Ebolavirus (EBOV) is a negative sense, single stranded RNA virus that causes Ebola hemorrhagic fever. This disease causes substantial morbidity and mortality in humans, with death occurring in 50-90% of cases. Despite years of intensive research, much of the molecular mechanism underlying the entry of EBOV remains unknown. We performed a bioinformatics screen to identify novel entry cofactors by correlating mRNA expression in a panel of human cancer cell lines with permissivity to the EBOV entry glycoprotein. This assay identified several known EBOV entry cofactors such as actin and the tyrosine kinase Axl. In addition, several genes involved in macropinocytosis and endosomal maturation were also correlated with EBOV permissivity. Subsequent evaluation of plasma membrane proteins correlated by this screen showed T-cell immunoglobulin and mucin domain-1 (TIM-1) mRNA expression correlated extremely well with EBOV pseudovirion transduction. Depletion of TIM-1 from highly-permissive cells inhibits EBOV pseudovirion transduction. Conversely, expression of TIM-1 in poorly-permissive cells significantly and specifically enhances EBOV pseudovirion transduction and infection. TIM-1 binds to EBOV GP and this binding is important in the initial interaction between the virus and the host cell. ARD5, a TIM-1 mAb, significantly inhibits EBOV GP-mediated entry into several cell lines and primary human airway epithelia in a dose and time-dependent manner. Therefore, TIM-1 is the first receptor identified for EBOV. Additionally, AMP-activated protein kinase (AMPK) mRNA correlated strongly with EBOV pseudovirion transduction. Compound C, a specific AMPK inhibitor, inhibited EBOV pseudovirion transduction and infection in a time and dose-dependent manner into several cell lines and primary human monocyte derived macrophages. Mouse embryonic fibroblasts (MEFs) lacking functional AMPK were significantly less permissive to EBOV GP-mediated infection that WT MEFs. Visualization of virus entry into these cells revealed that EBOV causes actin polymerization independently of AMPK, but AMPK-/- cells do not form lamellipodia in the presence of EBOV and, consequently, cannot internalize virus into cells by macropinocytosis.

This dissertation is a tale of two emerging human pathogens. The first is a genus of viruses, orthoebolaviruses, which periodically cause outbreaks in humans in central and western Africa following spillover from animal reservoirs. Outbreaks of orthoebolaviruses have high rates of morbidity and mortality and can cause symptoms ranging from vomiting and diarrhea to hemorrhage. Understanding both how the virus evolves to fit its host as well as how the host reacts to viral infection is paramount to understanding what determines whether an infected patient will die or survive orthoebolavirus infection. To understand how orthoebolavirus genomic plasticity allows the virus to optimize itself to its host, I analyzed viral genomic sequencing data from two Orthoebolavirus species during serial passage in tissue culture: Ebola virus and Sudan virus. In low-passage Sudan virus, I discovered a true viral quasispecies in which three to four viral genotypes circulated within the same stock. I then examined how that quasispecies reacted when put into a nonhuman primate model (NHP) of infection; unexpectedly, we saw that the mix of genotypes in the challenge stock matched the mix of genotypes seen at clinical endpoint. To begin to understand what a successful immune response to orthoebolavirus infection entails, I characterized the circulating transcriptomic response in two survival models of Ebola virus disease. In a uniform survival model where NHPs were challenged with Bombali virus, I showed that NHPs have a clear and robust response to infection despite varying symptom severity. In a Taï Forest virus challenge model with ~44% survival, I showed that NHPs that succumb do so in a uniform manner consistent with other models of Ebola virus disease. In contrast, survivors were highly variable in their response to infection: some mimicked the non-survivor response but recovered in time, while others hardly responded at all. After covering orthoebolavirus genomic plasticity and the host response to infection in the first