microRNAs (miRNAs) are a class of noncoding RNAs that regulate gene expression. This class of 18-25 nucleotide-long non-coding RNAs has been found to play critical roles in the modulation of a wide spectrum of cellular processes including immunity, development, and metabolism. They modulate their interactions by binding to the 3’ untranslated region of the target messenger RNA to mediate the repression of gene expression. Given their emerging critical roles in the regulation of biological processes, it is not surprising that miRNAs play a significant part in modulating host-virus interactions. Viruses are obligate parasites that hijack the host cellular machinery and processes to promote their life cycle and their propagation. Emerging evidence suggests that miRNAs add an extra regulatory layer to fine-tune viral pathogenesis. This offers novel opportunities not only to delineate the crosstalk between the host and the virus but also allows for the development of novel therapeutics and the identification of novel potential biomarkers of viral infection. Herein, we examine the roles of various miRNAs in the modulation of host-virus interactions. In this thesis, we identify a polycistronic miRNA cluster (miR-183, miR-96, and miR-182) to possess antiviral properties against RNA viruses by augmenting innate immune responses to viral infection. We as well identify miR-383 to possess novel antiviral potential against Dengue virus (DENV), through its targeting of PLA2G4A, a pro-viral host factor essential for the production of infectious particles. Finally, we examine miR-185’s role in the modulation of SARS-CoV-2 infection where we show that miR-185’s regulation of fatty acid and cholesterol metabolism suppresses the virus’s entry and propagation in lung and liver cells. Collectively, the findings in this thesis demonstrate the critical role that miRNAs play in the modulation of host-virus interaction through modifying the host’s cellular environment essential for the regulation of viral pathogenesis.

Doctor of Philosophy / Department of Biology / Stefan Rothenburg / Vaccinia virus is the prototypic poxvirus that has been widely used as a model for investigating poxvirus biology and genetics. Like several members of the Poxviridae family, vaccinia virus can infect several different species including mice, cows and humans. Because the entry of poxviruses into a host cell relies on ubiquitously expressed surface molecules, which are found in many species, the ability of poxviruses to infect and replicate in different host cells primarily depends on their ability to subvert the host’s innate immune response. One critical barrier to infection is overcoming the general shutdown of protein translation initiated by the cellular protein kinase PKR. PKR detects cytoplasmic double-stranded (ds) RNA generated during infection by the replicating virus, which activates it to phosphorylate the alpha-subunit of the eukaryotic translation initiation factor 2 (eIF2) and suppress general translation. Poxviruses are large viruses with dsDNA genomes that encode around 200 genes. Several of these genes are known as host range genes and are important for replication in different host species and many interact with components of the host immune response to promote viral replication. Two genes in vaccinia virus, called E3L and K3L, are known inhibitors of PKR and have previously been shown to be important for virus replication in cells from different species. The molecular explanation behind their host range function, however, is unknown. The main goal of the research presented in this thesis is to determine the molecular mechanisms for the host range function of vaccinia virus E3L and K3L, particularly in different hamster host cells. Along with an analysis of vaccinia virus host range genes, we have used genome-wide comparisons between host-restricted poxviruses in the Leporipoxvirus genus to parse out the potential genomic determinants of host range restriction in this clade of poxviruses. The overarching aim of this thesis work is to better understand the molecular mechanisms for host range in poxviruses.

Seasonal and pandemic Influenza virus infections cause about three to five million cases of severe illness and about 250,000 to 500,000 deaths world-wide annually according to the WHO. Although investigated intensively, Influenza virus pathogenesis is still not very well understood and hard to predict. Influenza A viruses contain a segmented, single-(-) stranded RNA genome encoding at least 10 different proteins and are highly diverse due to hypermutation and reassortment. In previous work, 56 viral genes from six different influenza A virus isolates had been cloned and genome-wide screened for virus-host protein interactions using yeast-two hybrid technology and several human and chicken cDNA libraries, leading to the identification of 127 high-confidence cellular interactors of which 40 have also been identified by RNA interference in other studies. In this thesis, two of the cellular interactors identified which both bound to the viral multifunctional protein NS1, TRBP and PACT, were further investigated with regard to their role in virus life cycle. These two proteins are known to be involved in miRNA silencing and PKR regulation. Both interactions between NS1 and TRBP and NS1 and PACT were confirmed by co-immunoprecipitation, and both TRBP and PACT co-localized with NS1 in a cytosolic compartment. NS1 was also found to be present in the RISC complex in pull-down assays with the RISC core component Ago2. In functional assays, NS1 dose-dependently inhibited RNA silencing. Although no differences in TRBP-binding between NS1 proteins of various different influenza strains could be detected in direct mating Y2H assays, they varied with regard to their inhibitory activity on RNA silencing. TRBP and PACT alone were unable to restore NS1-induced inhibition of RNA silencing activity, however both together restored RNA silencing. Moreover, the siRNA knockdown of PACT abolished the association of NS1 with Ago2, and NS1 competitively inhibited the binding of TRBP and PACT to Ago2. The depletion of either TRBP or PACT led to an inhibition of influenza virus replication. The depletion of TRBP also lifted cellular IFNβ level without infection. However, the knockdown of TRBP but not PACT blocked IFNβ production and increased cell viability post infection. These results indicate that NS1 inhibits the binding of PACT and TRBP to the RISC complex and thereby inhibits miRNA-induced gene silencing. The hypothesis that TRBP supports influenza replication potentially by regulating PKR regulation and IFNβ induction requires further investigation. In conclusion, this study provides evidence for the complexity of virus-host interactions and the dual role of viral proteins in activating both positive and negative regulatory cellular mechanisms.

