Typ 1 Diabetes mellitus (IDDM) ist eine multifaktorielle Erkrankung deren Risiko zu 50-60% von genetischen Faktoren vermittelt werden, wobei dem HLA-System auf Chromosom 6 (IDDM1) eine Schlüsselrolle zugesprochen wird. Innerhalb dieser Genregion befinden sich die beiden solitären retroviralen "long terminal repeats" (LTRs) DQ-LTR3 und DQ-LTR13. Sie können theoretisch mit Hilfe ihrer regulatorischen Elemente die Expression benachbarter funktioneller Gene beeinflussen. Im Rahmen der Fragestellung nach einer möglichen Funktion retroviraler LTRs im Kontext des Typ 1 Diabetes mellitus ist das unmittelbar benachbarte DQB1 Gen ein Kandidatengen von besonderer Bedeutung. Im Rahmen einer Familienassoziationsstudie wurde DQ-LTR13 als potentieller genetischer Risikomarker charakterisiert und es konnte gezeigt werden, daß die Haplotypen DQ8/LTR13+ und DQ2/LTR13- signifikant häufiger transmittiert werden als bei einer zufälligen Vererbung erwartet. Gleichzeitig unterscheidet sich ihr Segregationsmuster signifikant von dem der Haplotypen DQ8/LTR13- und DQ2/LTR13+. Das Kopplungsungleichgewicht zwischen DQ-LTR3 und DQ-LTR13 ist so ausgeprägt, daß sie zwar präferentiell aber nicht ausschließlich zusammen auf einem Haplotypen vorkommen. Die Wahrscheinlichkeit einer Erkrankung an IDDM in Zusammenhang mit einem der beiden Risikohaplotypen DQ8/LTR13+ bzw DQ2/LTR13- liegt bei 76% bzw. 68%. Aufgrund dieser Ergebnisse ist DQ-LTR13 insgesamt als unabhängiger genetischer Risikomarker für Typ 1 Diabetes mellitus zu bewerten, zusätzlich zu den bekannten Risikohaplotypen DQ8 und DQ2 und unabhängig von DQ-LTR3. Im Rahmen funktioneller Analysen unter Verwendung eines Reportergen-Assays konnte nach Transfektion diverser EBV-transformierter lymphoblastoider B-Zellen für keine der beiden LTRs die Fähigkeit nachgewiesen werden, als eigenständiger Promotor auf die Expression des Reportergens Luciferase zu wirken. Diese Fähigkeit konnte auch nicht durch die Gabe von Hydrocortison bzw. ß-Estradiol induziert werden. DQ-LTR13 ist jedoch in vitro in der Lage, abhängig von transkriptioneller Orientierung und Position, über den Promotor des DQB1 Gens verstärkend auf die Expression des Luciferase-Gens zu wirken. Dieser Effekt ist darüber hinaus, bezüglich der beiden Risikoallele DQB1*0302 und DQB1*0201, differentiell ausgeprägt. Eine Verifizierung des beobachteten Effekts in vivo war im Rahmen der vorliegenden Doktorarbeit leider nicht möglich

The role of Human Endogenous Retrovirus K10 (HERV-K10) in the pathogenesis of rheumatoid arthritis via molecular mimicry

Rheumatoid arthritis (RA) is a chronic joint disease of unknown aetiology. The autoimmune nature of RA is underlined by abundant generation of rheumatoid factor (RF) autoantibodies to IgG1 Fc, and anti-citrullinated protein antibodies (ACPA) to citrullinated autoantigens such as fibrinogen. Although RA pathogenesis has not been elucidated, genetic predisposition, environmental insults and viral pathogens are considered contributory factors. Human endogenous retrovirus K10 (HERV-K10) is one such virus as it retained the capacity to produce viral particles in RA synovium. This study set out to explore how HERV-K10 Gag matrix region could contribute to RA pathogenesis and perpetuation, with particular emphasis on its ability to mimic host autoantigens. We showed that Gag region exhibits high levels of sequence and structural homology to IgG1 Fc and it could provide a key epitope important for auto-reactivity in RA. Analysis of how HERV-K10 may evoke immune responses in RA was broadened by investigation of serological cross-reactivity of novel anti-K10 polyclonal antibody (PAbMAG) with IgG1 Fc. We showed that PAbMAG cross-reacted with linear and conformational epitopes on IgG1 Fc. In a further development, we showed a significantly elevated mean IgG response to HERV-K10 epitopes in serum samples from RA patients when compared to other arthritides. These data suggest that molecular mimicry between viral and host proteins has the potential to lead to antigen-driven high-affinity RF IgG immunological reactivity in RA. Finally, we broadened our study of mimicry in RA by the investigation of citrullinated autoantigens. Structural studies demonstrated high levels of homology between citrullinated fibrinogen, IgG1 Fc and HERV. We further explored how protein citrullination affects the cross-reactivity of autoantibody responses in RA. These experiments revealed that generation of neoepitopes through citrullination of HERV-K10 and autoantigens IgG1 Fc and fibrinogen enhanced the reactivity of RA sera to these targets. Moreover, we showed that RF autoantibodies could mediate responses to a classical ACPA target fibrinogen, only when it is citrullinated, in the absence of ACPAs. These data provide a new insight into the initiation and propagation of immunological responses in RA and how viral/host molecular mimics and citrullination could modulate serum cross-reactivity profiles in RA.South Staffordshire Medical Foundation, Rotha Abraham Bequest, The Royal Wolverhampton Hospital Charity, New Cross Kidney Patients Association, James Beattie Charitable Trust.A thesis submitted in partial fulfilment of the requirements of the University of Wolverhampton for the degree of Doctor of Philosoph

