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1
Zwart, Lizahn. „Investigating two AHSV non-structural proteins : tubule-forming protein NS1 and novel protein NS4“. Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/62198.
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African horse sickness is an equid disease caused by African horse sickness virus (AHSV). AHSV produces seven structural proteins that form the virion and four non-structural proteins with various roles during replication. The first part of this study investigated the intracellular distribution and co-localisations of NS1 with other AHSV proteins to facilitate its eventual functional characterisation. Confocal microscopy revealed that NS1 formed small cytoplasmic foci early after infection that gradually converged into large fluorescent NS1 tubule bundles. Tubule bundles were more organised in AHSV-infected cells than in cells expressing NS1 alone, suggesting that tubule bundle formation requires the presence of other AHSV proteins or regulation of NS1 expression rates. NS1 occasionally co-localised with VP7 crystalline structures, independently of other AHSV proteins. However, when NS1-eGFP, a modified NS1 protein that contains enhanced green fluorescent protein (eGFP) near the C-terminus, was co-expressed with VP7, co-localisation between these proteins occurred in most co-infected cells. It is not clear how the addition of eGFP to NS1 induces this co-localisation and further investigation will be required to determine the function of NS1 during viral replication.
The second part of the study focused on characterising the novel non-structural AHSV protein NS4. The NS4 open reading frame (ORF) occurs on segment 9, overlapping the VP6 ORF in a different reading frame. In silico analysis of segment 9 nucleotide and NS4 predicted amino acid sequences revealed a large amount of variation between serotypes, and two main types of NS4 were identified based on these analyses. These proteins differed in length and amino acid sequence and were named NS4-I and NS4-II. Immunoblotting confirmed that AHSV NS4 is translated in AHSV infected insect and mammalian cells, and also in Sf9 insect cells infected with recombinant baculoviruses that overexpress the genome segment 9 proteins, VP6 and NS4. Confocal microscopy showed that NS4 localised to both the cytoplasm and nucleus, but not the nucleolus, in AHSV-infected cells and recombinant baculovirus infected Sf9 cells. Nucleic acid protection assays using bacterially expressed purified NS4 showed that both types of NS4 bind dsDNA, but not dsRNA. This was the first study to focus on AHSV NS4. Future work will focus on determining the role of non-structural proteins in viral pathogenesis, and will involve the use of a reverse genetics system for AHSV.Dissertation (MSc)--University of Pretoria, 2013.
Genetics
MSc
Unrestricted
2
Tai, Hung, und 戴雄. „The role of the non-structural protein, NS1, in influenza virus replication“. Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44660303.
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3
Evans, Johanna. „Characterisation of the NS1 and the NS2 non-structural protein genes of human respiratory syncytial virus (HRSV)“. Thesis, University of Warwick, 1994. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.283482.
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4
Thompson, Catherine Isabelle. „Protein interaction studies on the rotavirus non-structural protein NSP1“. Thesis, University of Warwick, 1999. http://wrap.warwick.ac.uk/80266/.
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Rotavirus encodes six structural and six non-structural proteins. In contrast to the structural proteins, the functional roles of the non-structural proteins are not well defined beyond a realisation that they must have a role in the viral replication cycle. A fuller understanding of the replication cycle must therefore rest on determining the specific roles played by the non-structural proteins. Non-structural protein NSP1 shows high levels of sequence divergence. A generally well conserved cysteine-rich region at the amino-terminus may form a zinc finger structure. It has been shown to possess non-specific RNA-binding activity, and has been found associated with the smallest of three replication intermediates (RIs) found in infected cells, together with the viral proteins VP1, VP3 and NSP3. VP2 and VP6 are added sequentially to the pre-core RI to form the core RI and single-shelled RI respectively. The function of NSP1 in the replication cycle and the importance of its presence in early replication complexes has not been determined. The intermolecular interactions that occur between the components of the RIs have not been defined. Protein-protein interactions between NSP1 and VP1, VP2, VP3, and NSP3, from the UKtc strain of bovine rotavirus, were investigated using a variety of approaches, the first of which was the yeast two-hybrid system. In this assay a self-interaction of NSP1 was not detected. Protein-protein interactions between NSPl and VPl, VP2, VP3, and NSP3, were also not detected. Both the full-length protein and a truncated NSPl, consisting of only the amino terminal third of the protein, were tested. A direct self-interaction of NSP3 was shown and quantified. Radio-immunoprecipitation analysis of in vitro translated viral proteins using specific anti-NSP1 serum was also employed. However, it failed to detect direct protein-protein interactions between NSP1 and VPI, VP2, and VP3. Immunoprecipitation of UKtc rotavirus-infected celllysates with anti-NSP1 serum showed the co-precipitation of viral proteins VPl, VP2, VP3NP4, VP6 and NSP3, with NSP1. It was proposed that NSP1 formed a previously unrecognised complex with these proteins. Immunoprecipitation of nuclease-treated infected cell lysates showed a reduction in the co-precipitation of VP2, VP3NP4 and NSP3 with NSP1. No reduction in the co-precipitation of VP6 was seen. The association of the complex proteins may be mediated by RNA binding. Immunoprecipitation with an anti-VP6 monoclonal antibody reciprocally precipitated small amounts of NSP1, VP2, VP3/VP4, and NSP3, with VP6.
5
Whittington, Christi Leigh. „Molecular Dynamics of the RNA Binding Cavity of Influenza A Non-structural Protein 1 (NS1) RNA Binding Domain“. Scholar Commons, 2012. http://scholarcommons.usf.edu/etd/4256.
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Molecular dynamics simulations were performed on the influenza A non-structural protein 1 (NS1) RNA binding domain (RBD), a homodimer. Fourteen simulations were performed at 298K, nine ionized with 0.1M KCl and five with no ions. Several analysis techniques were employed to study RBD residue flexibility. The focus of the study was the RNA binding cavity formed by side chains of helix 2 (chain A) and helix 2’ (chain B) and cavity intermonomeric salt bridges. Opening of the salt bridges D29–R46’ and D29’–R46 was observed in several of the trajectories. The RNA binding cavity has large flexibility, where the dimension and shape change during the dynamics. One pair of residues surrounding the cavity and necessary for RNA binding, residues R38 and R38’, have motions during the simulations which cover the top of the cavity. There is correlation between the salt bridge breaking, flexibility of R38 and R38’, and the cavity size and shape changes. Possible RBD small molecule drug targets are these two salt bridges and the pair R38 and R38’. Disrupting the events that occur around these areas could possibly inactivate RNA binding function of the domain. These results could have implications in searching for potential molecules that effectively treat influenza A.
6
Monastyrskaya, Katherine Valerie. „Characterisation and modification of non-structural protein NS1 of BTV-10 in relation to virus-specified tubule formation“. Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.282517.
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7
To, Thuan. „PDZ Binding Motif of NS1 Proteins of Influenza A Viruses: : A Virulent Factor in the Expression of Interferon-β?“ Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-177337.
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Background: The PDZ domain is a peptide sequence of 80-90 amino acids and can be found in e.g. bacteria, animals and plants. These domains are commonly part of the cytoplasmic and membrane adapter proteins and its function are important in protein-protein interactions. The NS1 proteins of influenza A viruses play an important role in inhibiting the IFN-β production in many ways. In the C-terminus of the NS1 protein, a peptide sequence of four amino acids had been demonstrated to bind to the PDZ domain termed as PDZ binding motif (PBM). Objective: The aim of this study is to determine whether the PBM sequence of the NS1 protein of influenza A virus plays a key roll in the expression of interferon-β. Methods: The open reading frame of the NS1 protein was amplified and cloned into expressing vector and transfected into A549 cells along with a reporter plasmid containing ISRE promoter, driving expression of firefly luciferase. Dual luciferase reporter assay was performed to measure luciferase activity which represented expression of IFN-β. The assay was performed only once and unfortunately the result can not be trusted since the negative control showed positive value. Therefore, to understand the interaction between the PBM sequence of NS1 proteins and the production of IFN-β, further experiments are needed.
8
Lymperopoulos, Konstantinos. „Functional characterisation of the bluetongue virus non-structural protein NS2-protein and RNA-protein interactions“. Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424733.
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9
Horscroft, Nigel John. „Orbivirus non-structural protein NS2 : its role in virus replication“. Thesis, University of Oxford, 1997. http://ora.ox.ac.uk/objects/uuid:9b550db6-dd9d-4127-941f-93eab2b6e038.
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10
Lacheiner, Karen. „Tubules composed of non-structural protein NS1 of african horsesickness virus as a system for the immune display of foreign peptides“. Pretoria : [s.n.], 2006. http://upetd.up.ac.za/thesis/available/etd-07092008-103908.
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11
Rezelj, Veronica Valentina. „Characterization of the non-structural (NSs) protein of tick-borne phleboviruses“. Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8149/.