and second sections, respectively, I will then shift to the other focus of my dissertation work: SARS-CoV-2 and molecular epidemiology. SARS-CoV-2 swept the globe in 2020 following spillover into humans from an animal reservoir in late 2019, and surveillance sequencing of viral genomes early in the pandemic showed the virus was rapidly adapting to its new host. I leveraged this high mutation rate to spin up a molecular epidemiology operation for Boston Medical Center (BMC) and Boston University (BU). From mid-2020 through spring 2022, I catalogued, processed, sequenced, and analyzed samples and viral genomes from over 7,000 SARS-CoV-2 patient swabs. I worked with contact tracing teams, physicians, and infection control from BU and BMC to quantify viral introductions, identify transmission chains, and integrate the genetic linkages with traditional epidemiological data. / 2025-01-11T00:00:00Z

Les virus appartenant à l’ordre des Mononegavirales possèdent une « large » protéine L, responsable du cycle réplication/transcription et de maturation des ARNs. Six domaines conservés portent les différentes activités de cette protéine dont le site catalytique d’une activité méthyltransférase (MTase) de la coiffe. La coiffe est une structure chimique constituée d’une guanosine méthylée en position N7 reliée à l’extrémité 5’ des ARNm par une liaison 5’-5’ triphosphate. Une seconde méthylation est également présente en position 2’O du ribose du premier nucléotide de l’extrémité 5’ de l’ARNm. Ces méthylations ont un rôle critique chez les virus car elles permettent la traduction efficace des ARNm mais permettent également aux ARNs viraux d’échapper à leur détection par l’immunité innée de l’hôte. Ainsi, la caractérisation de ce domaine chez le virus Ebola serait un point clé pour une meilleure compréhension de la réplication des filovirus et un pas vers l’élaboration d’une nouvelle stratégie thérapeutique. Nous avons donc produit le domaine MTase du virus Soudan (SUDV) afin de caractériser son activité. Il a été démontré que le domaine C-terminal de la protéine L joue un rôle dans le recrutement de l’ARN, crucial pour l’activité MTase. Nous avons pu identifier une activité A-2’O MTase interne originale. Le domaine MTase de SUDV est également capable de méthyler les positions N7 et 2’O de la coiffe mais une caractérisation plus approfondie est nécessaire. Enfin, nous avons identifié des molécules inhibant l’activité MTase des filovirus. Une analyse biochimique plus poussée permettra d’initier le développement d’une nouvelle stratégie antivirale contre le virus Ebola. / In the Mononegavirales order, viruses encode a large protein (L), which is responsible for replication/transcription and RNA modifications. This protein harbours six conserved domains accountable of these different activities. Among these domains, the conserved region VI (CRVI) has been predicted to support cap-methyltransferase (MTase) activity. The cap consists in a N7-methylated guanosine linked to the first nucleotide at the mRNA 5'-end by a 5'-5' triphosphate bond. This structure can also be methylated at the 2'O position of N1 ribose. These methylations play a critical role in virus life cycle as N7 methylation triggers efficient viral RNA translation and 2'O methylation hampers the detection of viral RNA by the host innate immunity. Thus, the characterization of this domain in Ebola virus is a key point to understand replication of mononegaviruses and design new antiviral strategies. We produced the MTase domain of Sudan ebolavirus (SUDV) to characterize its MTase activity. We demonstrated that the protruding C-terminal domain is essential for MTase activity as this domain is a key for the RNA recognition. Using synthetic short RNAs holding different cap structures, we discovered that SUDV MTase harbours an unconventional A-2’O MTase activity. Besides this, the MTase domain is able to methylate the cap structure at N7 and 2'O positions but further characterization would be necessary to fully understand the cap synthesis. Finally, we identified compounds limiting the Ebola virus MTase activity. Further biochemistry and compounds characterization results will thus pave the way towards the development of an innovative antiviral strategy.

