Master of Science in Biomedical Sciences / Department of Anatomy and Physiology / Yongming Sang / Animal metabolic and immune systems integrate and inter-regulate to exert effective immune responses to distinct pathogens. The signaling pathway mediated by mechanistic target of rapamycin (mTOR) is critical in cellular metabolism and implicated in host antiviral responses. Recent studies highlight the significance of the mTOR signaling pathway in the interferon (IFN) response. Type I IFNs mediate host defense, particularly, against viral infections, and have myriad roles in antiviral innate and adaptive immunity. In addition to their well-known antiviral properties, type I IFNs also affect host metabolism. However, little is known about how animal type I IFN signaling coordinates immunometabolic reactions during antiviral defense. Therefore, understanding the interaction of mTOR signaling and the type I IFN system becomes increasingly important in potentiating antiviral immunity. Tissue macrophages (MФs) are a primary IFN producer during viral infection, and their polarization to different activation statuses is critical for regulation of immune and metabolic homeostasis. Using porcine reproductive and respiratory syndrome virus (PRRSV) as a model, we found that genes in the mTOR signaling pathway were regulated differently in PRRSV-infected porcine alveolar MФs at different activation statuses. Therefore we hypothesize that: 1) the mTOR signaling pathway involves host anti-PRRSV regulation; 2) mTOR signaling interacts with IFN signaling to modulate the antiviral response; and 3) different type I IFN subtypes (such as IFN-α1 and IFN-β) regulate mTOR signaling differently. We show that modulation of mTOR signaling regulated PRRSV infection in MARC-145 cells and porcine primary cells, in part, through regulating production and signaling of type I IFNs. In addition, expression and phosphorylation of two key components in the mTOR signaling pathway, AKT and p70 S6 kinase, were regulated by type I IFNs and PRRSV infection. Taken together, we determined that the mTOR signaling pathway, a key pathway in regulation of cell metabolism, also mediates the type I IFN response, a key immune response in PRRSV infection. Our findings reveal that the mTOR signaling pathway potentially has a bi-directional loop with the type I IFN system and implies that some components in the mTOR signaling pathway can serve as targets for augmentation of antiviral immunity and therapeutic designs.

RNA interference (RNAi) is a pathway, which employs Dicer to process long double stranded RNAs (dsRNA) from endogenous or exogenous sources into short interfering RNAs (siRNA). siRNAs are loaded onto Argonaute proteins to mediate sequence-specific post-transcriptional RNA targeting resulting in regulation of protein-coding genes and retrotransposons or antiviral immune response. Another small RNA pathway - PIWI-associated RNA (piRNA) pathway is suppressing retrotransposons in the germline. In mice, canonical RNAi pathway activity is negligible in somatic cells where a full-length Dicer produces gene-regulatory microRNAs (miRNA) but RNAi is highly active in oocytes, which express a truncated oocyte-specific Dicer isoform (DicerO ). DicerO lacks an N-terminal DExD helicase domain and has higher cleavage activity of long dsRNAs. Deletion of oocyte specific DicerO promoter leads to transcriptome aberrations, which include upregulation of putative RNAi targets and MT retrotransposons and, consequently, to meiotic spindle defects and female sterility. In contrast, the piRNA pathway is non-essential in mouse oocytes, potentially because of overlapping functions of RNAi. The PhD thesis aims to understand biological significance of mammalian endogenous RNAi and to explore consequences of re-activated RNAi...