Protéines à motif tripartite (TRIM) chez le porc (Sus scrofa) et réplication du rétrovirus endogène porcin

Les études des interactions entre cellules hôtes et rétrovirus ont conduit à définir le concept de restriction virale dont les facteurs constituent une part de l'immunité innée des cellules hôtes. Ces facteurs contribuent au contrôle des rétrovirus endogènes (ERV) dont l'émergence peut être associée à certaines pathologies telles que des leucémies ou des immunodéficiences. Chez le porc, certains ERV (PERV) sont réplicatifs, pourtant aucune pathologie ne leur a, à ce jour, été associée. Les mécanismes de restriction virale impliqués dans ce phénomène ont fait l'objet de nombreuses études. Elles n'ont cependant concerné que certains facteurs. Les protéines porcines à motif tripartite (poTRIM) n'ont ainsi fait l'objet que de peu d'études. Pourtant, de nombreux membres de cette famille participent à la restriction virale chez d'autres organismes que le porc. La présente étude s'intéresse par conséquent aux orthologues porcins de ces protéines et à leur relation avec les PERV. L'élaboration d'une stratégie d'expression de ces protéines dans un modèle humain, sensible à l'infection par le PERV nous a permis d'évaluer et de caractériser les effets des TRIM sur le cycle infectieux du PERV. Cette stratégie a mis en évidence une activité de restriction par TRIM8 tandis que TRIM44 semble au contraire agir en faveur de la réplication virale. En ce qui concerne poTRIM11, elle favorise l'entrée du PERV tout en inhibant son expression. L'étude a également confirmé l'insensibilité du PERV vis-à-vis de poTRIM5a. L'ensemble de ces résultats contribuent à la compréhension de la relation entre la réplication des PERV et le contrôle mené par son hôte.From studies of pathogens and their host interaction has emerged the concept of viral restriction considered to be part of an innate immune system. These factors contribute to the control of endogenous retroviruses (ERV) whose emergence may be associated with several diseases such as leukemia or immunodeficiency. Three subgroups of the porcine ERV-g-1 group (PERV) are replicative. Nevertheless, these PERVs are not associated with any pathology in the pig. Several studies have been performed on viral restriction mechanism capabilities of the pig but these covered a very limited number of restriction factors. Regarding the porcine tripartite motif-containing (poTRIM) proteins, knowledge is weak although several members of this family have proved to be implicated in the viral restriction of other species. The purpose of this study is to investigate the relationship between these orthologous poTRIMs proteins and replicating PERVs. In order to explore this potential interaction, a TRIM protein expressing model in human cells, known to be sensitive to the PERV infection, has been developed. It has enabled us to assess and characterize potential TRIMs effects on the PERV infection cycle. We equally identified poTRIM8 as a restriction factor. Conversely, poTRIM44 seems to act as an enhancer of the PERV infection, while, TRIM11 displayed ambiguous effects including an enhancer effect of the early infectious stages and an inhibitor activity of the late infectious stages. In this study, we also confirmed the PERV insensitivity to the porcine TRIM5a protein. Finally, this work aims at contributing to the understanding of the relationship between PERV replication and their control leading by the host cells.RENNES1-Bibl. électronique (352382106) / SudocSudocFranceF