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In recent years, a number of newly discovered tick-borne viruses exhibiting a wide spectrum of diseases in humans have been ascribed to the Phlebovirus genus of the Bunyaviridae family. These viruses have a tripartite RNA genome composed of two negative-sense RNA segments (medium and large) and one ambisense segment (small), which encode four structural proteins and one non-structural protein (NSs). The NSs protein is the major virulence factor of bunyaviruses, and acts as an antagonist of a key component of the first line of defence against viral infections: the interferon (IFN) system (Bridgen et al., 2001; Weber et al., 2002). The work presented herein describes the characterization of tick-borne phlebovirus NSs proteins as IFN antagonists. The development of a reverse genetics system for the apathogenic tick-borne Uukuniemi phlebovirus (UUKV) enabled the recovery of infectious UUKV entirely from cDNA. A recombinant UUKV lacking NSs induced higher amounts of IFN in infected cells compared to wild-type UUKV, suggesting a role of NSs in modulating the IFN response. The weak IFN antagonistic activity of UUKV NSs was evident using transient transfection reporter assays in comparison to the NSs protein of either pathogenic Heartland virus (HRTV) or Severe fever with thrombocytopenia syndrome virus (SFTSV). The sensitivity of UUKV, HRTV and SFTSV to exogenous and virus-induced IFN, as well as their growth kinetics in IFN-competent cells were examined. The molecular mechanisms employed by UUKV, HRTV and SFTSV NSs proteins to evade antiviral immunity were investigated using reporter assays, immunofluorescence, and immunoprecipitation studies. Collectively, these assays showed that UUKV NSs was able to weakly inhibit IFN induction but not IFN signalling, through a novel interaction with MAVS (mitochondrial antiviral signalling protein). On the other hand, HRTV and SFTSV NSs proteins potently inhibited IFN induction through an interaction with TBK1, and type I but not type II IFN signalling via an interaction with STAT2. Finally, the development of a minigenome system for HRTV in conjunction with minigenomes developed for UUKV and SFTSV (Brennan et al., 2015) provided preliminary data to assess possible outcomes of tick-borne phlebovirus reassortment. In summary, the results described in this thesis offer insights into how tick-borne phlebovirus pathogenicity may be linked to the capacity of their NSs proteins to block the innate immune system. The data presented also illustrate the plethora of viral immune evasion strategies utilized by emerging phleboviruses, and provide an insight into the possibility of tick-borne phlebovirus reassortment.
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Thomas, Claire Philippa. „The expression of bluetongue virus non-structural protein NS2 and its structure-function relationship“. Thesis, University of Oxford, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292328.
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13
Furnon, Wilhelm. „La protéine non-structurale NS1 du virus West Nile : étude fonctionnelle et cible potentielle de nouvelles molécules antivirales“. Thesis, Lyon, 2018. http://www.theses.fr/2018LYSE1008/document.
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Parmi les virus émergents transmis par des moustiques (arbovirus), le genre flavivirus est fortement représenté avec les virus Dengue, Zika, et le virus West Nile (WNV). Le WNV est responsable de nombreux cas de maladies neuroinvasives sévères, parfois mortelles, chez l'humain et les chevaux. Ce virus représente donc un problème de santé publique humaine et animale. Il n'existe pour le moment aucun vaccin humain ni aucun traitement spécifique anti-WNV.Parmi les déterminants viraux essentiels à l'infection par les flavivirus, la glycoprotéine non-structurale NS1 possède des propriétés multifonctionnelles. La forme sNS1, sécrétée dans le milieu extracellulaire, est fortement impliquée dans la dérégulation du système immunitaire de l'hôte. Ces mécanismes participent à l'évasion du virus à la réponse antivirale et, paradoxalement, à la pathogenèse observée dans les formes sévères de la maladie. L'essentiel de ces données concernant le virus de la Dengue, nous souhaitions étudier les propriétés fonctionnelles, in vitro, de la protéine sNS1WNV au cours de l'infection de cellules épithéliales, gliales et neuronales de mammifères. En effet, la structure des protéines sNS1 de flavivirus étant très similaire, notre hypothèse suppose un rôle de sNS1WNV dans les infections neuroinvasives.Si la protéine sNS1WNV ne semble pas moduler les étapes de l'infection virale, elle est cependant à l'origine d'un remodelage du cytosquelette d'actine dans les cellules épithéliales. Elle est aussi impliquée dans l'activation de voies antivirales chez les cellules neuronales non infectées. D'autre part, en ciblant sNS1 et la protéine d'enveloppe E du WNV, nous avons pu isoler, par criblage de molécules aRep (protéines artificielles à motifs répétés), des ligands de haute affinité pour ces déterminants viraux. Ces nouvelles molécules, capables de se lier spécifiquement aux protéines sNS1 et E, ont le potentiel pour servir de base au développement de nouveaux outils de diagnostics et d'agents thérapeutiques antivirauxAmong emerging mosquito-borne viruses (arboviruses), flaviviruses like Dengue, Zika and West Nile virus (WNV) are very often involved in outbreaks. WNV causes several neuroinvasive diseases, which can be lethal, in humans and horses each year. This virus is a threat for both, human and animal public health. Furthermore, there is no human vaccine currently or any specific antiviral treatments against WNV.Among viral factors which are essential for flavivirus infection, the nonstructural glycoprotein NS1 is a multifunctional protein. The secreted form sNS1, is released in the extracellular medium from infected cells and is strongly involved in immune system dysregulation. The functions of sNS1 play roles in immune escape and, paradoxically, in pathogenesis which is observed in severe forms of the disease. Because most of this data are about Dengue Virus, we would like to study, in vitro, functional properties of the sNS1WNV during infection of epithelial, glial and neuronal mammalian cells. Based on the high sNS1 protein structure similarities among flaviviruses, our hypothesis suggests a role of sNS1WNV in neuroinvasive infections.The sNS1WNV protein doesn’t seem to modulate viral infection steps. However, it is involved in actin cytoskeleton remodeling in epithelial cells. sNS1WNV is also involved in the activation of antiviral response pathways in non-infected neuronal cells. On the other hand, by targeting sNS1 and envelope protein E of WNV, we performed a screening of aRep molecules (artificial proteins with alphahelicoïdal repeats) and isolated ligands with high affinity for these viral factors. Because this new type of molecules is able to specifically bind to sNS1 and E, they have potential to be used for the development of new diagnostic tools and antiviral therapeutic agents
14
Wardell, Andrew D. „Expression and characterisation of the hepatitis C virus non-structural protein 3“. Thesis, Imperial College London, 1999. http://hdl.handle.net/10044/1/11239.
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15
Beaton, Andrew Robert. „Towards understanding the release of BTV : functional analysis of the non-structural protein NS3“. Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.312299.
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16
KHALIL, JUMANA A. T. „The Non-structural Protein NSs of SFTSV Causes an NF-κB dependent cytokine storm“. Doctoral thesis, Kyoto University, 2021. http://hdl.handle.net/2433/265212.
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京都大学新制・課程博士
博士(生命科学)
甲第23440号
生博第461号
新制||生||61(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 千坂 修
学位規則第4条第1項該当
Doctor of Philosophy in Life Sciences
Kyoto University
DFAM
17
Hatherell, Tracey-Leigh. „An investigation into the subcellular localisation of non-structural protein NS3 of African horsesickness virus“. Diss., University of Pretoria, 2007. http://hdl.handle.net/2263/26279.
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African horsesickness virus (AHSV) is a double-stranded RNA virus belonging to the Orbivirus genus in the Reoviridae family (Bremer et al., 1990; Calisher and Mertens, 1998). The virus is highly pathogenic and its mortality rate in horses, the most susceptible species, may be as high as 95% (House, 1993). S10, the smallest genome segment of AHSV, codes for two proteins (NS3 and NS3A) from in-phase overlapping reading frames. The C-terminal sequences of these proteins are identical, but NS3A lacks the first 10 amino acids present on the N-terminal of NS3 (Van Staden and Huismans, 1991). Nonstructural protein NS3 is a membrane protein, associated with both smooth intracellular membranes and the plasma membrane. NS3 has pleiotropic roles in the viral life cycle including the transport and release of mature virions and viroporin-like alteration of cell membrane permeability. NS3 is cytotoxic when expressed in bacterial or insect cells, and is speculated to play a vital role in viral virulence and disease pathogenesis (Stoltz et al., 1996; Van Staden et al., 1995). A number of different domains that could mediate the membrane interaction or intracellular trafficking of NS3 have been identified. The relative contributions of these domains in insect and mammalian cells are not known, but could differ, as there are distinct differences in NS3 expression levels, cytopathic effects and virus release mechanisms in these two cell types. In order to investigate the subcellular localisation of NS3, a number of full-length, truncated or mutant versions of AHSV-3 NS3 were constructed as C-terminal eGFP (enhanced green fluorescent protein) fusion proteins. These proteins were used to generate recombinant baculoviruses for expression in Spodoptera frugiperda (Sf9) insect cells and were compared in terms of their subcellular localisation by conventional fluorescence microscopy. Confocal laser microscopy was used to investigate co-localisation with the nucleus, the Golgi apparatus and the Endoplasmic Reticulum (ER). Subcellular fractionations and membrane flotation analyses were used to confirm membrane interactions and to identify detergent-resistant membrane fractions. NS3 as well as a C-terminal deletion of NS3 targeting a putative dileucine motif both localised to cellular/nuclear membrane components. In contrast, site-specific mutations to either of the transmembrane domains abolished membrane association and resulted in cytoplasmic localisation. NS3A showed mixed results, displaying both membrane localisation and a cytoplasmic distribution. The 11 amino acid region unique to NS3 and absent from NS3A, which has been shown to bind to cellular exocytosis proteins in bluetongue virus (Beaton et al., 2002), did not display membrane interaction. These results indicate that both of the hydrophobic domains as well as the N11 region are required to be present for NS3 to be properly targeted to the plasma/nuclear membrane. In addition, NS3 was shown to be present in detergent-resistant membrane fractions, indicative of a possible localisation within lipid rafts. The above results indicate that the NS3 protein contains specific signals involved in membrane targeting, confirming a potential role for NS3 in viral localisation and release in the AHSV replication cycle.Dissertation (MSc (Genetics))--University of Pretoria, 2009.