Honey bees are economically important pollinators of a wide variety of crops that have attracted the attention of both researchers and the public alike due to unusual declines in the numbers of managed colonies in some parts of the world. Viral infections are thought to be a significant factor contributing to these declines, along with exposure to agricultural and apicultural pesticides, but viruses have proven a challenging pathogen to study in a bee model and interactions between viruses and the bee antiviral immune response remain poorly understood. Recent studies have demonstrated an important role for inwardly-rectifying ATP-sensitive potassium (KATP) channels in the cardiac regulation of the fruit fly antiviral immune response, but no information is available on their role in the heart-specific regulation of bee immunity. The results of this work demonstrate that KATP channel modulators have an observable effect on honey bee heart rate that supports their expected physiological role in bee cardiac function. Here, it is also reported that the entomopathogenic flock house virus (FHV) infects adult bees, causing rapid onset of mortality and accumulation of viral RNA. Furthermore, infection-mediated mortality can be altered by pre-exposure to KATP channel modulators. Finally, this work shows that exposure to environmental stressors such as commonly used in-hive acaricides can impact bee cardiac physiology and tolerance to viral infection. These results suggest that KATP channels provide a significant link between cellular metabolism and the antiviral immune response in bees and highlight the significant impact of environmental stressors on pollinator health. / Ph. D.

PIDD has previously been described as a death domain (DD)-containing protein that is inducible upon p53 activation and plays a role in programmed cell death. It has previously been shown that PIDD interacts with RAIDD (RIP-associated ICE/CED3 homologous protein with a death domain) in a cytoplasmic complex known as the PIDDosome, which results in the activation of capsase-2 and ultimately in cell death in response to DNA damage. Despite earlier studies on PIDD, however, the physiological role of PIDD has not been elucidated. Thus, we have generated PIDD-deficient mice and examined its in vivo functions particularly in cell death and in antiviral innate immunity. The first major aim of the thesis is to determine whether or not PIDD is required in cell death. PIDD mice are developmentally normal and do not display a pronounced phenotype. Surprisingly, PIDD deficiency perturbed neither DNA damage-induced nor stress-induced cell death in a variety of cell types, suggesting that PIDD may not play a critical role in cell death. In addition, caspase-2 processing occurred normally in the absence of PIDD in response to ionizing irradiation or etoposide treatment, indicating that PIDD is dispensable in the cleavage of caspase-2. The second major aim is to examine the role of PIDD and RAIDD in LCMV-induced innate immunity. To study the role of PIDD and RAIDD in antiviral immune responses, I have generated PIDD/RAIDD double-deficient mice and challenged them with lymphocytic choriomeningitis virus (LCMV). Interestingly, I observed that ablation of both PIDD and RAIDD together resulted in defective viral clearance in the spleen, but not in other organs including the lung, liver, and kidney. In addition, the production of type I IFN was also decreased in the mice deficient in both PIDD and RAIDD. However, the cytotoxicity of the T lymphocytes was largely intact in the absence of both PIDD and RAIDD. Collectively, our results suggest that PIDD is dispensable in cell death, yet PIDD and RAIDD together have a synergistic effect in LCMV-induced antiviral innate immunity. The findings presented in this thesis provide a better understanding of the physiological role of PIDD and may ultimately contribute to the novel therapeutic strategies for the proper control of viral infection.

Dans les organismes multicellulaires, la régulation de l’expression des gènes par les microARNs est un mécanisme essentiel pour le développement cellulaire et l’homéostasie. De plus, le rôle des microARNs a été démontré dans de nombreux processus immunitaires, tels que l’inflammation. Les virus évoluant conjointement avec leurs hôtes, ils ont appris à détourner la machinerie cellulaire pour leur propre bénéfice. Ainsi, des microARNs codés par certains génomes viraux ont été mis en évidence, mais leurs fonctions, ainsi que leurs cibles, restent encore largement inconnues. En utilisant une lignée de souris présentant une mutation hypomorphe pour le gène dicer, caractérisée par une diminution de la production des microARNs, et son hôte naturel, le cytomégalovirus murin, un virus membre de la famille des β-Herpesvirus, nous avons étudié le rôle potentiel des microARNs d’origine cellulaire et virale dans la pathogénèse de ce virus. Lors de l’infection aigüe, nos résultats montrent un rôle dominant et protecteur des microARNs cellulaires, comparé à celui des microARNs viraux, prédits pour être des facteurs de pathogénicité. / In multicellular organisms, gene expression regulation by microRNAs is an essential mechanism for cell development and homeostasis. Moreover, several immune-related processes, such as inflammation, have been demonstrated to require specific microRNAs. As viruses have coevolved with their host, they have learned to hijack the cellular defenses for their own benefit. Thus microRNAs-encoding genes were also recently discovered in the genome of Herpesviruses, but up to now, the function and the targets of most microRNAs of viral origin are still largely unknown. Using a hypomorphic mouse mutant line, characterized by a diminished production of microRNAs, and the Mouse Cytomegalovirus, a natural pathogen of mice which belongs to the family of β-Herpesviruses, we investigated the potential roles of microRNAs of both cellular and viral origin in the pathogenesis of this virus. Our results point toward a dominant role of cellular microRNAs as protective factors compared to virally-derived microRNAs which are usually predicted to carry pathogenic functions in acute infections.

