Mammals are constantly challenged by numerous pathogens that pose a threat to their health. Upon infection, retroviruses quickly integrate their genome into that of their host thereby permanently modifying it. Protein members of the APOBEC3 (A3) family exhibit cytidine deaminase activity that specifically acts on single-stranded DNA to deaminate deoxycytidine bases into deoxyuridines. This process is potentially mutagenic because uracil directs the insertion of adenine on the opposite DNA strand. High levels of mutations induced by A3 proteins in the retroviral genome ultimately inactivate progeny viruses. However, under conditions where low levels of A3 proteins are present, sub-lethal mutagenesis can occur and is generally believed to be beneficial for the virus. Powerful and affordable techniques designed to detect rare deamination events generated by these deaminases along the full length of retroviral genomes are therefore essential. Through the course of my studies, I developed such a new tool that I called HyperHRM which was instrumental to my project’s success. In addition to the antiretroviral affects of their catalytic activity, some members of the A3 family have the ability to hinder reverse transcription independently of their enzymatic properties. Yet, the details underlying the deamination-independent restriction by the proteins remain unclear. Through my work, I have advanced our current understanding of this elusive process by defining the essential role for RNA-binding in the inhibition of the early steps of infection by APOBEC3G (A3G). I also demonstrate that the ability to bind RNA is important for the selection of DNA dinucleotides targeted for deamination by A3 enzymes. Based on the premise that the DNA context for deamination may alter viral fitness in various ways, I then investigated the gene inactivation potency of different A3 based on their preferred DNA substrate. My experiments showed that mutations introduced in a 5'CC context by A3G are much more lethal for the virus because of the high frequency of termination codons that are generated. I therefore clearly established that deamination target specificity has a strong influence on the overall restriction potency of A3 proteins and demonstrated that such specificity was linked to the ability of A3 proteins to bind RNA. Finally, in addition to retroviruses, mobile elements such as retrotransposons can also lead to genomic instability if not properly controlled. The A3 protein family has been shown to play a crucial role in the restriction of these elements through a mechanism that is not believed to require the enzymatic activity of the proteins, although the details of the restriction mechanism are not yet understood. Here, I provide molecular insights on the potential mechanism of retrotransposon restriction by showing that the RNA-binding properties of the enzymes are not involved in the restriction of L1 retrotransposition. A complete elucidation of the modes of restriction employed by the A3 could lead to the development of a new generation of antiretroviral drugs. Overall, my research has led to the design of a new research tool to detect and quantify A3-induced mutations in retroviruses, but more importantly, it has enabled a better understanding of how the RNA-binding abilities of A3 proteins play an essential role in the overall restriction potency of retroviruses and retrotransposons.

Human APOBEC3 proteins are host-encoded intrinsic restriction factors that can prevent the replication of a broad range of human and animal retroviruses such as HIV, SIV, FIV, MLVs and XMRV. The main pathway of the restriction is believed to occur as a result of the cytidine deaminase activity of these proteins that converts cytidines into uridines in single-stranded DNA retroviral replication intermediates. Uridines in these DNA intermediates disrupt the viral replication cycle and also alter retrovirus infectivity because of the C-to-T transition mutations generated as a result of the deaminase activity on the minus strand DNA. In addition, human APOBEC3 proteins also exhibit a deamination-independent pathway to restrict retroviruses that is not currently well understood. Although the restriction of retroviruses by human APOBEC3 proteins has been intensely studied in vitro, our understanding of how the murine APOBEC3 (mA3) protein restricts retroviruses and/or prevents zoonotic infections in vivo is very limited. In contrast to humans and primates that have 7 APOBEC3 genes, mice have but a single copy. My study of the function and structure of mA3 revealed that it has an inverted functional organization for cytidine deamination in comparison to the human A3G catalytic sites. I have also found that disruption of the integrity of either of these catalytic sites substantially impedes restriction of HIV and MLV. Interestingly, our data shows that mA3 induces a significant decrease in retroviral activity of HIV and MLVs by exploiting both deamination-dependent and -independent pathways. However, the deaminase activity of mA3 is essential to confer long-term restriction of retroviral infection. My observations suggest that mA3 has dual activities, both deamination-dependent and -independent, that work cooperatively to restrict a broad range of human and animal retroviral pathogens. In the context of the intrinsic immune system, APOBEC3 proteins provide a powerful block to the transmission of retroviral pathogens that very few have found ways to evade.