Genetics
unrestricted
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Dong, Jiawei. „Etude in vitro des interactions entre la protéine NS1 du virus respiratoire syncytial et la sous-unité MED25 du Médiateur humain“. Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASQ077.
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Le virus respiratoire syncytial (VRS) est une cause majeure d'infections respiratoires sévères, notamment chez les nourrissons et les jeunes enfants. Il parvient à échapper au système immunitaire inné notamment grâce à ses deux protéines non structurales NS1 et NS2. La protéine NS1 joue le rôle d'antagoniste des interférons. Elle inhibe la production d'interférons ainsi que leurs voies de signalisation. Cependant, une nouvelle hypothèse suggère qu'elle pourrait également contribuer à réguler l'expression des gènes de l'hôte via une interaction avec la sous-unité MED25 du complexe médiateur, un coactivateur de la transcription par l'ARN polymérase II. Mon travail de thèse porte sur la caractérisation structurale de cette interaction in vitro.Dans un premier temps, j'ai voulu m'assurer que la protéine NS1 était sous forme native dans les conditions où sont faites les expériences d'interaction. En effet, NS1 peut être produite sous forme de protéine recombinante dans E. coli et une structure cristallographique de NS1 est disponible : elle révèle un domaine globulaire "NS1core" et une hélice C-terminale "NS1α3" située à l'interface d'un dimère. Mais NS1 est difficile à étudier en solution en raison de sa propension à s'auto-associer. J'ai donc analysé le comportement de NS1 dans différentes conditions expérimentales et avec différentes techniques biophysiques : fluorimétrie différentielle à balayage pour la stabilité thermique, dichroïsme circulaire pour la structure secondaire, diffusion de la lumière pour la taille. Cela a permis de mettre en évidence un équilibre entre monomère et dimère de NS1. Un mutant de délétion de NS1, NS1∆α3 qui correspond à NS1core, a pu être étudié à l'échelle du résidu par résonance magnétique nucléaire (RMN). J'ai attribué les fréquences de résonance du squelette peptidique ce qui m'a permis de montrer qu'il était structuré en solution. Les largeurs de raie importantes et les mesures de relaxation 15N pointent vers des phénomènes d'échange. L'attribution de NS1∆α3 m'a permis ensuite d'attribution partiellement la protéine NS1 entière et d'analyser des expériences d'interaction par RMN.La seconde partie de ma thèse s'intéresse à l'interaction entre NS1 et le domaine ACID de MED25. Des études par RMN de la protéine MED25-ACID marquée aux isotopes 15N et 13C avec un peptide correspondant à NS1α3 ont d'abord révélé que NS1α3 interagit avec MED25-ACID. Des expériences de calorimétrie ont montré que la protéine NS1 entière avait une affinité bien supérieure à celle de NS1α3, suggérant une interaction potentielle via le domaine globulaire NS1core en plus de l'hélice NS1α3. Des données obtenues par interférométrie de bio-couches (BLI) ont ensuite confirmé cette interaction. Elles montrent que NS1∆α3 se lie à MED25-ACID avec une affinité plus faible que NS1, en présentant deux modes de fixation. Des modélisations par AlphaFold2 n'ont pas produit de modèle de complexe fiable avec NS1∆α3 ou NS1α3. Mais elles ont permis de prédire avec une certaine fiabilité la structure du complexe MED25-ACID−NS1. Des mutants de NS1, basés sur cette prédiction, ont été testés par BLI, montrant une réduction de l'interaction avec MED25-ACIDRespiratory syncytial virus (RSV) is a major cause of severe respiratory infections, particularly in infants and young children. It evades the innate immune system notably thanks to its two non-structural proteins, NS1 and NS2. The NS1 protein functions as an interferon antagonist, inhibiting both the production of interferons and their signaling pathways. However, a new hypothesis suggests that NS1 could also contribute to the regulation of host gene expression via an interaction with the MED25 subunit of the mediator complex, a coactivator of transcription by RNA polymerase II. My thesis focuses on the structural characterization of this interaction in vitro.In a first step, I wanted to ensure that NS1 was in its native form under the conditions used for interaction experiments. NS1 can indeed be produced as a recombinant protein in E. coli, and a crystallographic structure of NS1 is available: it reveals a globular domain "NS1core" and a C-terminal helix "NS1α3" located at the interface of an NS1 dimer. However, NS1 is challenging to study in solution due to its propensity to self-assemble. I thus analyzed the behavior of NS1 under different experimental conditions and by different biophysical techniques: differential scanning fluorimetry to assess stability, circular dichroism to assess secondary structure, and light scattering to assess size. This allowed providing evidence for an NS1 monomer-dimer equilibrium. A deletion mutant of NS1, NS1∆α3 corresponding to NS1core, was amenable to nuclear magnetic resonance (NMR) for structural analysis at the single residue scale. I performed backbone assignment, and showed that it was well folded in solution. Large line-widths and 15N relaxation measurements pointed at exchange phenomena. Assignment of NS1∆α3 then permitted to partially assign full-length NS1 and to analyze NMR interaction experiments.The second part of my thesis focuses on the interaction between NS1 and the ACID domain of MED25. NMR studies using 15N- and 13C-labeled MED25-ACID protein and a peptide corresponding to NS1α3 first revealed that NS1α3 interacts with MED25-ACID. Additionally, calorimetry experiments showed that full-length NS1 had a much higher affinity than NS1α3, suggesting a potential interaction via the globular NS1core domain in addition to the NS1α3 helix. Data obtained from biolayer interferometry (BLI) then confirmed this interaction. These data showed that NS1∆α3 binds to MED25-ACID with lower affinity than NS1, exhibiting two binding modes. AlphaFold2 modeling did not produce reliable complex models with NS1∆α3 or NS1α3. But it allowed reasonably accurate prediction of the structure of the MED25-ACID−NS1 complex. NS1 mutants based on this prediction were tested by BLI, showing a reduction in interaction with MED25-ACID
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Barski, Michał S. „Structural studies of bunyavirus interferon antagonist proteins“. Thesis, University of St Andrews, 2016. http://hdl.handle.net/10023/8408.
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Bunyaviridae is one of the biggest known viral families, and includes many viruses of clinical and economic importance. The major virulence factor of most bunyaviruses is the non-structural protein (NSs). NSs is expressed early in infection and inhibits the innate immune response of the host by blocking several steps in the interferon induction and signalling pathways. Hence, NSs significantly contributes to the establishment of a successful viral infection and replication, persistent infection and the zoonotic capacity of bunyaviruses. Although functions and structures of many viral interferon antagonists are known, no structure of a bunyavirus NSs protein has been solved to date. This strongly limits our understanding of the role and the mechanism of interferon antagonism in this large virus family. In this work the first structure for a bunyavirus interferon antagonist, the core domain crystal structure of NSs from the Rift Valley fever virus (RVFV) is presented. RVFV is one of the most clinically significant members of the Bunyaviridae family, causing recurrent epidemics in Africa and Arabia, often featuring high-mortality haemorrhagic fevers. The structure shows a novel all-helical fold. The unique molecular packing of NSs in the crystal creates stable fibrillar networks, which could correspond to the characteristic fibrillation of NSs observed in vivo in the nuclei of RVFV infected cells. This first NSs structure might be a useful template for future structure-aided design of drugs that target the RVFV interferon antagonism. Attempts at characterising other bunyavirus NSs proteins of other genera were made, but were hampered by problems with obtaining sufficient amounts of soluble and folded protein. The approaches that proved unsuccessful for the solubilisation of these NSs proteins, however, should inform future experiments aimed at obtaining recombinant NSs for structural studies.
20
Brown, Heather Piehl. „Homology-based Structural Prediction of the Binding Interface Between the Tick-Borne Encephalitis Virus Restriction Factor TRIM79 and the Flavivirus Non-structural 5 Protein“. University of Toledo Health Science Campus / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=mco1481304908426729.
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21
Miller, Cathy Lea. „Investigation of the role of minute virus of mice (MVM) small non-structural protein NS2 interactions with host cell proteins during MVM infection“. free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3025638.
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Bakhache, William. „Interactions de la protéine nsP1 du virus Chikungunya avec les membranes de l’hôte et conséquences fonctionnelles“. Thesis, Montpellier, 2020. http://www.theses.fr/2020MONTT008.
