Le virus de la dengue (DENV) et le virus Zika (ZIKV) sont deux virus transmis par le moustique et responsables de maladies importantes chez l’Homme. En absence de vaccin et de traitements antiviraux efficaces, ces pathogènes représentent des problèmes de santé publique majeurs. Les bases moléculaires des interactions qu’établissent le DENV et ZIKV et la cellule hôte lors de l’entrée virale sont peu connues. Notre laboratoire a récemment identifié, les protéines TIM (TIM-1 et TIM-4) et TAM (Tyro3 et Axl), deux familles de récepteurs à la phosphatidylsérine (PtdSer) impliqués dans la reconnaissance et l’élimination des cellules apoptotiques par phagocytose, comme de nouveaux facteurs d’entrée du DENV. Les récepteurs TIM et TAM permettent l’infection par le DENV en interagissant avec la PtdSer associée aux virions selon un mécanisme similaire à la reconnaissance des cellules apoptotiques (mimétisme apoptotique). L’objectif général de mon travail de thèse a été d’explorer les mécanismes moléculaires et cellulaires par lesquels TIM-1 et Axl médient l’entrée des flavivirus. A l’aide de techniques d’imagerie en temps réel nous avons montré que TIM-1 et DENV sont co-internalisés et que TIM-1 joue un rôle actif dans l’entrée du DENV. Notamment, nous avons montré que deux résidus lysine présentes dans le domaine cytoplasmique de TIM-1 sont importantes pour l’ubiquitination du récepteur et pour l’endocytose du virus. La recherche de partenaires de TIM-1 par des études de spectrométrie de masse a permis d’identifier STAM, un membre du complexe ESCRT-0 impliqué dans le trafic des récepteurs ubiquitinés, comme facteur important pour l’infection. Collectivement, nos résultats suggèrent très fortement que TIM-1 est le premier récepteur bona fide caractérisé pour le DENV.Identifier les facteurs d’entrée du ZIKV représente un enjeu majeur dans la compréhension du tropisme et de la pathogénèse associée à ce virus. Nous avons montré que le récepteur Axl est essentiel pour l’entrée du ZIKV dans les cellules microgliales, les astrocytes du cerveau humain en développement ainsi que dans les fibroblastes de la peau. Nos études ont démontré un double rôle du récepteur Axl dans l’infection par ZIKV. Axl lie et permet l’internalisation des particules virales, mais aussi, contribue à l’établissement d’un environnement favorable à la réplication virale en inhibant la réponse immunitaire innée. En conclusion, ce travail a contribué à améliorer notre compréhension des mécanismes d’entrée des virus DENV et ZIKV. Nos résultats indiquent que ces deux virus exploitent plusieurs récepteurs aux phospholipides pour initier leur cycle infectieux, ce qui pourrait contribuer à l’élargissement de leur tropisme. / Dengue virus (DENV) and ZIKA virus (ZIKV) are two mosquito-borne viruses responsible for important diseases in humans. Since there is currently no vaccine neither antiviral treatment available against these human pathogens, they are two major health concerns. The molecular basis of DENV and ZIKV host cell interactions leading to virus entry are poorly understood, hampering the discovery of new targets for antiviral intervention. Our laboratory recently discovered that TIM (TIM-1 and TIM-4) and TAM (Tyro3 and Axl) proteins, two receptor families that contribute to the phosphatidylserine (PtdSer)-dependent phagocytic removal of apoptotic cells, are DENV entry factors. TIM and TAM receptors mediate DENV infection by interacting with virion-associated PtdSer through a mechanism similar to the recognition and engulfment of apoptotic cells by phagocytes (viral apoptotic mimicry). The general objective of my PhD was to establish a detailed understanding of the molecular mechanisms by which TIM-1 and Axl mediated infection. Using live imaging, we demonstrated that TIM-1 and DENV are co-internalised and TIM-1 play an active role during DENV endocytosis. We showed that TIM-1 cytoplasmic domain is essential for DENV internalization, especially, we identified two lysine residues that are essential for TIM-1 ubiquitination and DENV endocytosis. Proteomic analysis of TIM-1 interacting partners identified STAM, a member of the ESCRT-0 complex involved in intracellular sorting of ubiquitinated cargos, as an essential host factor for DENV infection. Collectively our results establish TIM-1 as the first identified DENV bona fide receptor.Identifying ZIKV entry factors represents a major challenge in the understanding of ZIKV tropism and pathogenesis. We showed that Axl is responsible for ZIKV infection of microglial cells and astrocytes in the human developing brain and primary fibroblasts in human skin, suggesting an important role of this receptor during ZIKV life cycle. We also highlighted the dual role of the Axl receptor in ZIKV infection, which simultaneously promotes viral entry and dampens the innate immune response to facilitate a post entry step of the ZIKV life cycle. In conclusion, this work provided new insights in our understanding of the DENV and ZIKV entry program. Both viruses engage phospholipid receptors for their infectious entry, providing a rational to ascertain therapeutic strategies targeting virion-associated phospholipids.