Dans une ère où les moustiques modifiés commencent à être utilisés ou envisagés pour contrôler les épidémies de Dengue ou malaria, le manque de connaissance sur l’immunité des insectes vecteurs envers certains pathogènes se fait cruellement ressentir. Pourtant la possibilité de changements de vecteurs, dû à un changement de leur immunité, provoquée par l’Homme est réelle. Pour déterminer la contribution de l’immunité dans différents compartiments du vecteur contre divers pathogènes avons étudié la réponse antivirale dans la première barrière de transmission chez le moustique vecteur de la malaria après une infection par un repas sanguin. Nous montrons que les réponses antivirales sont différentes entre compartiments, et proposons un modèle où des interactions tripartites entre le virus, l’immunité du moustique et la flore entérique interagissent pour contrôler l’infection précoce du moustique après le repas sanguin. De façon surprenante, nous avons également montré que la voie de l’ARN interférence n’a pas d’effet antiviral dans ce compartiment. Nous suggérons que cette voie est utilisée par le parasite Plasmodium pour détourner la réponse antiparasitaire médiée par Toll, grâce à un facteur de virulence de nature ARN double brin. Nous avons également montré que des biais expérimentaux lors de l’infection des insectes ont conduit à l’élaboration d’un dogme disant que la voie de l’ARN interférence est la voie antivirale principale des insectes, mais nos resultats suggèrent que malgré l’importance de cette voie pour controler l’intensité de la réplication virale lors de l’infection disséminée, cette voie n’a aucune fonction antivirale lors de l’infection initiale du tube digestif. Néanmoins, le séquençage des produits de cette voie permet d’assembler de-novo des génomes de virus commensaux. Les résultats de ces travaux montrent très clairement qu’il faut évaluer le rôle et l’impact de toute modification d’insectes vecteurs pour plusieurs classes de pathogènes. Cela ouvre également de nombreux nouveaux champs de recherches et pose de nombreuses nouvelles questions. / In an era where modified mosquitoes are starting to be used in nature for controlling malaria and Dengue, lack of knowledge about immunity of mosquito vectors to some pathogen classes are becoming more evident. The risks for human-provoked vector shifts of pathogens transmission have not been examined. To fill these gaps, we assessed the antiviral immunity of the malaria vector, Anopheles gambiae, in the strongest mosquito bottleneck for pathogens, the midgut infection barrier after an infective bloodmeal. This work shows that the antiviral responses are different and highly compartmentalized between the midgut and systemic immunity. We propose a model where tripartite interactions between virus, mosquito immunity and enteric flora control early arboviral infection in the midgut. Surprisingly, we showed that while the siRNA pathway had no evident anti-arbovirus activity in the midgut, it was used by Plasmodium to evade mosquito immunity. A virus-like elicitor of double strand RNA nature is transferred from the parasite at the ookinete stage to the mosquito midgut cells, resulting in a shift of immune balance from anti-parasite response to an antiviral-like response. Importantly, our work shows that biases in experimental infection methods led to the misconstruction of a dogma stating that siRNA is the main antiviral pathways in insects, at least in the midgut compartment. And that the use of the pathway products can be successfully used to de-novo assemble previously unknown viruses from the virome. This work indicates that immune modifications in vectors need to be evaluated for changes of vectorial competence to different pathogens. It also opens numerous avenues of research and raises a lot of interesting questions that will need to be investigated in the future.