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Les virus à ARN de polarité positive (ARN(+)) partagent la capacité de réorganiser les membranes cellulaires en organelles vésiculaires. Ces compartiments, appelés organelles de réplication (OR), fournissent un environnement approprié permettant d’héberger la machinerie de réplication virale, ses cofacteurs cellulaires et les ARN viraux néo-synthétisés. En raison de leur rôle indispensable à la réplication virale, ces compartiments et les mécanismes moléculaires nécessaires à leur assemblage ont suscité un réel intérêt ces dernières années. Alors que des progrès significatifs ont été réalisés dans ce domaine pour d’autres virus à ARN(+), peu de données relatives au mécanisme de formation des ORs des Alphavirus ont été produites. Ces virus ont pourtant été associés à des enjeux majeurs de santé publique au cours de la dernière décennie, en particulier avec la propagation récente du virus Chikungunya (CHIKV). CHIKV est en effet un virus réémergent transmis par les moustiques et à l’origine d’épidémies ayant des conséquences socio-économiques dévastatrices dans les pays où il se propage. Les symptômes se caractérisent par une forte fièvre et une éruption cutanée, avec un pourcentage significatif de patients qui souffrent de douleurs articulaires à long terme, souvent invalidantes. À l’heure actuelle, il n’existe aucun vaccin ou traitement antiviral pour ce virus.Les OR de CHIKV se présentent comme des sphérules de 50 à 60 nm résultant d’une courbure négative de la membrane plasmique. À l’intérieur de ces compartiments, la machinerie de réplication est ancrée à la membrane par l’interaction directe de la protéine non structurale 1 (nsP1) avec la bicouche lipidique. Cette protéine virale, exprimée de façon isolée, conduit à des déformations membranaires de type filopodes. Ainsi, nsP1 apparait comme un acteur majeur du remodelage membranaire au cours de l’infection par les Alphavirus. Dans ce contexte, le but de cette thèse, centrée sur nsP1, est de caractériser les interactions de nsP1 avec les membranes cellulaires et de définir les conséquences fonctionnelles de ces interactions dans la réplication virale. Nous avons mis en évidence le rôle du métabolisme lipidique dans l’ancrage membranaire de nsP1 et dans l’infection virale. Nos résultats indiquent que la production d’acides gras est nécessaire au cycle infectieux et favorise l’interaction de nsP1 avec les membranes. Ils mettent en évidence le rôle complètement nouveau des acides gras insaturés dans l’étape de réplication des Alphavirus. Nous avons également démontré l’affinité de la forme palmitoylée de nsP1 pour les microdomaines lipidiques riches en cholestérol de la membrane plasmique. Nous avons établi les conséquences fonctionnelles de cette affinité sur la localisation des autres protéines non structurales et sur la réplication virale. Enfin, nous avons défini l’interactome fonctionnel de nsP1, de façon à identifier les cofacteurs cellulaires pouvant contribuer aux déformations membranaires induites par cette protéine virale. Ce travail permet de mieux comprendre les mécanismes de déformation membranaires observés au cours de l’infection par les AlphavirusPositive strand RNA ((+) RNA) viruses share the common capacity to rearrange cellular membranes into vesicular organelles. These membranous compartments referred to as replication organelles (ROs), are seen as providing an appropriate environment recruiting all viral components and cofactors required for replication. Because of their strict necessity for viral replication, these compartments and the molecular mechanisms required for their assembly have generated an intense interest in recent years. Contrasting with the consequential advances made in this field for other (+)RNA viruses, virtually no mechanistic data has been produced on the formation of ROs by Alphaviruses which in the last decade have proven to be medically paramount viruses, especially with the recent spread of Chikungunya virus (CHIKV). CHIKV is a re-emerging virus transmitted by mosquitoes that has caused outbreaks with devastating socio-economic impact in countries where it propagates. Symptoms include high fever and rash, with a significant percentage of patients suffering of long-term, often incapacitating, joint pain. Currently there is no vaccine or anti-viral treatment for this virus.CHIKV ROs appear as 50-60 nm electron translucent bulb-shaped spherules resulting from negative curvature at the plasma membrane. Inside these compartments, the replication machinery is anchored to the membrane through the direct interaction of the non-structural protein 1 (nsP1) with the lipid bilayer. When expressed as an isolated protein nsP1 dramatically remodels cellular membranes into filopodia-like protrusions. Therefore, this designated nsP1 as a critical factor in cellular membrane reshaping observed during infection. In this context, the aim of this thesis, with nsP1 at its centerpiece, is to characterize nsP1 interactions with cellular membranes and to define their functional consequences on viral replication. In this investigation, we have demonstrated the role of host cell lipid metabolism in nsP1 membrane anchoring and viral infection. Our results indicate that fatty acid synthesis is required for viral life cycle and favors nsP1 interaction with membranes. We also provide the very first information on the role of unsaturated fatty acids in Alphavirus replication. In-depth studies on the role of cholesterol revealed that palmitoylated nsP1 anchored CHIKV non-structural proteins to cholesterol-rich microdomains with functional consequences on replication. Finally, we have identified nsP1 interactome in order to identify host-cofactors required for the membrane deformation induced by this viral protein. Taken together, this thesis provides new information on nsP1/membrane lipids and host cofactors interplay. This work will allow the further comprehension of the mechanisms behind membrane deformation observed during Alphavirus replication
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Chung, Keun-Taik. „Interactions and functions of rotavirus non-structural proteins NSP1 and NSP3“. Thesis, University of Warwick, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414314.
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Essien, Thomas [Verfasser], Frank Torsten [Akademischer Betreuer] Hufert, Sabine [Akademischer Betreuer] Mihm und Martin [Akademischer Betreuer] Oppermann. „The Inhibition of RNA-Polymerase II-Mediated Expression by the Non-Structural Protein NSs of the Oropouche Virus and Establishing an Oropouche Virus Minireplicon System / Thomas Essien. Gutachter: Sabine Mihm ; Martin Oppermann. Betreuer: Frank Torsten Hufert“. Göttingen : Niedersächsische Staats- und Universitätsbibliothek Göttingen, 2015. http://d-nb.info/1072293250/34.
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Ross-Smith, Natalie. „The role of Non-structure protein 2 (NS2) in Bluetongue virus replication“. Thesis, University of Oxford, 2008. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.510212.
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Schwardt, Malte. „BDV X protein is a non-structural protein“. [S.l. : s.n.], 2006. http://nbn-resolving.de/urn:nbn:de:bsz:25-opus-60677.
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Schiller, Christian Bernd. „Structural and functional analysis of the eukaryotic DNA repair proteins Mre11 and Nbs1“. Diss., lmu, 2011. http://nbn-resolving.de/urn:nbn:de:bvb:19-134006.
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Fritz, Matthieu. „Analyse interactomiques et fonctionnelles de la protéine NS2 du virus de l'hépatite C et d'hepacivirus non-humains“. Thesis, Sorbonne Paris Cité, 2017. http://www.theses.fr/2017USPCC310/document.
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L’émergence récente de nouvelles thérapies antivirales efficaces est une avancée considérable pour lutter contre l'infection chronique par le virus de l'hépatite C (VHC). Cependant, un pic de carcinomes hépatocellulaires, représentant l'atteinte hépatique ultime liée à l'infection, est attendu dans la prochaine décennie. Approfondir les connaissances des différentes étapes du cycle viral et de l’interférence du VHC avec l'hépatocyte hôte permet de mieux comprendre la pathogénèse associée à ce virus. Les travaux présentés dans cette thèse ont eu pour objectif d'identifier le réseau de partenaires cellulaires et viraux de la protéine non-structurale NS2 du VHC et de mieux comprendre les mécanismes d'action et de régulation de cette protéine transmembranaire multi-fonctionnelle, qui est un acteur clé du clivage protéolytique de la polyprotéine virale et de la morphogénèse des virions. Dans une première partie, nous avons analysé comparativement les mécanismes moléculaires de l’activité enzymatique des protéines NS2 du VHC et de plusieurs hepacivirus non-humains, qui infectent des primates du Nouveau Monde (GBV-B) ou qui ont été récemment identifiés chez plusieurs autres espèces animales (NPHV, RHV, BHV et GHV). Des analyses phylogénétiques, des modèles structuraux tridimensionnels et des Études dans un contexte d'expression transitoire de précurseurs polypeptidiques viraux ou dans des modèles d'infection ont montré que l’activité des protéases NS2 de divers hepacivirus (1) s'exerce à la jonction NS2/NS3 sous la forme d'homodimères formant deux triades catalytiques composites ; (2) est régulée dans le contexte de la polyprotéine virale par quelques résidus de surface du domaine N-terminal de NS3 (NS3N) nécessaires à son activation ; (3) est efficace en l'absence complète de NS3N, suggérant un rôle négatif ou régulateur, plutôt qu'activateur de NS3N, contrairement au dogme en vigueur actuellement. Ces travaux soulignent l'importance fonctionnelle des mécanismes protéolytiques de NS2 conservés parmi les différents hepacivirus. Dans une deuxième partie, nous avons identifié un réseau de facteurs cellulaires et viraux interagissant avec NS2 au cours du cycle infectieux par un crible interactomique reposant sur la purification par affinité et l'analyse par spectrométrie de masse des complexes protéiques isolés de cellules hépatocytaires infectées, ainsi que par un test de complémentation enzymatique fonctionnelle. Par une approche d'ARN interférence, nous avons ensuite montré qu'un nombre limité de facteurs cellulaires interagissant avec NS2 sont impliqués dans la production et la sécrétion de particules virales infectieuses, incluant des protéines du complexe de la peptidase signal (SPCS) au sein du réticulum endoplasmique, des protéines chaperonnes (DNAJB11, HSPA5) et une protéine impliquée dans le transport intracellulaire (SURF4). Notamment, nos Études suggèrent que plusieurs membres du SPCS forment un complexe multi-protéique avec NS2, impliquant Également la glycoprotéine virale E2, qui jouerait un rôle dans une Étape précoce de l'assemblage ou lors de l’enveloppement de la particule virale. En conclusion, mes travaux de thèse ont permis d'identifier pour la première fois une série limitée de facteurs hépatocytaires interagissant spécifiquement avec la protéine NS2 du VHC au cours de l'infection et de déterminer parmi ceux-ci les facteurs essentiels la morphogenèse virale. Par ailleurs, nos résultats ont permis d’enrichir les connaissances naissantes des hepacivirus non-humains récemment identifiés et de montrer que ceux-ci partageaient avec le VHC des mécanismes clés mis en jeu au cours du cycle viral, ce qui contribue consolider leur intérêt comme modèles animaux de substitutionThe recent emergence of a panel of direct acting antivirals will certainly help combat chronic hepatitis C in the future. However, in the current context worldwide, a peak of hepatitis C virus (HCV)-induced hepatocellular