Thesis advisor: Welkin Johnson / Endogenous Retroviruses (ERVs) are “fossilized” retroviruses of a once exogenous retrovirus located in the genome of extant vertebrates. Retroviral infection results in a provirus integration into the host genome. An infection of a germline cell could lead to the provirus potentially being inherited by the offspring of the infected individual. Once in the genome, the provirus becomes subject to evolutionary processes and can become either lost or fixed in a population, remaining as “fossils” long after the exogenous retrovirus has gone extinct23. Notably, 8% of the human genome consists of ERVs30. Human Endogenous Retrovirus Type K (HERV-K)(HML-2) family is of particular interest. HERV-K integrations are as old as 30-35 million years, endogenizing before the separation of humans and Old World Monkeys. However, there are human specific insertions, some as young as 150,000 – 250,000 years, making them the youngest insertion in the human genome. There are over 90 insertions in the human genome; the bulk is shared by all humans44,47. Transcripts of HERV-K genes are upregulated in multiple cancer and tumor cell lines 14,39,46, as well as in HIV-1 infected patients 7,11,29. Just as there are human specific insertions of ERV-K, there are also Old World Monkey specific insertions44. I have identified an intact endogenous retroviral envelope open reading frame on chromosome 12 of the rhesus macaque genome. This viral envelope-encoding sequence, which I refer to as rhERV-K env, retains all the canonical features of a retroviral Env protein. An alignment between rhERV-K env and a consensus sequence of HERV-K, HERV-Kcon env, shows a 70% amino acid sequence identity. For experimental purposes, reconstructed HERV-K envelopes have been incorporated into virions of Human Immunodeficiency virus (HIV-1)19,26,49, Murine Leukemia Virus (MLV)12, and Vesicular stomatitis Virus (VSV)26,41,49. While these approaches have illuminated some aspects of HERV-K Env-mediated entry, to date a cell-surface receptor has not been identified for any ERV-K Env. This could be due to its low infectivity levels12,26,49, its seemingly broad cell tropism limiting identification of null cell lines26,49, or possibly the HERV-K consensus reconstructions are not an accurate representation of the progenitor HERV-K virus. I am interested in understanding how the ERV-K retrovirus accessed the human germline (some 150,000 – 250,000 years ago). To do this, I focused specifically on the envelope proteins of HERV-K and rhERV-K, with the goal of analyzing the ERV-K entry process. The identification and inclusion of rhERV-K Env in this study is meant to circumvent the possibility that the previously described consensus reconstructions of human HERV-K Env are not representative, and may also provide a means to compare the endogenization process in the human/ape and old-world monkey lineages. I focused on developing two systems for single-cycle infection, one based on Mason-Pfizer Monkey Virus (MPMV) (which has not been done before), and a second based on MLV, which has previously been reported on. MPMV, like HERV-K, is a betaretrovirus, and I reasoned that possibly using a betaretrovirus would overcome some of the low-infectivity issues associated with prior attempts using HIV and MLV. To develop a system for examining function of the ERV-K Env proteins, I addressed 3 issues: 1. Are the HERV-K Env and rhERV-K Env proteins expressed and properly processed? 2. Can they be incorporated into virions of a heterologous virus? 3. Are ERV-K pseudotyped virions infectious? I have answered these questions in the following thesis. / Thesis (MS) — Boston College, 2017. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Biology.