Les arbovirus (virus transmis par des arthropodes) sont à l'origine de maladies humaines telles que la dengue, le chikungunya ou encore le Zika. Le moustique Aedes aegypti, est le vecteur majeur de ces trois arbovirus. La faible efficacité des méthodes de contrôle des populations de moustiques, principalement réalisées au moyen d'insecticides chimiques ouvre un champ de développement de nouvelles approches en lutte antivectorielle. Le moustique, hôte vecteur, contrôle la réplication virale en limitant les réponses immunitaires antivirales. La machinerie RNA interférence (RNAi) est la voie jouant un rôle majeur dans l'immunité antivirale chez le moustique. Alors que le rôle des deux voies, siRNA (" small interfering RNA ") et piRNA (" piwi-interfering RNA "), est de mieux en mieux compris dans les réactions antivirales du vecteur, peu de connaissances sont disponibles à ce jour en ce qui concernent les interactions entre la voie miRNA (" micro RNA ") et les arbovirus. Ainsi, nous proposons une analyse détaillée des mécanismes par lesquels les miARN tentent de réguler la réplication virale chez le moustique. Dans la première partie de la thèse, nous avons effectué une analyse génomique pour identifier les miRNAs pouvant interagir chez Ae. aegypti avec divers lignées/génotypes des virus chikungunya (CHIKV), de dengue (DENV) et de Zika. Avec l'aide d'outils de prédiction faisant appel à divers algorithmes, plusieurs sites de liaison de miARN avec différents lignées/génotypes de chaque arbovirus ont été identifiés. Nous avons ensuite sélectionné les miARN pouvant cibler plus d'un arbovirus et nécessitant un faible seuil d'énergie lors de la formation des complexes entre l'ARNm. / Mosquito-borne arboviruses cause some of the world’s most devastating diseases and are responsible for recent dengue, chikungunya and Zika pandemics. The yellow-fever mosquito. Aedes aegypti, plays an important role in the transmission of all three viruses. The ineffectiveness of chemical control methods targeting Ae. aegypti makes urgent the need for novel vector-based approaches for controlling these diseases. Mosquitoes control arbovirus replication by triggering immune responses. RNAi machinery is the most significant pathway playing a role on antiviral immunity. Although the role of exogenous siRNA and piRNA pathways in mosquito antiviral immunity is increasingly better understood, there is still little knowledge regarding interactions between the mosquito cellular miRNA pathway and arboviruses. Thus further analysis of mechanisms by which miRNAs may regulate arbovirus replication in mosquitoes is pivotal. In the first part of the thesis, we carried out genomic analysis to identify Ae. aegypti miRNAs that potentially interact with various lineages and genotypes of chikungunya (CHIKV), dengue (DENV) and Zika viruses. By using prediction tools with distinct algorithms, several miRNA binding sites were commonly found within different genotypes/and or lineages of each arbovirus. We further analyzed the miRNAs that could target more than one arbovirus and required a low energy threshold to form miRNA-vRNA (viral RNA) complexes and predicted potential RNA structures using RNAhybrid software. Thus, we predicted miRNA candidates that might participate in regulating arboviral replication in Ae. aegypti. In the second part of the thesis, we developed a miRNA-based approach that results in a dual resistance phenotype in mosquitoes to dengue serotype 3 (DENV-3) and chikungunya (CHIKV) viruses for stopping arboviruses spreading within urban cycles. The target viruses are from two distinct arboviral families and the antiviral mechanism is designed to function through the endogenous miRNA pathway in infected mosquitoes. Ten artificial antiviral 4 miRNAs capable of targeting ~97% of all published strains were designed based on derived consensus sequences of CHIKV and DENV-3. The antiviral miRNA constructs were placed under control of either an Aedes PolyUbiquitin (PUb) or Carboxypeptidase A (AeCPA) gene promoter triggering respectively expression ubiquitously in the transgenic mosquitoes or more locally in the midgut epithelial cells following a blood meal. Challenge experiments using viruses added in blood meals showed subsequent reductions in viral transmission efficiency in the saliva of transgenic mosquitoes as a result of lowered infection rate and dissemination efficiency. Several components of mosquito fitness, including larval development time, larval/pupal mortality, adult lifespan, sex ratio, and male mating competitiveness, were examined: transgenic mosquitoes with the PUb promoter showed minor fitness costs at all developing stages whereas those based on AeCPA exhibited a high fitness cost. Further development of these strains with gene editing tools could make them candidates for releases in population replacement strategies for sustainable control of multiple arbovirus diseases.