carcinoma is expected in the next decade. Deepening our understanding of HCV life cycle and HCV interference with host cells may help monitor HCV-associated pathogenesis. The aim of my PhD work was to identify the network of host and viral interactors of HCV nonstructural protein 2 and to unravel the mechanisms of action and regulation of this multifunctional, transmembrane protein, which is key both for the viral polyprotein cleavage and virion morphogenesis.In the first part of the work, we comparatively characterized molecular mechanisms underlying the enzymatic activity of NS2 proteins from HCV and from various non-human hepaciviruses that infect small New World primates (GBV-B) or that were recently identified in the wild in several mammalian species (NPHV, RHV, BHV, GHV). A combination of phylogenetic analyses, tridimensional structural models, and studies relying on the transient expression of viral polypeptide precursors or on infection models showed that NS2 proteases of the various hepaciviruses (1) act as dimers with two composite active sites to ensure NS2/NS3 junction cleavage, (2) are regulated in the polyprotein backbone via a hydrophobic patch at the surface of NS3 N-terminal domain (NS3N) that is essential to activate NS2 protease, and (3) are efficient in the complete absence of NS3N, which is unprecedented and suggests that NS3N has rather a negative or regulating role on NS2 activity. These data underline the functional importance of NS2 proteolytic mechanisms that are conserved across hepaciviruses.In the second part, we identified a network of cellular factors and viral proteins that interact with NS2 in the course of HCV infection using an interactomic screen based on affinity purification and mass spectrometry analysis of protein complexes retrieved form HCV infected hepatoma cells, as well as a split-luciferase complementation assay. Next, using a gene silencing approach, we found that a limited set of NS2 interactors among these host factors were involved in HCV particle assembly and/or secretion. This includes members of the endoplasmic reticulum signal peptidase complex (SPCS), chaperone proteins (DNAJB11, HSPA5) and a factor involved in intracellular transport (SURF4). Notably, our data are in favor of the existence of a multiprotein complex involving NS2, several members of the SPCS, and the viral E2 glycoprotein, which likely plays a role in an early step of HCV particle assembly or during particle envelopment. Altogether, my PhD work allowed us to identify a limited set of hepatocyte factors interacting with HCV NS2 during infection and to pinpoint those that are essential for HCV morphogenesis. Additionally, our results contributed to the molecular characterization of the recently identified non-human hepaciviruses and revealed that these hepaciviruses share with HCV key mechanisms in the course of their infectious life cycles. This highlights the value of non-human hepaciviruses as surrogate animal models of HCV infection
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Nethisinghe, Suran. „Further characterisation of bluetongue virus non-structural protein 2“. Thesis, London School of Hygiene and Tropical Medicine (University of London), 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.497276.
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Ward, Rebecca. „Bluetongue virus non-structural protein 1 : virus-host interactions“. Thesis, London School of Hygiene and Tropical Medicine (University of London), 2006. http://researchonline.lshtm.ac.uk/4646527/.
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Bluetongue virus (BTY) is an orbivirus of the Reoviridae family that infects sheep and other ruminants. BTY has three non-structural proteins, NS I, NS2 and NS3/3A. NS I forms tubular structures and its function is currently unknown. To investigate the role of NS I in BTY infection, the interactions of NS I with mammalian and insect cellular proteins, and BTY viral proteins, were examined. BTY NS I was identi tied as interacting with aldolase A, NUBP 1, Pyruvate kinase M2, cathespin B, SUM 0-1 and peptide TY7 using the yeast two-hybrid system, ELISA and immunofluorescence analysis. TY7 and NS I caused extensive cell death within 24h of co-expression; this cell death was not apoptosis and reduced BTY yield by 37%. The interaction of NS I with SUMO-I and its importance in BTY infection was confinned using siRNA to knockdown SUMO-I during BTY-IO infection. Knockdown of SUMO-I elicited a dramatic reduction in virus yield by 73%. NS I interactions with proteins of the insect vector Culicoides were also examined. A putative interaction between NS 1 and the ubiquitin activating enzyme El (UBA EI) ofCulicoides was identified during screening of a phage library, this has not been confirmed by other means. NS 1 interactions with other BTY proteins were analysed using immunoprecipitation and a strong interaction between NS 1 and YP7 was identified; this was confim1ed using the yeast two-hybrid system and immunoflourescence. Two main roles have been hypothesised for NS I from this data; firstly it is likely that NS I interaction with SUMO-I and UBA E I allows the targeting of specific proteins for sumoylation and ubiquitination allowing NS 1 to modify the host response to BTY infection. Secondly it is possible that NS I serves as an anchor for YP7 and virus cores allowing the build up of cores at the cytoskeleton in close proximity to YP2 for subsequent assembly and release. RNAi against NS J eliminated tubule formation but did not affect virus yield or YP7 and SUMO-J distribution and expression. It is therefore likely that the function of NS I does not rely on tubule fom1ation and that tubules are a form of storage for the active monomer of NSI.
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Bhatt, Veer Sandeep. „Non-lectin type Protein-carbohydrate Interactions: A Structural Perspective“. The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1306858684.
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Butan, Carmen Crina. „Structural characterization of the RNA binding domain of BTV non-structural protein 2“. Thesis, Birkbeck (University of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414198.
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Jacobs, Michael Graham. „Membrane association of dengue 2 virus non-structural protein 1“. Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325917.
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Chauché, Caroline Marie. „Molecular evolution of equine influenza virus non-structural protein 1“. Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/8877/.
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Influenza A viruses (IAVs) are common infections of certain avian reservoir species, and they periodically transfer to mammalian hosts. These cross-species jumps are usually associated with sporadic outbreaks, and on rare occasions lead to the establishment of a lineage in the new host species. The immune pressure exerted by the new host on the emergent virus forces it to evolve and adopt strategies to evade immunity in order to survive in nature. Understanding the biological mechanisms that allow successful inter-species transmission and adaptation to mammals is crucial to develop the theoretical tools required to predict and/or control emergence of new viruses in humans and animals. H3N8 equine influenza virus (EIV) represents an interesting model to study the dynamic of within-host variation of an avian-origin IAV. Indeed, this virus has emerged from birds in 1963 and has circulated in horse populations for more than fifty years despite the availability of vaccines. Evidence of evolution of EIV virulence factor non-structural protein 1 (NS1) also exists. NS1 is the main viral antagonist of the host interferon (IFN) response, and it relies on different strategies for overcoming these responses, which varies depending on the viral strain. While some NS1 proteins effectively block the induction of IFN and IFN stimulated genes (ISGs), others block general gene expression at a post-transcriptional level, and therefore reduce the synthesis of IFN and ISGs indirectly. Importantly, little is known about the contribution of these NS1 functions to EIV infection phenotype and adaptation to horses. In this work, we characterised NS1 proteins spanning the entire EIV lineage and showed that NS1s from different time periods after EIV emergence counteract the IFN response using different and mutually exclusive mechanisms. While EIVs circulating in the early 1960s blocked general gene expression by a NS1-mediated blockade of the cleavage and polyadenylation specificity factor 30 (CPSF30), NS1s from contemporary EIVs specifically inhibit the induction of ISGs by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially during EIV evolution, E186K substitution and C-terminal truncation. These changes in NS1 allowed contemporary EIVs to replicate in the presence of high levels of IFN. The results shown here with EIV indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation.
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Poonsiri, T. „Structural study of the C-terminal domain of non-structural protein 1 and capsid protein from Japanese encephalitis virus“. Thesis, University of Liverpool, 2018. http://livrepository.liverpool.ac.uk/3021941/.
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Japanese encephalitis virus (JEV) is a mosquito-transmitted Flavivirus that is closely related to other emerging viral pathogens including dengue (DENV), West Nile (WNV) and Zika viruses (ZIKV). JEV infection can result in meningitis and encephalitis, which in severe cases cause permanent brain damage and death. JEV occurs predominantly in rural areas throughout South East Asia, the Pacific islands, and the Far East, causing around 68,000 cases worldwide each year. There is no specific treatment for JEV. This study aims to determine the molecular structure of new potential drug targets for JEV. In this study, the JEV non-structural protein 1 C-terminal β-ladder domain (C-NS1) is presented at 2.1 Å resolution. The crystal structure of C-JEVNS1 shares a conserved fold with flavivirus C-NS1 domains. The surface charge distribution of C-JEVNS1 is similar to WNV and ZIKV but is significantly different from DENV. Analysis of the C-JEVNS1 structure, in silico molecular dynamics simulations and experimental solution small angle X-ray scattering, indicate extensive loop flexibility on the exterior of the protein. It is proposed that this together with charge distribution on the exterior of the protein influence NS1-host protein interaction specificity which may impact on pathogenicity. These factors may also affect the interaction with the monoclonal antibody, 22NS1, which is protective against WNV infection. Liposome and heparin binding assays indicate that only the N-terminal region of NS1 participates in the interaction with lipidic membranes and that sulphate binding sites are not the glycosaminoglycans binding interfaces. For the first time, the crystal structure of the JEV capsid protein at 1.98 Å is also reported and compared to the existing flavivirus capsid protein. JEV capsid shows helical secondary structure (α helixes 1-4) and protein folding similar to DENV and WNV capsid proteins. It forms a homodimer by antiparallel pairing with another subunit (‘), α helix 1-1’, 2-2’, and 4-4’. The capsid dimer is believed to be the building block of the nucleocapsid. The flexibility of the N-terminal α helix 1 of the capsid could be important for its function. This dimer model agrees with a previous suggestion that the capsid protein interacts with RNA via the basic rich C-terminal, α4-α4’, and associates with lipid bilayers at the opposite hydrophobic, α2-α2’.
