IFITM1, 2 et 3 sont des protéines transmembranaires qui sont régulées à la hausse après stimulation interféron. Ces protéines sont capables d’inhiber un large spectre de virus. Le mécanisme d’action admis indique que la présence des IFITMs dans la membrane lipidique des cellules cibles diminue l’entrée des virus en bloquant la fusion de la membrane virale avec la membrane cellulaire.J’ai pris part en début de thèse à un travail qui a permis à notre équipe de mettre en évidence une deuxième configuration antivirale des protéines IFITMs contre le VIH-1 (Virus de l’Immunodéficience Humaine). En effet la présence des IFITMs dans les cellules productrices de virus et non seulement dans les cellules cibles permet deux choses: l’incorporation des IFITMs dans les particules virales et la baisse d’infectivité des virus produits. Suite à cette première étude, nous nous sommes posés deux problématiques: 1) comprendre le mécanisme d’inhibition du VIH-1 par les IFITMs et 2) déterminer le niveau de conservation de cette nouvelle configuration. Mon travail de thèse s’est concentré sur la première et l’utilisation d’un panel de mutants d’IFITM3 a permis: de dissocier l’activité anti VIH-1 et l’incorporation virale et d’identifier des domaines protéiques régulant l’habilité d’IFITM3 à interférer avec la production de particules virales infectieuses. J’ai également participé à travail collaboratif mis en place par notre équipe qui nous a permis de montrer que le mécanisme d’inhibition que nous avons mis en évidence pour le VIH-1 était un mécanisme conservé qui permettait de réduire l’infectivité de nombreux autres virus. / IFITM1, -2 and -3 are transmembrane proteins, upregulated after type I interferon response and have been shown to inhibit a broad spectrum of viruses. The commonly admitted restriction in the field denotes that the presence of IFITM proteins in the lipidic membranes of target cells decreases viral entry by impeding the viral to cell membrane fusion, essential for the liberation of the core viral into the cytoplasm.I took part at the beginning of my thesis to a teamwork that allowed us to discover a new antiviral mechanisms for these proteins, at least for HIV-1. According to this mechanism, the presence of IFITMs in virus producing cells results in the production of viral particles that incorporate IFITMs and display decreased infectivity.Since then, my PhD work has consisted in: 1) understanding the molecular mechanism by which IFITMs inhibit HIV virion particles and 2) determine the conservation of this novel mechanism of inhibition against other viruses.First, I focused on IFITM3 and tested a large panel of mutants to identify the protein domain(s) required for either incorporation into virions and/or for the antiviral activity. This work allowed me to identify unknown domains in IFITM3 important for the antiviral effect of IFITM3 in virus-producing cells. Second, I have participated to a large collaboration initiated by our team to analyze the antiviral effects that IFITMs exerted on several viruses. Our results indicate that the novel mechanism of inhibition by IFITMs that we have described for HIV is conserved among different classes of viruses.

In the past few years, interferon-induced transmembrane (IFITM) proteins have been identified as important antiviral factors. The current understanding of IFITMs suggests that they localise within distinct cellular compartments from where they exert their broad antiviral role. For example, IFITM1 localises to the plasma membrane and restricts viruses that do not require endocytosis to infect host cells. In contrast, IFITM2 and IFITM3 are found in the early and late endosomes, respectively, and are potent inhibitors of viruses that depend on endosomal pathways for infection. I begin this dissertation by providing some background on the biology and function of IFITM proteins, including details of in vitro assays that have helped elucidate IFITMs role as antiviral factors. I also describe some early candidate-gene association studies that have attempted to correlate genetic variation within these genes with variation in viral restriction. I also describe how genetic association studies have been used more broadly to understand the biology underlying both infectious and non-communicable diseases. Evidence from in vitro, and in vivo work has demonstrated the IFITMs role as potent antiviral factors, however, no genome-wide association study has reported any significant associations to genetic variant in or around these genes. In Chapter 2, I explore reasons why this may be the case and calculate the coverage of IFITM genes by commercially available genotyping arrays. I show that IFITM2 and IFITM3 are amongst the 7% of all protein coding genes with less than 25% common variant (minor allele frequency > 5%) coverage across all arrays. Poor coverage of genetic variation is therefore one explanation for the lack of IFITM associations in GWAS. The lack of coverage in the genotyping arrays led me to explore other tools to capture variation in the IFITM region. I employ a targeted sequencing method using two different sequencing technologies: short-read sequencing (Illumina MiSeq) and single molecule, real-time sequencing (PacBio RS). Conventional pulldown protocols for targeted sequencing have not been designed for single molecule, real-time sequencing at the time, thus in Chapter 3, I provide some details of the optimisation work required to adapt the targeted method for PacBio sequencing. I then assess the performance of the method for both Illumina and PacBio sequencing. In Chapter 4, I apply the targeted sequencing method described in Chapter 3 to test genetic variants in and around IFITM1, IFITM2 and IFITM3 for association with rapid disease progression in HIV. I also explore the contribution of rare genetic variants (MAF < 1%) to this phenotype by testing for a differential enrichment between cases and controls across each of the three genes. Studies in vitro have also reported that IFITM proteins are potent restrictors of dengue virus infection. In Chapter 5, I use genotype data across a cohort of 2,008 Vietnamese children diagnosed with dengue haemorrhagic fever (DHF) and 2,018 cord blood controls to test if common variants are associated with the disease.