Biological systems, their physical structure and their functions, are built, maintained, and controlled by the activity of enzymes. Understanding how enzymes contribute to the regulation of various pathways and processes allows us to gain a deeper understanding of the entirety of the biological system. As changes in enzyme activity are often essential for the pathogenesis of multiple and varied diseases, identifying these changes represents a crucial step to both understanding the disease and preventing its progression within the individual. Enzymes’ functional output can be controlled by numerous different mechanisms, including control of transcription and translation, subcellular localisation, co-factor interactions, or chemical modification to specific amino acids. Activity-based protein profiling allows the potential for activity of target enzymes to be measured, thereby gaining a more accurate representation of the functional state of the biological system. In this work, profiling differential enzyme activity allows the discovery of previously unknown links between metabolic regulatory enzymes and infection by the hepatitis C virus (HCV). The novel probe wortmannin-yne is described and is shown to be able to report on the activity multiple kinases, including MAPK1, whose activity is dysregulated during HCV replication. Novel probes designed to target a smaller selection of kinases, phosphatidylinositol kinases, are reported and are shown to be capable of measuring HCV-induced changes to not only kinase activity but also regulatory protein-protein interactions with the phosphoinositide kinases. Lastly, the role of microRNA-27b in the HCV-induced dysregulation of lipid metabolic enzymes is examined. Three novel targets of microRNA-27b are identified, and their dysregulation is shown to have an effect on the life cycle of HCV. Altogether, this work has developed new tools for the study of metabolic enzymes and identified new avenues of investigation into the dysregulation of lipid metabolism.

<p>  </p> <p>Infection by Marburgvirus (MARV) or the closely related Ebolavirus (EBOV) results in potentially lethal hemorrhagic fever in humans characterized by uncontrolled viremia, a systemic pro-inflammatory response, and multi-organ failure. Currently, there are no approved countermeasures to treat or prevent MARV infection, which leaves a critical need for development of antiviral therapies. One approach to develop antiviral therapies is exploit a virus’s dependency on the host cell and disrupt critical human-viral interactions. While multiple studies identified host-viral interactions involved in EBOV infection, we are currently limited in our knowledge of host-viral interactions that occur during MARV infection and how these interactions influence the viral replication cycle. Thus, the purpose of this research was to identify and further characterize the biological significance of human-MARV protein-protein interactions that occur during infection.</p> <p>Here, we used genome-wide yeast two-hybrid (Y2H) screens to identify directly interacting human-viral proteins. We identified 431 putative interactions with MARV and used a combination of a novel NanoLuc Y2H assay and confidence criteria to prioritize a final set of 396 interactions. Bioinformatic analysis revealed that the molecular functions of the interacting human genes were significantly enriched in RNA binding, cell adhesion, and cytoskeleton binding. </p> <p>MARV and EBOV have many similarities in their genomic organization, sequence, and protein structures that could facilitate interactions to common host factors during infection. Thus, to identify shared interactions between these related viruses, we compared the MARV interactions to EBOV interactions identified in a parallel Y2H screen. We identified 145 human proteins targeted by both MARV and EBOV. The majority (77%) of shared interactions occurred between homologous viral proteins. Additional bioinformatic analyses comparing MARV and EBOV interactions revealed that these viruses interact with different host factors with similar molecular functions (RNA binding, DNA binding, actin and microtubule binding. Together, these data support the notion that while MARV and EBOV target common host factors there are still differences in protein interactions that support functions specific to each virus. </p> <p>To investigate the biological significance of the identified interactions, we focused on host interactions with the viral matrix protein, VP40. VP40 is a multifunctional protein that facilitates viral assembly and budding from the host cell. Y2H assays using VP40 mutants revealed that the WW-containing host protein MAGI1 interacted with the late domain of VP40. This interaction was validated in mammalian cells using coimmunoprecipitation and GFP complementation assays. Based on multiple reports of WW-containing host proteins interacting with VP40, we predict that the interaction between MAGI1 and VP40 regulates viral budding.</p> <p>In conclusion, the work presented here successfully identified 396 novel human-MARV interactions, which furthers the field’s understanding of host factors involved in MARV infection. Additionally, we identified interactions shared by MARV and EBOV, which could be beneficial in the development of a broad antiviral therapy against filoviruses. Lastly, we validate the interaction between MAGI1 and VP40, which has a potential role in viral budding </p>