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Yang, Weiming. „Functional studies of the group A rotavirus non-structural protein NSP4“. Thesis, University of Warwick, 2010. http://wrap.warwick.ac.uk/35683/.
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NSP4, encoded by rotavirus genome segment 10 has been shown to be a transmembrane, endoplasmic reticulum (ER) specific N-linked glycoprotein. Consistent with its localization to the ER membrane, NSP4 was first shown to have a role in the morphogenesis of the infectious virion. The protein has also been reported to have cytotoxic activity when applied extracellularly to cells. Consequently it has been earmarked as an enterotoxin being secreted from virus-infected cells to cause early cellular pathology in the gut. The effect of expressing the NSP4 protein of group A rotaviruses in cells has been studied. It led to the rapid appearance of long cytoplasmic extrusions. Site-directed mutagenesis was used to block N-linked glycosylation at both of the known glycosylation sites near the amino terminus of NSP4. This revealed that the NSP4 induced formation of the cytoplasmic extrusions was dependent on the protein’s ability to become fully glycosylated. The cytoplasmic extrusions seen in cells expressing glycosylated NSP4 were also evident in virus-infected cells. Using real-time confocal microscopy a dynamic elongation of the cytoplasmic extrusions with a growth speed of 2 μm/min was observed in virus-infected cells. The cytoplasmic extrusions were found to contain β-tubulin and F-actin. Inhibiting their polymerization prevented the formation of the extrusions from virus-infected cells. Functional studies using Cell Tracker dyes showed that the cytoplasmic extrusions could disseminate vesicles from virus-infected cells onto the plasma membrane surface of uninfected cells. The vesicles were then found in the interior of the uninfected cells. Mono-specific antibody to NSP4 revealed the presence of the protein in the vesicles suggesting that the cytoplasmic extrusions facilitated the direct cell-cell spread of NSP4. The effect of NSP4 expression on the microtubular network of cells was analysed. It was found that NSP4 de-polymerized the microtubular network from the centre of cells and promoted the assembly of microtubules at the periphery of the cells in a glycosylation independent manner. Similar de-polymerization and re-assembly of the microtubules was observed in the virus-infected cells. Interestingly in the presence of nocodazole, tubular structures containing tubulin and viral proteins excluding NSP4 were found in virus-infected cells. A YFP-PCA assay was established to screen for cellular partners of NSP4. The functionality and the sensitivity of the assay were examined, but only two false positive colonies were isolated in the first screening. In conclusion, the function of glycosylated and unglycosylated NSP4 was examined with the former possessing the ability to promote the formation of the cytoplasmic extrusions from cells and both being capable of disrupting the microtubular network indicating that two forms of NSP4 play different roles in NSP4 function. The cytoplasmic extrusions seen in our studies may be relevant to rotavirus infection and pathogenesis.
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Sung, Po-yu. „Functional analysis of the non-enzymatic properties of the dengue virus non-structural protein 5“. Thesis, University of Bristol, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.556732.
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The four serotypes of dengue virus (DENV 1-4) cause the most important arthropod-borne viral disease of humans. The DENV RNA genome is translated into a polyprotein that is cleaved into three structural and seven nonstructural proteins. Nonstructural protein 5 (NSS) has enzymatic activities required for capping and replication of the viral genome. NSS is phosphorylated and with some serotypes can accumulate in the nucleus of infected cells. However the role of NSS phosphorylation or nuclear trafficking in the virus lifecycle, is not well understood. Using a combination of proteomic and bioinformatic approaches, potential NSS phosphorylation sites were identified and analysed for their role in the virus lifecycle using a DENV reverse genetic system. Mutation of the NSS amino acid Thr-39S was found to decrease NSS nuclear localisation and viral replication in a cell specific manner. Mutations preventing and mimicking phosphorylation at Thr-39S had similar effects on NSS localisation and viral growth, showing that the effects were not caused by phosphorylation. Mutation of Thr-39S did not influence NSS mediated suppression of interferon signalling. For the first time this thesis revealed that there are serotype specific differences in NSS localisation. DENV-1 and DENV-4 NSS are predominantly localised to the cytoplasm, whereas DENV-2 and DENV-3 NSS accumulate in the nucleus. The role of the NSS nuclear localisation signal (NLS) in mediating these differences was investigated. NSS of all DENV serotypes bound to importin-a, the protein responsible for active nuclear import, but exchanging the NLSs of DENV-2 and 4 NSS didn't fully confer the properties of the NLS to the background protein, suggesting that protein context was also important for NSS nuclear localisation. Chimeric DENV-2 containing sequences encoding the DENV-3 NSS or polymerase subdomain were produced, whilst DENV-2 containing the DENV-4 NSS sequence failed to replicate. The two DENV-2/3 chimeric viruses showed decreased replication especially in the insect cell line C6/36; continued passaging resulted in the accumulation of mutations in NS1 and NS2B that increased viral replication.
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Richard, Audrey. „Oncolytic H-1 parvovirus NS1 protein : identifying and characterizing new transcriptional and posttranslational regulatory elements“. Phd thesis, Université du Droit et de la Santé - Lille II, 2011. http://tel.archives-ouvertes.fr/tel-00826936.
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H 1 parvovirus (H-1PV) is a little single stranded DNA virus that preferentially replicates in a lytic manner in transformed cells due to their expression profile that meets the requirements for the activation of H¬ 1 PV life cycle unlike normal cells. This feature is known as oncotropism. H 1PV genome is constituted by two transcriptional units. The first one is driven by the proliferation and transformation dependent P4 promoter and allows the expression of both non structural proteins NS1 and NS2, and the second one controls the expression of both capsid proteins VP1 and VP2 through the activation of P38 promoter. H-1PV life cycle tightly depends on NS1 protein that is involved in crucial events, including viral DNA replication, P38 promoter activation as well as cytotoxicity.NS1 protein is regulated at both transcriptional and post translational levels.My thesis aimed at identifying new determining elements for both of these regulations and characterizing their involvement in both H-1PV life cycle and oncotropism.
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Le, May Nicolas. „Mécanismes de pathogenèse de la protéine non structurale NSs du virus de la Fièvre de la Vallée du Rift“. Paris 7, 2005. http://www.theses.fr/2005PA077205.
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Le virus de la fièvre de la Vallée du Rift (RVFV) est un arbovirus appartenant à la famille des Bunyaviridae et au genre Phlebovirus. Il est transmis par les moustiques et infecte l'homme et les ruminants. De graves épidémies/épizooties ont eu lieu en Afrique et le virus circule maintenant en Arabie Saoudite ainsi qu'au Yémen. Le génome viral est composé de trois segments d'ARN simple brin: les segments L. M sont de polarité négative et le segment S utilise une stratégie ambisens. Bien que le cycle viral ait heu dans le cytoplasme, la protéine NSs (256 acides aminés, 31 kDa), codée par le segment S, a une localisation nucléaire où elle forme des filaments. NSs est un facteur de pathogenèse du virus qui inhibe la synthèse des ARNm du gène codant pour l'IFN bêta sans perturber pour autant l'enhanceosome (NF-KB, IRF3 et ATF2/cjun). Pour comprendre les mécanismes par lesquels NSs inhibe la réponse anti-virale, nous avons recherché ses partenaires cellulaires. Nous avons montré que l'infection par le VFVR a pour conséquences : i) une rapide diminution de la synthèse des ARN cellulaires parallèle à la décroissance de la concentration du facteur de transcription TFIIH, ii) l'inhibition du recrutement de CBP et de l'acétylation des histones au niveau du promoteur IFNp et iii) la protéolyse de STAT1. Par les techniques du double hybride, d'immunoprécipitations de protéines ou de la chromatine et en microscopie confocale, nous avons démontré que chacun des événements est lié aux interactions de la protéine NSs avec respectivement - la sous unité p44 du TFIIH, la sous unité SAP30 de certains co-répresseurs dont Sin3 et N-coR et la protéine Socs 1 présente dans une E3 ligase : ces différents partenaires de NSs sont présents dans les filaments nucléaires. NSs en interagissant avec p44 empêche la néo-formation du TFIIH. NSs, via SAP30 et son association avec le facteur de transcription YY1, stabilise des co-répresseurs responsables de la déacétylation des histones et empêche l'interaction entre CBP et YY1 au niveau du promoteur IFNp. Enfin NSs provoque l'accumulation de Socs 1 qui recrute un complexe E3 ligase CBCSo' responsable de la dégradation de STAT1 inhibant l'induction par l'IFNy. Ainsi la protéine multifonctionnelle NSs inhibe par trois mécanismes indépendants la réponse anti-virale de l'hôteThe Rift Valley fever virus is a phlebovirus of the Bunyaviridae family transmitted by mosquitoes and affecting cattle, sheep, goats and humans. It causes many dramatic epidémies and epizootics in Africa and recently it was introduced in Yemen and in Saudi Arabia with a high mortality rate. The viral genome is composed of three segments of RNA: the L and M segments are of negative polarity and encode respectively for the RNA polymerase RNA dependent and the precursor of envelope glycoproteins. The S segment utilises an ambisense strategy and codes for the nucleoprotein N and the non structural protein NSs. Although the viral cycle is cytoplasmic, the NSs protein (256 amino acids, 31 kDa) is nuclear and forms filament. Moreover, it was shown that NSs is the major pathogenicity factor, inhibiting IFN beta messenger RNA synthesis but do not disturb the formation of the enhanceosome (NF-KB, IRF3 and ATF2/cjun). We found that infection by RVFV leads to i) a rapid and drastic suppression of host cellular RNA synthesis that parallels a decrease of the TFIIH transcription factor concentration, ii) an inhibition of CBP recruitment and histones acetylation on IFNp promoter and iii) STAT1 proteolysis. Using yeast two hybrid System, immunoprecipitations, Chips and confocal microscopy, we further demonstrated that each event is linked to the association of the nonstructural viral NSs protein with respectively the TFIIH subunit p44, co-repressors subunit SAP30 and Socs 1 in the nuclear filaments. NSs prevents the assembly of newly synthesized TFIIH subunits. NSs, through the interaction between SAP30 and YY1 transcription factor, stabilizes co-repressors like N-coR or Sin3 responsible of histones deacetylation on IFNp promoter and preventing the association between CBP and YY1. Finally NSs provokes Socs 1 accumulation and, through a Socs 1 containing-E3 ligase complex, it degrades STAT1 and inhibes induction by IFNy. These observations shed light on the mechanisms utilized by RVFV to evade the host response
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Bürckstümmer, Tilmann. „Identification of cellular targets of hepatitis C virus non-structural protein 5A“. [S.l. : s.n.], 2005. http://www.diss.fu-berlin.de/2005/150/index.html.