Ebola viruses, and other members of the family filoviridae, are enveloped, negative sense, RNA viruses that can cause hemorrhagic fever. Currently, there are no antivirals or approved vaccines available that target or protect from Ebola virus infection. However, recently, T-cell immunoglobulin and mucin domain-1 (TIM-1) has been identified as an epithelial-cell receptor for filoviruses and could be a potential target for antivirals. However, little is known about how TIM-1 enhances virus entry and the role of TIM-1 during in vivo infection. In order to determine the key residues of TM-1 involved in interaction with virus, we generated a panel of point-mutations in the immunoglobulin-like variable (IgV) domain of TIM-1. We determined that several residues within the IgV domain that are involved in binding of phosphatidylserine (PtdSer) are also critical for Ebola virus entry. Further, we found that TIM-1 interacts with Ebola virus through binding of PtdSer on the viral envelope. PtdSer liposomes, but not phosphatidylcholine liposomes, competed with TIM-1 for EBOV pseudovirion binding and transduction. In addition, annexin V (AnxV) substituted for the TIM-1 IgV domain, supporting a PtdSer-dependent mechanism. Our findings suggest that TIM-1-dependent uptake of EBOV occurs by apoptotic mimicry. We also determined that TIM-1 expression can enhance infection of a wide range of enveloped viruses, including alphaviruses and a baculovirus. As further evidence of the critical role of enveloped virion associated PtdSer in TIM-1-mediated uptake, TIM-1 enhanced internalization of pseudovirions and virus-like particles (VLPs) lacking a glycoprotein, providing evidence that TIM-1 and PtdSer-binding receptors can mediate virus uptake independent of a glycoprotein. These results provide evidence for a broad role of TIM-1 as a PtdSer-binding receptor that mediates enveloped virus uptake. The PtdSer-binding activity of the IgV domain is essential for both virus binding and internalization by TIM-1. However, another member of the TIM family, TIM-3, whose IgV domain also binds PtdSer, does not effectively enhance virus entry. These data indicate that other domains of TIM proteins are functionally important. We investigated the domains of the TIM family members that play a role in the enhancement of enveloped virus entry, thereby defining the features necessary for a functional PVEER. Using a variety of chimeras and deletion mutants, we found that in addition to a functional PtdSer binding domain PVEERs require a stalk domain of sufficient length, containing sequences that promote an extended structure. Neither the cytoplasmic nor the transmembrane domain of TIM-1 is essential for enhancing virus entry, provided the protein is still plasma membrane bound. Based on these defined characteristics, we generated a mimic lacking TIM sequences and composed of annexin V, the mucin-like domain of α-dystroglycan, and a glycophosphatidylinositol anchor that functioned as a PVEER to enhance transduction of virions displaying Ebola, Chikungunya, Ross River, or Sindbis virus glycoproteins. This identification of the key features necessary for PtdSer-mediated enhancement of virus entry provides a basis for more effective recognition of unknown PVEERs. Provided that expression of TIM-1 in cells enhances virus entry through binding of PtdSer on the viral membrane, we wanted to determine whether virus entry would still be enhanced if this interaction was reversed with TIM-1 present on the viral membrane. Further, we reasoned that this might allow for targeting of virus to cells with greater amounts of PtdSer exposed on their outer leaflet, such as cancer cells. In order to test this hypothesis, we generated virions in cells coexpressing a glycoprotein and one of the TIM family members. We found that expression of TIMs in virus-producing cells resulted in TIM proteins being released into the virus-containing medium and enhanced Ebola virus GP pseudovirion titers. Further, this enhancement was dependent on the amount of PtdSer exposed on the target-cell membrane. However, we also determined that TIMs were not being incorporated into virions and that coexpression of TIMs with non-ebolavirus glycoproteins in virus-producing cells resulted in virus stocks with both reduced titers and the quantity of virions.