Human APOBEC3 proteins are host-encoded intrinsic restriction factors that can prevent the replication of a broad range of human and animal retroviruses such as HIV, SIV, FIV, MLVs and XMRV. The main pathway of the restriction is believed to occur as a result of the cytidine deaminase activity of these proteins that converts cytidines into uridines in single-stranded DNA retroviral replication intermediates. Uridines in these DNA intermediates disrupt the viral replication cycle and also alter retrovirus infectivity because of the C-to-T transition mutations generated as a result of the deaminase activity on the minus strand DNA. In addition, human APOBEC3 proteins also exhibit a deamination-independent pathway to restrict retroviruses that is not currently well understood. Although the restriction of retroviruses by human APOBEC3 proteins has been intensely studied in vitro, our understanding of how the murine APOBEC3 (mA3) protein restricts retroviruses and/or prevents zoonotic infections in vivo is very limited. In contrast to humans and primates that have 7 APOBEC3 genes, mice have but a single copy. My study of the function and structure of mA3 revealed that it has an inverted functional organization for cytidine deamination in comparison to the human A3G catalytic sites. I have also found that disruption of the integrity of either of these catalytic sites substantially impedes restriction of HIV and MLV. Interestingly, our data shows that mA3 induces a significant decrease in retroviral activity of HIV and MLVs by exploiting both deamination-dependent and -independent pathways. However, the deaminase activity of mA3 is essential to confer long-term restriction of retroviral infection. My observations suggest that mA3 has dual activities, both deamination-dependent and -independent, that work cooperatively to restrict a broad range of human and animal retroviral pathogens. In the context of the intrinsic immune system, APOBEC3 proteins provide a powerful block to the transmission of retroviral pathogens that very few have found ways to evade.

In response to viral infection, host cells activate various antiviral defence mechanisms to inhibit virus replication. Therefore in order for a virus to replicate efficiently, it must counteract cellular antiviral defence. Promyelocytic leukemia protein (PML) is a cellular protein involved in intrinsic immunity. It inherently forms nuclear bodies (PML-NBs) that assemble at the site of viral genomes. Proteins related to epigenetic regulation are recruited to PML-NBs, and silence viral gene transcription. This study focuses on the role of two herpes simplex virus type 2 (HSV-2) proteins, ICP0 and Us3, in disrupting PML-NBs and counteracting cellular antiviral defence. En passant mutagenesis was used to create recombinant HSV-2 viruses lacking ICP0, Us3, or both ICP0 and Us3. Growth analysis of these recombinants indicates no growth defects for the ICP0Δ virus, while the Us3Δ virus grows to one log lower titres than wild type virus (WT). By contrast, the ICP0Δ virus displays a delay in PML-NB disruption, but the Us3Δ virus is as efficient as WT. However, Us3 is still important for PML-NB disruption, since the ICP0Δ/Us3Δ double mutant exhibits a greater delay than the ICP0Δ single mutant. Although PML is a mediator of the interferon (IFN) response and it was predicted that ICP0 and Us3 interfere with the IFN response through disruption of PML-NBs, my results show that only some HSV-2 Us3Δ clones are hypersensitive to the effects IFN, and others are resistant. Us3 affects more than one cellular pathway, and those cellular pathways are affected by more than one viral protein. I conclude that the activities of multiple viral proteins create a fine balance between activating cellular pathways to promote virus replication, and inhibiting cellular antiviral defence. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2014-01-23 10:55:16.715

Human APOBEC3 proteins are host-encoded intrinsic restriction factors that can prevent the replication of a broad range of human and animal retroviruses such as HIV, SIV, FIV, MLVs and XMRV. The main pathway of the restriction is believed to occur as a result of the cytidine deaminase activity of these proteins that converts cytidines into uridines in single-stranded DNA retroviral replication intermediates. Uridines in these DNA intermediates disrupt the viral replication cycle and also alter retrovirus infectivity because of the C-to-T transition mutations generated as a result of the deaminase activity on the minus strand DNA. In addition, human APOBEC3 proteins also exhibit a deamination-independent pathway to restrict retroviruses that is not currently well understood. Although the restriction of retroviruses by human APOBEC3 proteins has been intensely studied in vitro, our understanding of how the murine APOBEC3 (mA3) protein restricts retroviruses and/or prevents zoonotic infections in vivo is very limited. In contrast to humans and primates that have 7 APOBEC3 genes, mice have but a single copy. My study of the function and structure of mA3 revealed that it has an inverted functional organization for cytidine deamination in comparison to the human A3G catalytic sites. I have also found that disruption of the integrity of either of these catalytic sites substantially impedes restriction of HIV and MLV. Interestingly, our data shows that mA3 induces a significant decrease in retroviral activity of HIV and MLVs by exploiting both deamination-dependent and -independent pathways. However, the deaminase activity of mA3 is essential to confer long-term restriction of retroviral infection. My observations suggest that mA3 has dual activities, both deamination-dependent and -independent, that work cooperatively to restrict a broad range of human and animal retroviral pathogens. In the context of the intrinsic immune system, APOBEC3 proteins provide a powerful block to the transmission of retroviral pathogens that very few have found ways to evade.