La République du Congo (RC) ou nos travaux ont eu lieu, est un pays d’Afrique Centrale, il partage ses frontières avec la République démocratique du Congo, la République centrafricaine, le Gabon, le Cameroun, et l’Angola (Cabinda). Dans ces pays la circulation des arboviroses est documentée. En RC, il y avait peu ou pas de documentation sur les arboviroses avant nos travaux. Nous avons réalisé des études de séroprévalence des arboviroses de différentes familles chez des donneurs de sang Congolais. Nous avons aussi étudié l’épidémie de chikungunya ayant sévit en RC en 2011. Nos travaux ont permis de mettre en évidence des taux de séroprévalence élevés pour les pathogènes principaux incriminés: 47,2% pour Dengue, 27,8% pour Yellow fever, 24,4% pour West Nile, 38,8% pour Chikungunya et 7,9% pour Rift Valley fever. Ces taux de séroprévalence élevés prouvent la circulation de ces virus au Congo, bien qu’aucune épidémie n’ait été encore déclarée pour certains. Nous avons également isolé et caractérisé génétiquement une souche nommée "Brazza_MRS1", appartenant au lignage East Central and Southern African, issue de l’épidémie due au virus chikungunya de 2011. La RC a connu plusieurs épidémies dues au virus Ebola. Nous avons tenté de mieux caractériser la circulation des filovirus chez les donneurs de sang asymptomatiques par une étude de séroprévalence. Les taux de séroprévalence IgG anti Ebola virus, observés étaient de 2,5% en général (1,6% à Brazzaville, 4% à Pointe-Noire et 4% en milieux ruraux). Les facteurs de risques identifiés étaient l’exposition aux chauves-souris (p<0.001) et aux oiseaux (p = 0.04). Le taux de séroprévalence IgG anti Marburg virus était de 0,5%. / The Republic of Congo (RC) where our work took place is a Central African country, sharing borders with the Democratic Republic of Congo, Central African Republic, Gabon, Cameroon, and Angola (Cabinda). In these countries the circulation of arboviruses is documented. In RC, there was little or no documentation on arboviruses prior to our work. We conducted studies of arbovirus seroprevalence in Congolese blood donors for different virus families. We also studied the epidemic caused by the chikungunya virus that prevailed in RC in 2011.Our work have highlighted the high rate of seroprevalence for incriminated major pathogens: 47.2% for Dengue, 27.8% for Yellow Fever, 24.4% for West Nile, 38.8% for Chikungunya and 7.9% for Rift Valley fever. These high seroprevalence rates indicate that these viruses actively circulate in Congo, although no epidemic has yet been reported for some viruses. We have also isolated and genetically characterized a strain named "Brazza_MRS1", belonging to the East Central and Southern African lineage, after the chikungunya epidemic in 2011. The RC has experienced several outbreaks caused by the Ebola virus. We have performed a filovirus seroprevalence study to attempt to better characterize the circulation of filoviruses in asymptomatic Congolese blood donors. The observed rate of seroprevalence of anti Ebola IgG was 2.5% overall (1.6% in Brazzaville, 4% in Pointe-Noire and 4% in rural areas). Identified epidemiological risk factors were the exposure to bats (p <0.001) and birds (p = 0.04). The seroprevalence rate of Marburg virus IgG was low (0.5%).

To enter cells, the filovirus, ebolavirus (EBOV), must bind to target cells and internalize into an endocytic vesicle. The properties surrounding filoviral entry into permissive cells remain poorly studied. To date, the kinetics associated with filoviral-glycoprotein (GP)-mediated entry have never been investigated past 6 hours. Our initial entry studies with filoviral-GP pseudotyped retrovirions at 37˚C indicated that virions entered permissive cells with a half time (T50) of ~8 hours. We found that 10 to 20% of retroviral based virions bound to cells in over a one hour period at 4˚C suggesting that virion binding was relatively inefficient. Surprisingly, we also observed that less than half of the retroviral based pseudovirions pre-bound to the cell surface were internalized by 7 hours at 37˚C indicating that virion internalization was a slow process. Consistent with slow internalization of retroviral particles, we observed that, while virus entry lost sensitivity to ammonium chloride treatment with time, 50% of the virions remained sensitive to low pH neutralization for at least 7 hours. These