Interferon (IFN) regulatory factor 7 (IRF7) plays a key role in the production of IFN‐α in response to viral infection, and phosphorylation at IRF7 C‐terminal serine sites is prelude to its function. However, phosphatases that negatively regulate IRF7 phosphorylation and activity have not been reported. In this study, we have identified a conserved protein phosphatase 1 (PP1)‐binding motif in human and mouse IRF7 proteins, and shown that PP1 physically interacts with IRF7. Exogenous expression of PP1 subunits (PP1α, β, or γ) ablates IKKε‐stimulated IRF7 phosphorylation and dramatically attenuates IRF7 transcriptional activity. Inhibition of PP1 activity significantly increases IRF7 phosphorylation and IRF7‐mediated IFN‐α production in response to Newcastle disease virus (NDV) infection or Toll‐like receptor 7 (TLR7) challenge, leading to impaired viral replication. In addition, IFN treatment, TLR challenges and viral infection induce PP1 expression. Our findings disclose for the first time a pivotal role for PP1 in impeding IRF7‐mediated IFN‐α production in host immune responses.

No description available.

Mosquito-borne diseases remain a significant burden on global public health. Maintenance of mosquito-borne viruses in nature requires a biological transmission cycle that involves alternating virus replication in a susceptible vertebrate and mosquito host. Although infection of the vertebrate host is acute and often associated with disease, continual transmission of these viruses in nature depends on the establishment of a persistent, nonpathogenic infection in the mosquito vector. It is well known that invertebrates rely on small RNA pathways as an adaptive antiviral defense. The canonical antiviral response in these organisms involves dicer enzymes that cleave viral double-stranded RNA replicative intermediates (RIs) into small interfering RNAs (siRNAs; ~21-24 nucleotides). One strand of the siRNA duplex guides the targeting and destruction of complementary viral RNAs when loaded and retained in a multi-protein complex called the RNA-induced silencing complex. Here, we show that mosquito vectors mount a redundant double defense against virus infection mediated by two different small RNA pathways. Specifically, we demonstrate that in addition to a canonical antiviral response mediated by siRNAs, virus infection of the mosquito soma also triggers an antiviral immune pathway directed by ping-pong-dependent PIWI-interacting RNAs (piRNAs; ~24-30 nucleotides). The complexity of mosquito antiviral immunity has important implications for understanding how viruses both induce and modulate RNA-silencing responses in mosquito vectors. In mammals, viral RIs induce a range of relatively nonspecific antiviral responses. However, it remains unclear if viral RIs also trigger RNA silencing in mammals. Mosquito-borne viruses represent an ideal model for addressing this question as their transmission cycles involve alternating replication in mammalian and invertebrate hosts. Although we report identifying a subset of virus-derived small RNAs that appear to be products of RNA silencing in two mammalian cell lines infected with the mosquito-borne chikungunya virus (CHIKV), our studies suggest these small RNAs have little biological relevance in combating virus infections. Thus, while the accumulation of virus-derived siRNAs is essential to the survival of mosquitoes infected with CHIKV, they appear to have little functional significance in mammalian antiviral immunity. / Ph. D.