Human APOBEC3 proteins are host-encoded intrinsic restriction factors that can prevent the replication of a broad range of human and animal retroviruses such as HIV, SIV, FIV, MLVs and XMRV. The main pathway of the restriction is believed to occur as a result of the cytidine deaminase activity of these proteins that converts cytidines into uridines in single-stranded DNA retroviral replication intermediates. Uridines in these DNA intermediates disrupt the viral replication cycle and also alter retrovirus infectivity because of the C-to-T transition mutations generated as a result of the deaminase activity on the minus strand DNA. In addition, human APOBEC3 proteins also exhibit a deamination-independent pathway to restrict retroviruses that is not currently well understood. Although the restriction of retroviruses by human APOBEC3 proteins has been intensely studied in vitro, our understanding of how the murine APOBEC3 (mA3) protein restricts retroviruses and/or prevents zoonotic infections in vivo is very limited. In contrast to humans and primates that have 7 APOBEC3 genes, mice have but a single copy. My study of the function and structure of mA3 revealed that it has an inverted functional organization for cytidine deamination in comparison to the human A3G catalytic sites. I have also found that disruption of the integrity of either of these catalytic sites substantially impedes restriction of HIV and MLV. Interestingly, our data shows that mA3 induces a significant decrease in retroviral activity of HIV and MLVs by exploiting both deamination-dependent and -independent pathways. However, the deaminase activity of mA3 is essential to confer long-term restriction of retroviral infection. My observations suggest that mA3 has dual activities, both deamination-dependent and -independent, that work cooperatively to restrict a broad range of human and animal retroviral pathogens. In the context of the intrinsic immune system, APOBEC3 proteins provide a powerful block to the transmission of retroviral pathogens that very few have found ways to evade.

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 poTRIM5α. 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-γ-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 TRIM5α protein. Finally, this work aims at contributing to the understanding of the relationship between PERV replication and their control leading by the host cells.