slow entry kinetics for filoviruses have not been appreciated thus far, and could have significant implications in the timing and types of treatments that could be administered to filoviral infected individuals. We also determined the impact of specific carbohydrate linkages on host cell plasma membrane proteins involved in filoviral entry, by using a series of Chinese hamster ovary (CHO) cell lines deficient in one or more enzymes required for N- and O- linked glycosylation. The LdlD CHO cell line that expresses normal surface N-linked glycans but has abbreviated O-linked surface glycans showed a 50% reduction in transduction by both Zaire (ZEBOV) and Lake Victoria MARV-GP pseudotyped particles as compared to the control wild type parental CHO cell line (Pro5). Use of the novel O-linked inhibitor drug 1-68A allowed us to confirm the necessity of O-linked glycans in efficient ZEBOV entry into additional permissive cells types. Interestingly, loss of terminal sialic acids (Lec2 cells) or galactose (Lec8 cells) on both N- and O- linked sugars resulted in a 2-fold enhancement of filoviral GP mediated entry compared to control. However, Lec1 cells that have wild type O-linked glycans but highly abbreviated N-linked glycans had similar levels of transduction to control Pro5 cells. Further studies indicated that binding of ZEBOV pseudovirions to Pro5 and all mutant CHO cells was equal, indicating that a post-binding defect or enhancement in ZEBOV internalization may be occurring. These data identify the importance of host cell O-linked glycosylation during the initial steps of filovirus infection. While the receptor(s) used by filoviruses for productive binding and entry into cells remains to be identified, several proteins have been shown to enhance filoviral entry into cells. Axl, a plasma membrane associated Tyro3/Axl/Mer (TAM) family member, is necessary for optimal ZEBOV-GP-dependent entry into some permissive cells, but not others. To date, the precise role of Axl in virion entry is unknown. Through the use of biochemical inhibitors, RNAi, and dominant-negative constructs, we set out to characterize entry pathways used for ZEBOV uptake in cells that require Axl for optimal transduction (Axl-dependent cells) and to define the role of Axl in these processes. We demonstrate that ZEBOV-GP-dependent entry into Axl-dependent cells occurs through multiple pathways including both clathrin-dependent and caveolae/lipid raft-mediated endocytosis. Surprisingly, both dynamin-dependent and -independent fluid-phase uptake (FPU) pathways mediated ZEBOV-GP entry into the Axl-dependent cells as well. Reduction of Axl expression by RNAi treatment resulted in abrogation of ZEBOV entry by FPU-dependent pathways, but had no effect on receptor-mediated endocytosis mechanisms. Our findings demonstrate for the first time that Axl enhances FPU, thereby increasing productive ZEBOV entry, and providing insight into the mechanisms surrounding filoviral entry.

Ebolavirus (EBOV) is a single stranded RNA virus that causes haemorrhagic fever in humans and other mammals. The EBOV encodes 7 proteins, NP, L, VP30, VP35, VP40, GP and VP24. VP24 is believed to be one of the EBOV proteins that causes the extreme virulence of the pathogen. The protein blocks the interaction between PY-STAT1 and KPNA, a protein that is involved in the import of PY-STAT1 into the nucleus. The nuclear import of PYSTAT1 is therefore blocked. This leads to the inhibition of IFN signalling. The purpose of this study was to express and purify VP24 and KPNA5. The proteins recombinantly expressed as a fusion tag in E. coli in lysogeny broth. Purification of VP24 was done using immobilized metal ion affinity chromatography, size exclusion chromatography and ion exchange chromatography. Characterization of the protein was analysed using circular dichroism. The results obtained from this study showed that VP24 could be purified in pH 10 buffers with little loss of protein due to aggregation and the protein was folded with an alphahelical structure. The expression and purification of KPNA5 was more complicated and further evaluation is left for future studies. The established protocol for expression and purification of VP24 and the initial work on KPNA5 will go a long way to aiding future studies on the system, thus the answer to the question regarding the extreme virulence of EBOV will be closer.