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Marongiu, Michela. „Studies on the hyperphosphorylation of hepatitis C virus non structural protein NSSA“. Thesis, University of Southampton, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548251.
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Savage, Jason Eric. „Investigating a putative non structural protein of the birnavirus Drosophila-X virus“. College Park, Md. : University of Maryland, 2004. http://hdl.handle.net/1903/1841.
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Thesis (M.S.) -- University of Maryland, College Park, 2004.Thesis research directed by: Dept. of Cell Biology and Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Kohl, Alain. „La proteine non structurale nss du virus de la fievre de la vallee du rift-analyse de ses proprietes biologiques et biochimiques“. Paris 7, 1999. http://www.theses.fr/1999PA077127.
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Le genome du virus de la fievre de la vallee du rift (vfvr ; un arbovirus de la famille des bunyaviridae) est compose de trois segments d'arn ; les segments l et m sont de polarite negative, et le segment s utilise une strategie ambisens. Le segment s code pour deux proteines, la nucleoproteine n et la proteine non structurale nss, une phosphoproteine qui forme des filaments dans le noyau des cellules infectees. Afin de determiner si la phosphorylation influence les proprietes de la proteine nss, nous avons caracterise l'etat de phosphorylation de la proteine nss exprimee dans des cellules de mammiferes et de moustiques. Nous avons montre que la proteine nss phosphorylee est presente dans les compartiments nucleaires et cytoplasmiques des cellules infectees. Dans les deux types de cellules, la proteine est phosphorylee au niveau des serines 252 et 256 dans le noyau et le cytoplasme des cellules infectees. La forme diphosphorylee au niveau des serines 252 et 256 est majoritaire dans les cellules infectees, mais des formes monophosphorylees au niveau des serines 252 ou 256 peuvent egalement etre detectees. Les serines 252 et 256 sont localisees dans des motifs consensus pour la phosphorylation par la caseine kinase ii, et des essais de phosphorylation in vitro suggerent effectivement que la caseine kinase ii, presente chez tous les eukaryotes, phosphoryle la proteine nss. La proteine nss de la souche attenuee c13 est un cas particulier car le gene codant pour la proteine nss c13 est delete de 549 nucleotides et conserve les extemites amino-terminales et carboxyliques en phase. La proteine nss c13 n'avait jamais ete detectee in vivo. Au cours de cette these nous avons montre que la proteine nss c13 est rapidement degradee par le proteasome, qu'elle est egalement phosphorylee mais qu'elle n'est plus transloquee dans le noyau. Cela pourrait etre du a la deletion de deux motifs polyproline de sequence consensus pxxp qui interviennent dans la formation du filament nucleaire. Afin de creer un outil permettant de manipuler et de modifier le genome du vfvr, un virus de la foret de semliki recombinant exprimant la t7 arn polymerase a ete construit. Ce virus recombinant, capable d'infecter cellules de mammiferes et de moustiques, devrait etre utile pour le developpement d'un systeme de genetique inverse.
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behera, Jyoti R., und aruna Ranjan kilaru. „NOVEL STRUCTURAL CHARACTERISTICS OF OIL BIOSYNTHESIS REGULATOR PROTEIN IN AVOCADO“. Digital Commons @ East Tennessee State University, 2021. https://dc.etsu.edu/asrf/2021/presentations/21.
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Plants synthesize and store oil, mostly triacylglycerol (TAG), in various storage tissues that serves as a source of carbon and energy. The process is transcriptionally controlled by WRINKLED1 (WRI1), a member of the APETALA2 (AP2) class of transcription factors, that regulates most of the fatty acid biosynthesis genes. Among the four Arabidopsis WRI1 paralogs, only WRI2 is nonfunctional and failed to complement wri1-1 mutant seeds. The oleaginous Avocado (Persea americana) fruit mesocarp (60-70% DW oil) showed high expression levels for orthologs of WRI2, along with WRI1 and WRI3. While the role of WRI1 as a master seed oil biosynthesis regulator is well-established, the function of WRI1 paralogs in non-seed tissues is poorly understood. We conducted structural analyses to elucidate distinct features of avocado WRI paralogs compared to their orthologs in seed tissues. Comprehensive comparative in silico analyses of WRI1 paralogs from Arabidopsis (dicot), maize (monocot), and avocado revealed distinct features associated with their function. Our analysis showed the presence of only one AP2 domain in all WRI2 orthologs, compared to two AP2 in others. The highly conserved N-terminal region and the less conserved C-terminal regions make up the primary structure of the proteins, with amino acid composition bias characteristic of intrinsically disordered proteins (IDPs). Additionally, the avocado WRI2 showed a high proportion of random coil secondary structure, although it lacks a C-terminal intrinsically disordered region (IDR). Also, both WRI1 and WRI2 have distinct predicted phosphorylation target sites compared to their orthologs, whereas WRI2 lacks a PEST motif. Finally, through transient expression assays, we demonstrated that both avocado WRI1 and WRI2 are functional and drive TAG accumulation in Nicotiana benthamiana leaves. Our study showed that avocado WRI2 is structurally different and is functional, unlike its ortholog in Arabidopsis. This study provides us with new targets to enhance oil biosynthesis in plants.
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NESREEN, HAMAD ABDELGAWWAD HAMAD. „Structural analysis of the interaction between FUS/TLS protein and non-coding RNA“. Kyoto University, 2020. http://hdl.handle.net/2433/259065.
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Guzman, Efrain. „Molecular and functional characterization of the group B rotavirus enterotoxin non-structural protein 4“. Thesis, University of Warwick, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.412891.
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Yin, Chunhong. „Functional analysis of domain I of the hepatitis C virus non-structural NS5A protein“. Thesis, University of Leeds, 2018. http://etheses.whiterose.ac.uk/20605/.
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The research on hepatitis C virus protein NS5A has developed rapidly over the decades, primarily with the advent of the JFH1 cell culture infectious clone which allowed the study of all aspects of the virus lifecycle from entry to exit. As the important target of DAAs, the NS5A protein of hepatitis C virus (HCV) plays roles in both virus genome replication and assembly. NS5A comprises three domains, of these domain I is believed to be involved exclusively in genome replication. In contrast, domains II and III are required for the production of infectious virus particles and are largely dispensable for genome replication. Domain I is highly conserved between HCV and related hepaciviruses, and is highly structured, exhibiting different dimeric conformations. To investigate the functions of domain I in more detail, a mutagenic study of 12 absolutely conserved and surface-exposed residues were conducted within the context of a JFH1-derived sub-genomic replicon and infectious virus. Whilst most of these abrogated genome replication, three mutants (P35A, V67A and P145A) retained the ability to replicate but showed defects in virus assembly. Whilst P35A exhibited a modest reduction in infectivity, V67A and P145A produced no infectious virus. Using a combination of density gradient fractionation, biochemical analysis and high-resolution confocal microscopy, it was demonstrated that V67A and P145A disrupted the recruitment of NS5A to lipid droplets. In addition, the localisation and size of lipid droplets in cells infected with these two mutants were perturbed. Biophysical analysis revealed that V67A and P145A abrogated the ability of purified domain I to dimerize and resulted in an increased affinity of binding to HCV 3’UTR RNA. Taken collectively, we propose that domain I of NS5A plays multiple roles in assembly, binding nascent genomic RNA and transporting it to lipid droplets where it is transferred to Core. In parallel, this study also set out to investigate the interactions of NS5A domain I with cellular proteins by the approach of quantitative proteomic analysis. This study reveals novel functions of NS5A domain I in assembly of infectious HCV and provides new perspectives on the virus lifecycle.
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Aydin, Cihan. „Hepatitis C Virus Non-Structural Protein 3/4A: A Tale of Two Domains: A Dissertation“. eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/626.