La drosophile est un excellent modèle pour l’étude des mécanismes moléculaires de l’immunité innée, y compris les virus. Elle a permis la caractérisation de mécanismes de défense immunitaire conservés au cours de l’évolution, tel que les voies Toll et IMD qui régulent l’expression des peptides antimicrobiens induits en réponse aux infections fongiques et bactériennes. Deux types de réponse sont impliqués dans le contrôle des infections virales chez la drosophile : une réponse inductible et l’ARN interférence. Nous avons montré que l’ARN interférence est un mécanisme global de défense antivirale puisqu’il contrôle l’infection par un virus à ADN, en plus des virus à ARN tel que le virus C de la drosophile (DCV). Le virus DCV, apparenté aux Picornaviridae, est un pathogène naturel de la drosophile. Nous avons également observé que la résistance de mouches contrôles à l’infection par le virus DCV est dépendante du fond génétique. Elle est d’ailleurs corrélée à des polymorphismes présents dans un gène porté par le chromosome III : le gène pastrel. Nos expériences de perte et gain de fonction indiquent que ce gène code pour un facteur de restriction viral, bloquant l’infection par le virus DCV mais aussi par le virus de la paralysie du cricket (CrPV). Cette restriction apparait dans les premières heures après infection. La région C-terminale de la protéine Pastrel est nécessaire à son activité antivirale ainsi qu’à sa localisation dans les cellules. La protéine Pastrel co-localise avec le Rouge de Nil, un marqueur des gouttelettes lipidiques. Ainsi, nos résultats suggèrent un lien entre le métabolisme lipidique et le blocage d’une infection virale chez la drosophile. / Since the discovery of the evolutionarily conserved TOLL and IMD pathways, involved in antifungal and antibacterial immune responses, the fruit fly Drosophila melanogaster is used as a model to study the molecular mechanisms of innate immunity. To defend against viral pathogens, Drosophila relies on two main facets: the RNA interference (RNAi) pathway and virus specific inducible responses. We show that the RNAi pathway plays a role in the control of a DNA virus, in addition to RNA viruses. We also observe that the fly genetic background can modulate the resistance to infection by Drosophila C virus (DCV), a natural pathogen of Drosophila. This resistance to DCV infection is correlated with polymorphisms in a gene named pastrel,localized on the left arm of the third chromosome. Our loss- and gain-of-function experiments indicate that pastrel encodes a molecule opposing infection by picorna-like viruses DCV and also Cricket Paralysis virus (CrPV), raising the question of the mechanism involved. This restriction appears early after infection. The Cterminal region of Pastrel protein is important for its antiviral activity and its localization in vesicular structures co-localizing with Nile Red staining, a marker for lipid droplets. Altogether, our data suggest a connection between lipid droplets and restriction of viral infection in Drosophila, as already described in mammals between the restriction factor Viperin, present on lipid droplets, and the replication of the human pathogen Hepatitis C Virus.

Les interférons (IFN) constituent une famille de cytokines aux propriétés antiprolifératives et antivirales. Ils activent, via la voie Jak/STAT, des gènes spécifiques dont les produits sont les médiateurs des effets biologiques des IFN. C’est le cas de PML (Promyelocytic leukemia), appelée aussi TRIM19, qui joue un rôle central dans la défense antivirale. PML appartenant à la famille des protéines Tripartite Motif (TRIM), caractérisée par la présence en N-terminal d’un motif RBCC, constitué d’un domaine RING, d’une ou de deux boites B et d’un domaine coiled-coil. PML a été identifiée dans la leucémie aiguë promyélocytaire, une pathologie causée par la translocation chromosomique t(15 ;17) qui fusionne les gènes PML et RARA, aboutissant à la synthèse d'une protéine chimère PML-RARA. Le trioxyde d'arsenic (As2O3) cible la portion PML de la protéine oncogénique, entraînant sa dégradation et la rémission complète des patients. Dans les cellules saines, les transcrits PML issus d’un gène unique génèrent par épissage alternatif 7 isoformes principales de PML, dont six sont nucléaires (PMLI à PMLVI) et une cytoplasmique (PMLVIIb). Toutes possèdent la même extrémité N-terminale mais diffèrent au niveau de leur extrémité C-terminale, conférant à chaque isoforme des fonctions spécifiques.PML est l’organisatrice d’une structure multi-protéique appelée corps nucléaires (CN), impliquée dans divers processus cellulaires tels que l’apoptose, la dégradation des protéines ou encore la défense antivirale.PML est modifiée par SUMO de façon covalente au niveau de trois sites lysines (K65, K160, K490) et de façon non covalente, via son domaine SIM (pour « SUMO Interacting Motif »). Ces modifications sont requises pour