No description available.

Le VIH, comme tous les virus, nécessite la coopération de nombreuses protéines cellulaires pour réaliser son cycle viral. Néanmoins, un panel de protéines est spécifiquement dédié à la protection des cellules et ce, de façon autonome. Ces protéines sont nommées facteurs de restriction. Jusqu’à présent, un grand nombre de protéines ayant une action antivirale plus ou moins importante contre le VIH a été identifié. Seules quelques-Unes sont bien étudiées telles que APOBEC3G, Tétherine, TRIM5α ou SamHD1. Toutefois, ces facteurs de restriction ne permettent pas à eux seuls d’expliquer la permissivité de tous les types cellulaires. Notre étude a donc pour objet la découverte de nouvelles protéines agissant contre le VIH. Parmi un grand nombre de candidats, nous avons identifié une famille de protéines agissant contre le VIH : les IFITMs. Il a été précédemment décrit que l’expression des IFITMs protège les cellules de l’infection par le VIH mais aussi par de nombreux autres virus, filovirus, coronavirus, flavivirus ou encore paramyxovirus. Nous décrivons ici un nouveau mécanisme antiviral mis en place par les IFITMs. L’expression des IFITMs dans les cellules productrices de virus affecte la production de particules infectieuses. D’une part, moins de particules sont produites et d’autre part les particules sont moins infectieuses. Nous nous sommes focalisés sur le défaut d’infection et celui-Ci corrèle avec l’incorporation des IFITMs à la membrane des virions. Les virus produits et portant les IFITMs présentent un défaut lors du processus d’entrée dans la cellule cible au cours de l’étape de fusion. Ceci n’est pas dû à un défaut d’incorporation de l’enveloppe virale, mais le mécanisme précis n’est pas encore compris. Il apparaît donc que les IFITMs peuvent jouer un double rôle restrictif, protégeant les cellules où ils sont exprimés et diminuant la production de particules infectieuses. / HIV, as all viruses, hijacks the cellular machinery to its benefits. Nevertheless, a range of proteins protects cells by an autonomous way, they are called restriction factor and belong to a system called “the intrinsic immunity”. Up to now, a certain number of cellular proteins, most of which belong to this defense system, have been described to be more or less detrimental for HIV replication, as for example, APOBEC3G, Tetherin, TRIM5α and SamHD1. Given that a large number of cellular restriction factors are likely to be discovered, we started to screen to look for new ones. Among a lot of candidates, we have identified a family of proteins interfering with HIV: the IFITMs. Previously, studies described IFITMs as proteins protecting cell against viral entry and fighting a lot of viruses, including HIV. In this thesis, we described a new antiviral mechanism mediated by IFITMs against retroviruses. When IFITMs are expressed in cells producing new virions, there is a high defect of infectious particle release due to a defect of particle release and a defect of the infectivity of particles. We focus on the intrinsic defect of infectivity and we show that particles cannot anymore enter into target cells and this observation is correlated with the incorporation of IFITMs in viral membranes. The defect is located at the fusion step and is not explained by a defect of envelope incorporation.To conclude, IFITMs have a double restrictive effect, protecting cells from incoming viruses and decreasing the production of infectious particles.