The accessory protein Nef of human immunodeficiency virus type 1 (HIV-1) has long been known to enhance the infectivity of HIV-1 progeny virions. The multipass transmembrane proteins serine incorporator 3 (SERINC3) and SERINC5 were recently identified as novel antiviral proteins that restrict HIV-1 infectivity. Nef enhances HIV-1 infectivity by removing SERINCs from the plasma membrane, which prevents their incorporation into progeny HIV-1 virions. To exploit this potent intrinsic antiretroviral factor for potential therapy development, it is critical to explore the determinants in SERINC5 that govern its downregulation by Nef and its restriction on HIV-1 infectivity. Here I report that the ability to inhibit HIV-1 infectivity is conserved among vertebrate SERINC5 proteins, whereas the sensitivity to downregulation by Nef is not. However, a Nef-resistant SERINC5 became Nef-sensitive when its intracellular loop 4 (ICL4) was replaced by that of Nef-sensitive human SERINC5. Conversely, human SERINC5 became resistant to Nef when its ICL4 was replaced by that of a Nef-resistant SERINC5. In general, ICL4 regions from SERINCs that exhibited resistance to a given Nef conferred resistance to the same Nef when transferred to a sensitive SERINC, and vice versa. I demonstrate that human SERINC5 can be modified to restrict HIV-1 infectivity even in the presence of Nef. Moreover, by generating chimeras between SERINC5 and SERINC2, which does not exhibit antiretroviral activity, I demonstrate that SERINC5’s inhibitory function, unlike the sensitivity to Nef, requires the participation of more than one region. Helix 4 and extracellular loop 5 (ECL5) of SERINC5 are both required for the potent restriction of HIV-1 infectivity. In contrast, a large amino-terminal portion of SERINC5 is not required for its antiretroviral activity of SERINC5. The determinants in ECL5 disperse throughout the loop. Furthermore, the ECL5 of SERINC5 is a hotspot region that determines the Env-dependent antiretroviral activity of SERINC5.

Le virus du syndrome reproducteur et respiratoire porcin (VSRRP) est un pathogène de grande importance dans l'industrie porcine car il peut entraîner des pertes économiques. L'une des lignées cellulaires couramment utilisée pour la recherche et la production de vaccins est MARC-145 (cellules rénales de singe vert africain). Les interactions moléculaires entre les cellules hôtes et le virus sont essentielles pour comprendre le mécanisme par lequel le virus utilise la machinerie cellulaire pour se répliquer et infecter les cellules voisines. Notre objectif était d'analyser les changements protéomiques produits lors de l'infection par le VSRRP chez les cellules MARC-145, y compris la composition des virions et des exosomes produits par les cellules infectées. Les surnageants des cellules infectées et non infectées ont été purifiés pour obtenir les virions et exosomes des cellules hôtes. Par la suite, les protéines extraites ont été analysées par spectrométrie de masse à haute résolution quadripolaire-hybride-Orbitrap, et classées selon la fonction moléculaire et la localisation subcellulaire. Le besoin d'obtenir des données protéomiques fiables a conduit au prochain objectif : optimiser l'infection des cellules MARC-145 par le VSRRP. Pour évaluer l'efficacité de l'infection, nous avons synchronisé l'infection en utilisant un virus marqué avec une protéine fluorescente verte améliorée (eGFP) et en ajoutant des polycations à différentes concentrations pour stimuler la liaison des particules virales à la cellule. Pour vérifier le pourcentage de cellules infectées, nous avons utilisé la microscopie à fluorescence et la cytométrie en flux. Nos résultats suggèrent que les protéines cellulaires affectées au cours de l'infection par le VSRRP pourraient jouer un rôle important dans la réponse immunitaire de l'hôte et / ou le cycle de vie viral. L’efficacité de l'utilisation de polycations pour augmenter l'infection par le VSRRP a été démontrée. / Porcine reproductive and respiratory virus (PRRSV) is a pathogen of high importance in the porcine industry because it can lead to significant economic losses. One of the cell lines routinely used for research and vaccine production is MARC-145 (African green monkey kidney cells). Molecular interactions between the host cells and the virus are essential to understand how the virus uses the cell machinery to replicate and infect neighbouring cells. Our goal was to analyze the proteomic changes involved during the PRRSV infection in MARC-145 cells, including the composition of the infected cells' virions and exosomes. The infected and non-infected cells' supernatants were purified to obtain the host cells' virions and exosomes. Extracted proteins were further analyzed by High-Resolution-Quadrupole-Hybrid-Orbitrap mass spectrometry and classified according to molecular function and subcellular localization. The need for obtaining reliable proteomics data led to the next goal of optimizing the infection of MARC-145 cells with PRRSV. To assess the efficiency of the infection, we synchronized the infection, used a virus tagged with an enhanced green fluorescent protein (EGFP) and added polycations at different concentrations to stimulate the binding of the viral particles to the cell. To verify the percentage of infected cells, we used fluorescence microscopy and flow cytometry. Our findings suggest the cellular proteins affected during the PRRSV infection could play important roles in host immune response and/or the viral life cycle. The efficiency of the use of polycations was demonstrated to be effective in increasing PRRSV infection.