la formation de CN fonctionnels et le recrutement de protéines partenaires au sein de ceux-ci. Le but de ma thèse a été d’étudier le rôle différentiel des différentes isoformes de PML en réponse à l’As2O3 et suite à l’infection virale. Nous avons montré que le SIM de PML est nécessaire à sa dégradation en réponse à l'As2O3. Ce motif est présent dans toutes les isoformes de PML, hormis l’isoforme nucléaire PMLVI et l’isoforme cytoplasmique PMLVIIb. Le SIM de PML n’est pas requis pour sa SUMOylation et son interaction avec RNF4 (une E3 ubiquitine ligase responsable de la dégradation de PML via le protéasome). En revanche, ce motif est requis pour l’ubiquitination de PML, le recrutement des composants du protéasome et sa dégradation en réponse à l’As2O3. Concernant les propriétés antivirales de PML, l’étude que nous avons menée avec toutes les isoformes de PML a permis de montrer que seules PMLIII et PMLIV confèrent une résistance au Virus de la Stomatite Vésiculaire (VSV). L’effet antiviral de PMLIII n'est observé qu'à faible multiplicité d’infection (MOI) et est indépendant de la production d’IFN. Par contre, PMLIV exerce une puissante activité anti-VSV, y compris à forte MOI et s'exerce selon deux mécanismes distincts : (i) PMLIV inhibe la réplication du VSV par un mécanisme précoce indépendant de l’IFN, (ii) PMLIV augmente tardivement la production d’IFN-β via une plus forte activation d’IRF3 qui est due à la séquestration spécifique de Pin1 au sein des CN par PMLIV. Ces deux processus nécessitent la SUMOylation de PMLIV. Ces résultats montrent que PMLIV exerce une activité antivirale intrinsèque et est impliquée dans l’immunité innée en régulant positivement la voie de transduction conduisant à la synthèse d’IFN-β. / Interferons (IFNs) are a family of cytokines with antiproliferative and antiviral properties.They activate, via the Jak/Stat pathway, specific genes whose products are the mediators of the biological effects of IFNs. This is the case of PML (Promyelocytic leukemia), also known as TRIM19, which plays a central role in antiviral defense.PML belongs to the Tripartite Motif (TRIM) protein family, characterized by the presence of an N- terminal RBCC pattern, consisting of a RING domain, one or two B-boxes and a coiled-coil domain. PML was identified in acute promyelocytic leukemia, a disease caused by the chromosomal translocation t(15 ;17), which fuses the PML and RARA genes, leading to the synthesis of a chimeric protein PML-RARA . Arsenic trioxide (As2O3) targets the PML moiety of the oncogenic protein, resulting in its degradation and in the complete remission of patients.In healthy cells, PML transcripts derived from a single gene generate seven major isoforms of PML by alternative splicing, including six nuclear (PMLI to PMLVI) and one cytoplasmic (PMLVIIb). All share the same N-terminus but differ at their C-terminus, giving each isoform specific functions.PML is the organizer of a multi-protein structure called nuclear bodies (NBs) that are involved in various cellular processes such as apoptosis, protein degradation or antiviral defense.PML is covalently modified by SUMO at three lysine residues (K65, K160, K490) but also non-covalently via its SIM domain (for « SUMO Interacting Motif »). These modifications are required for the formation of functional NBs and the recruitment of partner proteins within them.The aim of my thesis was to study the differential role of the different PML isoforms in response to As2O3 and during viral infection.We have shown that the SIM PML SIM is necessary for its degradation in response to As2O3. This motif is present in all PML isoforms, except the nuclear PMLVI and the cytoplasmic PMLVIIb isoforms. The SIM of PML is not required for its SUMOylation and its interaction with RNF4 (the E3 ubiquitin ligase responsible for PML proteasome-dependent degradation). However, this motif is required for the ubiquitination of PML, the recruitment of proteasome components and the degradation of PML in response to As2O3.Concerning the antiviral properties of PML, the study that we conducted with all PML isoforms allowed us to show that only PMLIII and PMLIV confer resistance to Vesicular Stomatitis Virus (VSV). Whereas the antiviral activity of PMLIII is only observed at low multiplicity of infection (MOI) and is independent of IFN production, PMLIV has a potent anti-VSV activity, including at high MOI, which is mediated through two distinct mechanisms: (i) PMLIV inhibits the replication of VSV by an early and IFN-independent mechanism, (ii) PMLIV later increases the production of IFN-β via a stronger activation of IRF3, which is due to the specific sequestration of Pin1 by PMLIV within NBs. Both processes require the PMLIV SUMOylation. These results show that PMLIV has an intrinsic antiviral activity and is also involved in innate immunity by positively regulating the transduction pathway leading to IFN-β synthesis.