京都大学 / 新制・課程博士 / 博士(生命科学) / 甲第25450号 / 生博第521号 / 新制||生||69(附属図書館) / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 野田 岳志, 教授 朝長 啓造, 教授 鈴木 淳 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Les virus Ebola (EBOV) et Marburg (MARV) causent des fièvres hémorragiques chez les primates, y compris l’homme. Le taux de létalité peut atteindre 90% et il n’existe ni vaccin ni traitement contre ces virus. En raison de leurs caractéristiques moléculaires communes, EBOV et MARV sont regroupés au sein de la famille des Filoviridae. Le virion est composé de 7 protéines, dont la VP24, qui joue un rôle important dans l’assemblage et la condensation des nucléocapsides, et pour EBOV, elle est également responsable de l’inhibition de la réponse à l’IFN. Des mutations dans la séquence protéique de VP24 sont impliquées dans le processus d’adaptation chez un nouvel hôte. La protéine VP24 d’EBOV est donc multifonctionnelle. Pour MARV, cette protéine ne semble pas porter les fonctions décrites pour la VP24 d’EBOV. Afin de comprendre le rôle de la VP24 de MARV, nous avons identifié ses partenaires cellulaires par un crible double-hybride en levures. Nous avons mis en évidence l’interaction entre Keap1 et la VP24 de MARV, et confirmé ce résultat en cellules mammifères. Keap1 est une protéine impliquée dans le contrôle de la réponse au stress oxydatif, car elle inhibe le facteur de transcription Nrf2, qui régule l’expression d’enzymes impliquées dans la réduction des ERO. Nos résultats montrent que le domaine de Keap1 liant la VP24 est le même que celui liant Nrf2, et que la VP24 de MARV active Nrf2 pour la synthèse de molécules anti-oxydantes. Nous avons enfin évalué l’impact de la VP24 de MARV sur ERR, une autre cible de Keap1, et mesuré l’activité Nrf2 au cours de l’infection par EBOV. Nos résultats montrent des effets opposés des VP24 d’ EBOV et de MARV sur l’activité de Nrf2. / Ebola (EBOV) and Marburgvirus (MARV) are responsible for severe hemorrhagic syndrome in primates, including humans. The lethality rate can reach 90%, and no vaccine or treatment is available to counteract these diseases. EBOV and MARV have similar genomic organization and thus are placed in a distinct family, Filoviridae. VP24 is one of the 7 structural proteins which form the virion and has been shown to play an important role in assembly and condensation of viral nucleocapsids. VP24 of EBOV is responsible for prevention of cellular response to IFN. Mutations in EBOV VP24 gene are necessary for the adaptation to a new host. EBOV VP24 thus acts as a multifunctional factor. Available data suggest that MARV VP24 is not implicated in either the counteraction of IFN response, or in the adaptation process. In order to discover new functions for VP24 of MARV, we searched for its interaction with cellular proteins, using a yeast-double hybrid approach. We discovered an interaction between MARV VP24 and Keap1 protein and further confirmed this interaction in mammalian cells. Keap1 is a cellular protein involved in intracellular detection of Reactive Oxygen Species (ROS) and in the control of oxidative stress response. It inhibits the Nrf2 transcription factor, which regulates expression of antioxidant enzymes. Our results indicate that Keap1 binding domain for VP24 is the same as the one involved in Nrf2 binding, resulting in activation of transcriptional activity of Nrf2. Impact of MARV VP24 on ERRa, another target of Keap1, was also measured, as well as Nrf2 activity during EBOV infection. Our results showed that VP24 of EBOV and MARV have opposite effect on Nrf2 activity.