Membrane proteins are found in all kingdoms of life and are essential for cellular interactions with the environment. Although a large research effort have been put into this group many membrane proteins remains uncharacterized, both in terms of function and evolutionary history. We have estimated the component of α-helical membrane proteins within the human proteome; the membrane proteome. We found that the human membrane proteome make up 27% of all protein, which we could classify the majority of into 234 families and further into three major functional groups: receptors, transporters or enzymes. We extended this analysis by determining the membrane proteome of 24 organisms that covers all major groups of eukaryotes. This comprehensive membrane protein catalog of over 100,000 proteins was utilized to determine the evolutionary history of all membrane protein families throughout eukaryotes.  We also investigated the evolutionary history across eukaryotes of the antiviral Interferon induced transmembrane proteins (IFITM) and the G protein-coupled receptor (GPCR) superfamily in detail.  We identified ten novel human homologs to the IFITM proteins, which together with the known IFITMs forms a family that we call the Dispanins. Using phylogenetic analysis we show that the Dispanins first emerged in eukaryotes in a common ancestor of choanoflagellates and animals, and that the family later expanded in vertebrates into four subfamilies. The GPCR superfamily was mined across eukaryotic species and we present evidence for a common origin for four of the five main human GPCR families; Rhodopsin, Frizzled, Adhesion and Secretin in the cAMP receptor family that was found in non-metazoans and invertebrates, but has been lost in vertebrates. Here we present the first accurate estimation of the human proteome together with comprehensive functional and evolutionary classification and extend it to organisms that represents all major eukaryotic groups. Moreover, we identify a novel protein family, the Dispanins, which has an evolutionary history that has been formed by horizontal gene transfer from bacteria followed by expansions in the animal lineage. We also study the evolution of the GPCR superfamily throughout eukaryotic evolution and provide a comprehensive model of the evolution and relationship of these receptors.

No description available.

Ifitm基因是一群可被干擾素刺激表現的基因，目前已知這些基因參與細胞對病毒的防禦，然而其生化特性及這些基因參與的其他生物功能並不清楚。將這些基因敲除後小鼠會產生過度攝食的行為並會造成肥胖，同時在下視丘中黑皮質素的前驅物POMC的表現量顯著降低，此發現表明破壞Ifitm基因可導致下視丘功能被破壞。然而由於代謝的調控並非只由下視丘控制，Ifitm基因是否直接參與下視丘的功能並不清楚。本研究卽是在探討Ifitm基因是否在下視丘的功能中扮演重要的角色。我們首先透過與下視丘功能有關的行為分析測試是否有其他下視丘功能被破壞的證據。我們發現在IFITM基因剔除公鼠，3-6個月開始出現過度攝食的行為與體重明顯增加；然而IFITM基因剔除母鼠在18個月後才出現同樣的表現型。IFITM基因剔除公鼠，在OFT與FST的行為測試中行動力下降，IFITM基因剔除母鼠卻有較多的活動；在公鼠與母鼠中焦慮均顯著增加。我們進一步測試這些剔除鼠的下視丘組織及神經胜肽的表現，在組織分析中並沒有發現組織結構異常。在神經胜肽的分析中IFITM基因剔除鼠的CRF的表現量增加，與IFITM基因剔除鼠的焦慮行為顯示相同趨勢。POMC表現的減少與CRF表現的上升顯示下視丘的功能紊亂。我們同時發現IFITM基因剔除鼠中微膠細胞數增加，以及位於下視丘的細胞本體的體積增加。另外IFITM基因剔除鼠中的細胞激素TNF-α與IL-1β在周邊血清中的增加表明此剔除鼠的下視丘產生發炎。本研究發現剔除IFITM基因會造成多項下視丘功能受到影響，並發現此突變鼠有下視丘發炎的現象。此下視丘發炎是否為下視丘功能破壞的主因，及IFITM基因剔除鼠的下視丘功能異常的細胞分子機制將會進一步被分析。 / Ifitm genes are a group of interferon-inducible genes, best known for their antiviral roles. Their biological functions other than cellular antivirus and the biochemical properties of these proteins are not well-understand. Previously age-dependent hyperphagia and obesity were reported in IfitmDel mutant mice, in which 5 Ifitm genes were deleted. The levels of pro-opiomelanocortin (POMC) in hypothalamus of these mice were significantly reduced, suggesting that the hyperphagia phenotype of IfitmDel mutants is caused by defective central melanocortin signaling. In this study, we examine whether the hypothalamic dysfunctions are developed in these mutants. Whether hypothalamus associated behaviors are disrupted in IfitmDel mutants was first examined. We discovered that the hyperphagia and obesity phenotype in IfitmDel mutants are gender-dependent. These phenotypes in male mutants are detectable starting from 4-6 months of age, but not detected until one-year old in female mutants. Male mutants reveal the trends of decreased activities, while female mutants exhibit increased locomotor activity. Both gender develop anxiety-like phenotype. The behavioral abnormalities support the hypothesis of hypothalamic dysfunctions. As the Ifitm genes are expressed in various stage during development, the anatomy and the functions of the hypothalamus of these mutants were further tested. The histological analyses show no anatomical defects, neuron count differences and morphological alterations of neurons in IfitmDel mutant brains of both genders. These results indicate that these proteins are rather crucial for maintaining normal physiology in the developed central nervous system (CNS). The expression levels of neuropeptides related to stress response and anxiety or depression status, such as corticotrophin releasing factor (CRF), are altered in mutant hypothalamus. In contrast, Cell count and cell body size of microglia, whereas sera levels of pro-inflammatory cytokines, including TNF-α and IL-1β are elevated in IfitmDel mice, indicating hypothalamic inflammation and microglial activation phenotypes. The cellular mechanisms underlying the hypothalamic dysfunctions and the causative relationship of the hypothalamic inflammation with these phenotypes shall be further explored in the future studies.