Notre étude est consacrée à la caractérisation des mécanismes d'adaptation métabolique des neutrophiles au cours de l'inflammation et plus particulièrement au cours de leur recrutement dans les voies aériennes des patients atteints de mucoviscidose (cystic fibrosis ou CF en anglais). Dans la mucoviscidose, nous avons précédemment décrit que les neutrophiles présents dans la lumière du poumon sont viables et soumis à une reprogrammation, notamment via l'activation de la voie anabolique mTOR. Ce travail est fondé sur les propriétés spécifiques de ligands solubles dérivés des domaines liant le récepteur (RBD, pour Receptor-Binding Domain) des glycoprotéines d'enveloppes rétrovirales permettant leur utilisation pour la détection de transporteurs métaboliques à la surface des cellules. Dans un premier temps, nous avons validé l'utilisation de ce nouveau groupe de marqueurs pour identifier et caractériser le phénotype métabolique des leucocytes CF obtenus à partir d'échantillons de différents compartiments (sang et expectorât pulmonaire). Dans un deuxième temps, l'étude de l'expression de ces transporteurs métaboliques sur les neutrophiles sanguins de patients atteints de CF ou de polyarthrite rhumatoïde et de sujets contrôles, a permis la distinction de phénotypes métaboliques au niveau systémique selon différents états inflammatoires. Ensuite, nous avons comparé l'expression des transporteurs métaboliques entre les neutrophiles sanguins et pulmonaires de patients CF et révélé une adaptation métabolique de ces cellules lors de leur recrutement vers les poumons. Enfin, les neutrophiles présents dans les voies aériennes des patients CF présentent une modulation de leur activité transcriptionnelle. De façon surprenante, nos travaux démontrent que malgré leur reprogrammation, les neutrophiles recrutés vers les poumons CF sont fonctionnellement compétents, ajoutant ainsi un nouvel angle d'approche dans l'étude des neutrophiles au cours de l'inflammation, notamment dans le cas de la pathologie pulmonaire liée à la mucoviscidose. / The present study focuses on adaptive metabolic steps adopted by neutrophils during inflammation, particularly during their recruitment into the cystic fibrosis (CF) airways. In CF, we previously described that airway neutrophils are alive and undergo reprogramming, featuring notably the activation of the anabolic mTOR pathway. The present work is based on specific properties of soluble ligands derived from the receptor-binding domains (RBD) of retroviral glycoprotein envelopes, which can be used for the detection of metabolite transporters at the cell surface. First, we validated the use of this new set of markers for the identification and characterization of the metabolic phenotype of CF leukocytes obtained from distinct compartments (blood and sputum). Second, by studying the metabolite transporter expression on blood neutrophils from CF or rheumatoid arthritis patients and control subjects, we distinguished metabolic phenotypes characteristic of specific inflammatory states. Then, we compared metabolite transporter expression between CF blood and airway neutrophils and showed that neutrophils undergo significant metabolic adaptation upon recruitment into the lungs. Finally, we demonstrated that CF airway neutrophils display significant transcriptional modulation and that despite their metabolic reprogramming, they remain functionally competent, thus adding an additional angle of approach to neutrophil studies with regard to inflammation, notably during CF airway disease.

The present study focuses on adaptive metabolic steps adopted by neutrophils during inflammation, particularly during their recruitment into the cystic fibrosis (CF) airways. In CF, we previously described that airway neutrophils are alive and undergo reprogramming, featuring notably the activation of the anabolic mTOR pathway. The present work is based on specific properties of soluble ligands derived from the receptor-binding domains (RBD) of retroviral glycoprotein envelopes, which can be used for the detection of metabolite transporters at the cell surface. First, we validated the use of this new set of markers for the identification and characterization of the metabolic phenotype of CF leukocytes obtained from distinct compartments (blood and sputum). Second, by studying the metabolite transporter expression on blood neutrophils from CF or rheumatoid arthritis patients and control subjects, we distinguished metabolic phenotypes characteristic of specific inflammatory states. Then, we compared metabolite transporter expression between CF blood and airway neutrophils and showed that neutrophils undergo significant metabolic adaptation upon recruitment into the lungs. Finally, we demonstrated that CF airway neutrophils display significant transcriptional modulation and that despite their metabolic reprogramming, they remain functionally competent, thus adding an additional angle of approach to neutrophil studies with regard to inflammation, notably during CF airway disease.

The triggers for the development of multiple sclerosis (MS) have not been fully understood to date. One hypothesis proposes a viral etiology. Interestingly, viral proteins from human endogenous retroviruses (HERVs) may play a role in the pathogenesis of MS. Allelic variants of the HERV-K18 env gene represent a genetic risk factor for MS, and the envelope protein is considered to be an Epstein–Barr virus-trans-activated superantigen. To further specify a possible role for HERV-K18 in MS, the present study examined the immunogenicity of the purified surface unit (SU). HERV-K18(SU) induced envelope-specific plasma IgG in immunized mice and triggered proliferation of T cells isolated from these mice. It did not trigger phenotypic changes in a mouse model of experimental autoimmune encephalomyelitis. Further studies are needed to investigate the underlying mechanisms of HERV-K18 interaction with immune system regulators in more detail.