Les virus sont au centre de problème de santé publique. En raison de l'apparition de nouveaux virus et pour certains de leur résistance aux traitements existants il est toujours d’actualité de développement de nouveaux agents antiviraux. En général, la stratégie de lutte contre les infections virales est basée sur la vaccination ou sur l'activité des petites molécules, interférant avec un ou plusieurs processus biologiques participant au cycle de vie du virus. Dans ce contexte, nous avons conçu et synthétisé des petites bibliothèques de molécules visant des propriétés anti-filovirus. Dans ce projet de recherche, nous avons mis l'accent sur le développement de composés ciblant la protéine Niemann-Pick C1, les protéases cathepsine et le processus de réplication. Lors du développement des inhibiteurs de Neimann-Pick C1 plus de 70 composés ont été synthétisés, portant le squelette pipérazine. Afin d'obtenir des inhibiteurs de cystéine cathepsines pouvant être impliqués dans la réplication du virus Ebola, nous avons synthétisé une petite bibliothèque de composés porteurs de groupement 1,3,5-triazine et possédant des activité de l’ordre du nanomolaire sur les cathepsines B, K, L et S. Enfin, pour inhiber la réplication du virus en ciblant SAH hydrolase, nous avons proposé une série de C-nucléosides carbocyclic ayant motif de 4-aza-7,9-dideazaadenosine. / The viruses cause the problem of public health. Due to the appearance of new viruses and their resistance to existing treatments there is still relevant to develop new antivirals. Generally, the strategy to combat viral infections is based on vaccination or on the activity of small molecules, interfering with one or more biological processes participating in virus life cycle. In this context, we took an effort to design and synthesize the library of small molecules possessing anti-filovirus properties. In this research project, we were focused on the developing of compounds targeting Niemann-Pick C1 protein, cathepsin proteases and replication process. In our effort into the development of the inhibitors of Neimann-Pick C1 we prepared the series of about 70 compounds, having in common the piperazine moiety. Diverse 1,4-N,N - substituents of piperazine, differencing in a size and shape were studied. In order to obtain efficient cysteine cathepsins inhibitors, we synthesized the small library of compounds bearing 1,3,5-triazine moiety. Finally, to inhibit the virus replication by targeting SAH hydrolase, we proposed the series of carbocyclic C-nucleosides having motif of 4-aza-7,9-dideazaadenosine.

L’identification d’antiviraux à large spectre est un des défis majeurs de la rechercheactuelle en virologie. Une des stratégies les plus prometteuses consiste à cibler une interactionvirus/hôte conservée. Ainsi, avec la technique d’AlphaScreen® et le modèle d’interactionprotéine VI de l’Adénovirus (AdV)/Nedd4-2, nous avons réalisé un criblage biochimique àhaut débit contre l’interaction virus/hôte [LP]PxY/Nedd4, commune à différentes familles devirus. Nous avons trouvé des candidats inhibiteurs issus d’une banque de composés approuvéspar les agences de santé. Nous les avons testés, caractérisés et validé leur effet antiviral surdeux familles de virus totalement différentes. Ainsi, les composés C9 (Sulconazole) et C4(Flunarizine) que nous avons identifiés diminuent la réplication de l’AdV, un virus à ADNenveloppé et du virus de Marburg, un virus à ARN, non enveloppé de la famille desFiloviridae. Ces résultats ont permis de valider l’interaction [LP]PxY/Nedd4 comme unecible idéale d’un antiviral à large spectre et de proposer un repositionnement de ces moléculesC9 et C4 comme antiviraux potentiels. Nous avons également synthétisé de nouvellesmolécules analogues du composé C9 et démontré qu’elles étaient tout aussi efficaces que lecomposé lui-même sur la réplication de l’AdV. Ces résultats nous ont permis de présenter laclasse des dérivés imidazolés comme structure de base pour l’élaboration de nouveauxantiviraux, potentiellement à large spectre. / Broad-spectrum antiviral identification is considered as one of the major aims of theactual virology research and one strategy consists in targeting virus/host interaction. Using theAlphaScreen® technology and the adenoviral model protein VI/Nedd4-2, we performed highthroughputbiochemical screening targeting the [LP]PxY/Nedd4 interaction, a commoninteraction of different virus families. We identified candidate inhibitors from a librarycompound approved by health agencies. We tested, characterized and validated the antiviraleffect of those compounds on two very different virus families. Indeed, compounds C9(Sulconazole) and C4 (Flunarizine) decrease replication of the adenovirus, a DNA nonenvelopedvirus and the replication of the Marburg virus, an RNA enveloped virus from theFilovirus family. Taken together, those results permit us to validate the [LP]PxY/Nedd4interaction as good target for a broad spectrum antiviral and to propose the “repositioning” ofcompounds C4 and C9 as antivirals. Moreover, we have synthesized new analogues from C9showing similar effect on AdV replication compared to the original molecule (C9). Inconclusion, our work on developing new broad-spectrum antivirals highlights the possibilityto use imidazole derivatives as a new class of antiviral compounds.