No description available.

The egress pathway of herpes simplex virus-1 (HSV-1) is a complicated process mediated by co-ordinated activity of several virus glycoproteins. The virions are first assembled and enveloped at trans-Golgi-network (TGN) or endosome membranes and then travel through a guided pathway that is directed towards the cell adherent points for secretion. Once secreted the vast majority of virions remain associated with the extracellular membrane of cells and very few free virions are released into the culture medium (< 1%). The mechanisms that mediate both the targeted secretion of newly assembled virions at cell contact points and post-secretion attachment of virions with the extracellular surface of cells are poorly understood, and were the topics of this research. In this thesis, an HSV-1 passage mutant of increased virion secretion phenotype had been studied. Genome sequencing of the mutant virus identified mutations in three viral envelope proteins. Study of recombinant viruses that were constructed based on those three mutations revealed that a single amino acid change in glycoprotein I (gI) of glycine to arginine at residue 39 is responsible for the increased release of virus. The result suggests the principal effect of this mutation is to modify the secretory pathway used by virions during their release from infected cells. Data also suggests a role of gC in the attachment of virions to the extracellular surface of cells after egress. In the context of HSV-1 envelopment and egress glycoprotein E (gE), which forms a heterodimeric complex with gI (gE/gI), is known to be important. The gE/gI complex has been shown to interact with many tegument proteins and have a redundant role in secondary envelopment. The gE/gI complex has been also proposed to colocalise with various cellular components and sort the nascent virions to cell contact points. However, there is little understanding of the cellular proteins that gE/gI interact with, or the mechanisms that mediate targeted secretion of virions. This research has identified a novel interactome of gE/gI by mass-spectrometric analysis utilising stable isotope labelling with amino acids in cell culture (SILAC) medium. Among the cellular interactome obtained, Nipsnap1 was validated by co-precipitation assays from both infected and transfected cells, and furthermore using cell free systems, suggesting gE and Nipsnap1 directly interact. Nipsnap1 and its homologue Nipsnap2 have been proposed to contribute in vesicle transport and membrane fusion in cells. Using CRISPR-Cas9 technology these proteins were knocked out in a keratinocyte cell line (HaCaT) to investigate their role in HSV-1 egress. However, little or no effect on HSV-1 egress could be observed upon loss of either or both of these proteins suggesting the biological significance of gE-Nipsnap1 interaction may not be directly linked to any egress function of gE/gI. Two further interesting 'hits' from the gE/gI interactome were interferon-induced transmembrane protein type-2 (IFITM2), a virus restriction factor, and Myoferlin that has a putative role in endocytic vesicle recycling. This study could validate gE-Myoferlin interaction and co-localisation in infected or transfected cells however, functional significance of this interaction remains to be determined. Overall, the research of this thesis has provided a better understanding of the role of the gE/gI complex in HSV-1 egress and investigated the role of some interesting cellular proteins in the context of virion egress.