La plupart des virus ont développé des mécanismes de résistance ou de suppression du système immunitaire pour parvenir à infecter durablement leur hôte. Ces mécanismes demeurent encore imparfaitement connus. Un domaine immunosuppresseur (IS) a été identifié au niveau de la région transmembranaire des protéines d’enveloppe des rétrovirus endogènes ou infectieux. Ce domaine hautement conservé a été décrit par exemple comme inhibant l’activation lymphocytaire. Dans le laboratoire, il a été caractérisé en particulier via des expériences de rejet de cellules tumorales in vivo, ce qui a permis de définir des mutations inactivatrices. Afin de mieux comprendre les mécanismes de résistance des rétrovirus au système immunitaire, mes travaux de thèse ont porté sur l’identification de la ou des protéines capables d’interagir avec le domaine IS. Plusieurs approches cellulaires et moléculaires ont été développées, basées pour la plupart sur l’utilisation de sondes fluorescentes obtenues par synthèse chimique, constituées des domaines IS provenant de différents rétrovirus. Dans un premier temps, les cellules du système immunitaire qui lient les protéines virales ont été identifiées : les lymphocytes B et les cellules myéloïdes (monocytes, cellules dendritiques et macrophages). Dans un second temps, des expériences de co-immunoprécipitation et de chromatographie d’affinité couplées à la spectrométrie de masse ont été réalisées dans le but d’identifier sur ces cellules les protéines membranaires responsables de ces liaisons. Plusieurs agents de couplages chimiques ont été utilisés afin de maintenir les liaisons domaine IS - protéine de faibles affinités. En raison de résultats non-reproductibles obtenus au cours de ces expériences, des tests de liaison du domaine IS sur des cellules transfectées avec des banques d’ADNc, ou lors d’expériences de double hybride ont été réalisées. Ces deux approches ont permis d’identifier des protéines membranaires potentiellement impliquées dans la liaison du domaine IS : les protéines X1 et X2. Les co-transfections de vecteurs d’expression du domaine IS et de X2 ont mis en évidence des interactions protéiques au cours d’expériences de co-immunoprécipitation et de microscopie confocale, en particulier avec le domaine IS du rétrovirus HIV-1. Concernant X1, sa transfection induit la liaison cellulaire des domaines IS de HERV-W et MLV. En revanche, aucune interaction directe entre X1 et le domaine IS n’a pu être démontrée, notamment dans des expériences de co-immunoprécipitation et de microscopie confocale.La découverte des protéines membranaires qui interagissent avec le domaine IS demeure un enjeu critique pour la compréhension des voies de signalisation et de transcription qui permettent aux rétrovirus d’exercer leur effet sur le système immunitaire, l’objectif de ces travaux étant d’identifier à terme des nouvelles cibles thérapeutiques.En conclusion, même si des travaux complémentaires demeurent nécessaires, les protéines X1 et X2 pourraient contribuer à l’immunosuppression rétrovirale. / Most viruses have developed mechanisms of resistance or suppression of the immune system to achieve lasting infection of their host. These mechanisms are still imperfectly known. An immunosuppressive (IS) domain has been identified in the transmembrane region of envelope proteins of endogenous or infectious retroviruses. This highly conserved domain has been described, for example, as inhibiting lymphocyte activation. In the laboratory, it has been characterized by tumor cell rejection experiments in vivo, which has made it possible to define inactivating mutations. In order to better understand the mechanisms of resistance of retroviruses to the immune system, my thesis focused on the identification of the protein(s) interacting with the IS domain. Several cellular and molecular approaches have been developed, based for the most part on the use of fluorescent probes obtained by chemical synthesis, consisting of IS domains from different retroviruses. At first, immune system cells that bind viral proteins have been identified: B cells and myeloid cells (monocytes, dendritic cells and macrophages). In a second step, co-immunoprecipitation and affinity chromatography coupled to mass spectrometry were performed to identify on these cells the membrane proteins responsible for these bonds. Several chemical coupling agents have been used to prevent detachment of low affinity binding between proteins and the IS domain. Due to non-reproducible results obtained during these experiments, IS domain binding assays on cells transfected with cDNA libraries, or in double hybrid experiments were performed. These two approaches made it possible to identify membrane proteins potentially involved in the binding of the IS domain: the X1 and X2 proteins. Co-transfections of IS domain and X2 expression vectors demonstrated protein interactions in co-immunoprecipitation and confocal microscopy experiments, particularly with the IS domain of the HIV-1 retrovirus. Concerning X1, its transfection induces binding of the IS domains of HERV-W and MLV on cells membrane. On the other hand, no direct interaction between X1 and the IS domain could be demonstrated, especially in co-immunoprecipitation and confocal microscopy experiments.The discovery of membrane proteins that interact with the IS domain remains a critical issue for understanding the signaling and transcription pathways that allow retroviruses to exert their effect on the immune system, the aim of this work being to identify new therapeutic targets.In conclusion, although further work is still needed, the X1 and X2 proteins may contribute to retroviral immunosuppression.