Tim-1 (T cell immunoglobulin mucin domain 1) is a transmembrane protein expressed by many cell types, including activated T cells and B cells. Antibodies to Tim-1 have been shown to decrease severity of airway hyperreactivity and Th2 cytokine production in mice. Current literature suggests Tim-1 functions as a co-stimulatory molecule. We hypothesize that Tim-1 signals in lymphocytes, and that Tim-1 signaling modulates allergic airway disease. Chapter one provides a brief overview of current literature exploring identification of the Tim family of receptors, genetic associations between TIM-1 polymorphisms and human diseases, Tim-1 expression, Tim-1 ligands, studies of antibodies to Tim-1 in various mouse models of human disease, and signaling events downstream of Tim-1 engagement. Chapter two provides detailed experimental methodology. Chapter three details the characterization of Tim-1 deficient mice. Tim-1 deficient mice do not exhibit defects in lymphocyte or myeloid cell development, as determined by numbers of cells present in bone marrow, thymus, spleen, and lymph nodes. C57BL/6 Tim-1 deficient female mice appear to develop an increased number of lymph node cells and also develop anti-double stranded DNA antibodies. Chapter four explores the impact of Tim-1 deficiency in a murine allergic airway disease model, which demonstrated that Tim-1 deficient mice developed increased lung inflammation and increased antigen-specific Th2 cytokine production that was evident in mice backcrossed to both BALB/c and C57BL/6 backgrounds. These phenotypes were not evident using purified naïve CD4+ T cells polarized in vitro. As Tim-1 expression is not restricted to CD4+ T cells, adoptive transfer experiments were performed to determine whether the phenotype observed was due to the deficiency of Tim-1 on CD4+ T cells, non-CD4+ T cells, or Tim-1 deficiency on both CD4+ T cells and non-CD4+ T cells. Chapter five explores the impact of Tim-1 deficiency in a chronic Leishmania major intradermal infection model. Tim-1 deficient mice crossed to both BALB/c and C57BL/6 backrounds demonstrated similar parasite burden over the course of time, but in vitro restimulation of lymph node cells revealed a striking increase in cytokine production that extended to Th1, Th2, and Th17 lineages. Tim-1 signaling in murine B cell lines is explored in Chapter six. A Tim-1 monoclonal antibody conjugated to beads induces phosphorylation of Tim-1 and recruitment of the Src family kinase Fyn. This phosphorylation of Tim-1 is reduced in Fyn-deficient B cell lines. Chapter seven discusses the significance of these findings, relates current literature to these results, and provides some avenues for further exploration of Tim-1 function and signaling.

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20260号 / 医博第4219号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 前川 平, 教授 木原 正博, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Ebola virus (EBOV) is a member of the Filoviridae family of highly pathogenic viruses that cause severe hemorrhagic fever and is the causative agent of the 2014 West Africa outbreak. Currently, there are no approved filovirus vaccines or treatments to combat these sporadic and deadly epidemics. One target for EBOV antiviral therapy is to block viral entry into host cells. Recently, phosphatidylserine (PtdSer) receptors, primarily known for their involvement in the clearance of dying cells, were shown to mediate entry of enveloped viruses including filoviruses. The PtdSer receptors, T-cell immunoglobulin mucin domain-1 (TIM-1) and family member TIM-4, serve as filovirus receptors, significantly enhancing EBOV entry. TIM-dependent virus uptake occurs via apoptotic mimicry by binding to PtdSer on the surface of virions through a conserved PtdSer binding pocket within the amino terminal IgV domain. TIM-4 is expressed on antigen presenting cells (APCs), including macrophages and dendritic cells (DCs), which are critical in early EBOV infection. My studies are the first to define the molecular details of virion/TIM-4 interactions and establish the importance of TIM-4 for EBOV infection of murine resident peritoneal macrophages. In addition, previous work has utilized only in vitro models to establish the importance of the TIM proteins in EBOV entry. My studies are the first to demonstrate the importance of TIM-1 and TIM-4 for in vivo EBOV pathogenesis and to confirm them as relevant targets of future filovirus therapeutics. Macrophage phenotypes can vary greatly depending upon chemokine and cytokine signals from their microenvironment. Historically, macrophages have been classified into two major subgroups: classically activated macrophages (M1) and alternatively activated macrophages (M2). Macrophages are a critical early target of EBOV infection and my work primarily focused on interferon gamma-stimulated (M1) macrophages since this treatment profoundly inhibited EBOV infection of human and murine macrophages. Interferon gamma treatment blocked EBOV replication in macrophages, reducing viral RNA levels in a manner similar to that observed when cultures were treated with the protein synthesis inhibitor, cycloheximide. Microarray studies with interferon gamma-treated human macrophages identified more than 160 interferon-stimulated genes. Ectopic expression of a select group of these genes inhibited EBOV infection. These studies provide new potential avenues for antiviral targeting as these genes that have not previously appreciated to inhibit infection of negative strand RNA viruses including EBOV. In addition and most exciting, using MA-EBOV, we found that murine interferon gamma, when administered either 24 hours before or after infection, protects lethally challenged mice and significantly reduces morbidity. Our findings suggest that interferon gamma, an FDA-approved drug, may serve as a novel and effective prophylactic or treatment option.