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Two decades after the discovery of the Hepatitis C Virus (HCV), Hepatitis C infection still persists to be a global health problem. With the recent approval of the first set of directly acting antivirals (DAAs), the rate of sustained viral response for HCV-infected patients increased significantly. However, a complete cure has not been found yet. Drug development efforts primarily target NS3/4A protease, bifunctional serine protease-RNA helicase of HCV. HCV NS3/4A is critical in viral function; protease domain processes the viral polyprotein and helicase domain aids replication of HCV genome by unwinding double stranded RNA transcripts produced by NS5B, RNA-dependent RNA polymerase of HCV. Protease and helicase domains can be isolated, expressed and purified separately while retaining function. Isolated domains of HCV NS3/4A have been extensively used in biochemical and biophysical studies for scientific and therapeutic purposes to evaluate functional capability and mechanism. However, these domains are highly interdependent and modulate the activities of each other bidirectionally. Interdomain dependence was demonstrated in comparative studies where activities of isolated domains versus the full length protein were evaluated. Nevertheless, specific factors affecting interdependence have not been thoroughly studied.
Chapter II investigates the domain-domain interface formed between protease and helicase domains as a determinant in interdependence. Molecular dynamics simulations performed on single chain NS3/4A constructs demonstrated the importance of interface in the coupled dynamics of the two domains. The role of the interface in interdomain communication was experimentally probed by disrupting the domain-domain interface through Ala-scanning mutations in selected residues in the interface with significant buried surface areas. These interface mutants were assayed for both helicase and protease related activities. Instead of downregulating the activities of either domain, interface mutants caused enhancement of protease and helicase activities. In addition, the interface had minimal effect in RNA unwinding activity of the helicase domain, the mere presence of the protease domain was the main protagonist in elevated RNA unwinding activity. In conclusion, I suspect that the interface formed between the domains is transient in nature and plays a regulatory role more than a functional role. In addition, I found results supporting the suggestion that an alternate domain-domain arrangement other than what is observed in crystal structures is the active, biologically relevant conformation for both the helicase and the protease.
Chapter III investigates structural features of HCV NS3/4A protease inhibitors in relation to effects on inhibitor potency, susceptibility to drug resistance and modulation of potency by the helicase domain. Nearly all NS3/4A protease inhibitors share common features, with major differences only in bulky P2 extension groups and macrocyclization statuses. Enzymatic inhibition profiles of different drugs were analyzed for wildtype isolated protease domain and single chain NS3/4A helicase-protease construct, their multi drug resistant variants, and additional helicase mutants. Inhibitor potency was mainly influenced by macrocyclization, where macrocyclic drugs were significantly more potent compared to acyclic variants. Potency loss with respect to resistance mutations primarily depended on the P2 extension, while macrocyclization had minimal effect except for P2-P4 macrocyclic compounds which were up to an order of magnitude more susceptible to mutations A156T and, in lesser extent, D168A. Modulation by helicase domain was also dependent on P2 extension, although opposite trends were observed for danoprevir analogs versus others. In conclusion, this study provides a basis for future inhibitor development in both avoiding drug resistance and exploitation of the helicase domain for additional efficacy.
In this thesis, I have provided evidence further supporting and revealing the details of domain-domain dependency in HCV NS3/4A. Lessons learned here will aid future research for dissecting the interdependency to gain a better understanding of HCV NS3/4A function, which can possibly be extended to all Flaviviridae NS3 protease-helicase complexes. In addition, interdomain dependence can be exploited in future drug development efforts to create better drugs that will pave the way to an effective cure.
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De, Zorzi Rita. „Structural studies on molecular recognition in protein complexes and supramolecular systems“. Doctoral thesis, Università degli studi di Trieste, 2009. http://hdl.handle.net/10077/3082.
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2007/2008Il riconoscimento molecolare tra due o più specie chimiche mediante interazioni non covalenti è il principale argomento di studio della chimica supramolecolare. Individuare i fini meccanismi di complementarietà che presiedono il processo di associazione molecolare è di fondamentale importanza sia per la comprensione di come funzionano i sistemi biologici naturali sia per lo sviluppo di nuovi sistemi supramolecolari artificiali. Nel presente lavoro di tesi, l’analisi delle interazioni che governano il riconoscimento molecolare sia in sistemi supramolecolari artificiali che in complessi proteici naturali è stata condotta attraverso la tecnica di diffrazione di raggi X da cristallo singolo, che consente la precisa identificazione delle interazioni coinvolte e dei gruppi funzionali responsabili del riconoscimento molecolare. In particolare, sono state analizzate le differenze tra due forme cristalline del citocromo c da Cuore di Cavallo, ottenute rispettivamente in ambiente ossidante e riducente in presenza di ioni nitrato. Lo ione nitrato è stato utilizzato in questo lavoro biocristallografico come sonda ionica per analizzare le variazioni della superficie elettrostatica connesse con il processo ossidoriduttivo del citocromo e per individuare i principali passaggi del meccanismo di riconoscimento molecolare in cui è coinvolto questo trasportatore di elettroni. Nell’ambito dello studio di sistemi in grado di mimare i sistemi biologici, sono stati analizzati anche complessi supramolecolari artificiali contenenti porfirine. Un nuovo versatile materiale nanoporoso è stato ottenuto attraverso utilizzo di interazioni non covalenti sinergiche tra calixareni e porfirine. Questa struttura supramolecolare che ricorda le zeoliti è stata successivamente funzionalizzata attraverso la diffusione di ioni metallici nei canali della struttura. Il materiale nanoporoso così ottenuto, contenente un pigmento porfirinico assieme ad uno ione metallico, è molto promettente per il successivo sviluppo di sistemi artificiali che coniugano la capacità di raccogliere la radiazione elettromagnetica nel campo del visibile con centri catalitici in grado di immagazzinare tale energia in legami chimici. In questo lavoro di tesi, un complesso, costituito da un nucleo formato da 4 ioni rutenio legati da ponti ossigeno, che ha dimostrato elevate capacità catalitiche nella reazione di produzione di ossigeno a partire dall’acqua in presenza di cerio (IV), è stato caratterizzato strutturalmente. Lo studio cristallografico ha permesso di ottenere dettagli strutturali importanti per la comprensione del meccanismo di reazione di tale complesso. Sensori che si avvalgono delle caratteristiche di reversibilità dell’interazione e di specificità del substrato tipiche della chimica supramolecolare possono essere ottenuti mediante la progettazione razionale di opportuni recettori molecolari. In questa tesi, cristalli isomorfi di un cavitando tetrafosfonato sono stati ottenuti in presenza di diversi alcoli guest, permettendo il confronto delle interazioni che determinano la formazione del complesso. Successivamente, sono stati portati a termine esperimenti di cocristallizzazione in presenza di coppie alcoliche, al fine di studiare la competizione tra queste specie per il sito del cavitando. Molecole a cavità che presentano funzionalità di host possono essere utilizzate anche nella progettazione di polimeri supramolecolari. Questo tipo di sistemi è particolarmente interessante per la possibilità di attivare o disattivare la polimerizzazione in risposta ad uno stimolo esterno. In questa tesi, un approccio di questo tipo è stato applicato alla sintesi di un omopolimero e di un eteropolimero.
Molecular recognition of two or more molecules through non covalent interactions is the field of supramolecular chemistry. The evaluation of the subtle mechanisms of complementarity inducing the molecular association has a fundamental importance in order to both elucidate biological processes and develop new artificial supramolecular systems. In the present thesis, analyses on various, artificial and natural, supramolecular systems, have been carried out using X-ray diffractions techniques on single crystals, that allow the precise determination of interaction geometries of the functional groups involved. In particular, structural differences between two crystal forms of Horse Heart cytochrome c, obtained in presence of nitrate ions, in an oxidizing and in a reducing environment, respectively, have been analysed. In this biocrystallographic work, nitrate ions have been used as ionic probes to analyse variations on the electrostatic surface due to the oxidoreductive processes of cytochrome and to identify the main steps of the molecular recognition mechanism, involving this electron transport protein. In order to develop systems able to mimicking biological processes, supramolecular complexes containing porphyrins have been analysed. A highly flexible nanoporous material has been obtained by synergistic non-covalent interactions of calixarene and porphyrin building blocks. This supramolecular zeolite-like structure has been easily functionalized by diffusion and coordination of metal ions in the large void channels of the crystals. This new nanoporous material, containing a porphyrinic dye together with a metal ion, is very promising for the development of artificial systems combining visible light harvesting properties and catalytic centres, able to store energy in chemical bonds. In this thesis, a complex constituted by a core of four ruthenium atoms bound through oxygen bridges, that demonstrated catalytic properties in oxygen evolving reactions from water oxidation in presence of Ce (IV), has been characterized through X-ray diffraction. The crystallographic analysis has allowed the determination of important structural details in order to understand the reaction mechanism of this complex. Sensing systems, that exploit the characteristics of reversibility of interactions and specificity of the substrate, typical of supramolecular chemistry, can be achieved with a rational design of suitable molecular receptors. In this thesis, isomorphic crystals of a tetraphosphonato cavitand have been obtained in presence of different alcoholic guests, allowing the comparison of interactions responsible for the complex formation. Afterwards, cocrystallizzation experiments in presence of two alcoholic species have been carried out in order to investigate the competition of these molecules for the cavitand site. Hollow molecules with host functionalities can also be exploited in the design of supramolecular polymers. These systems have attracted particular interest for the possibility of switch on/off the polymerisation after an external stimuli. In this thesis, a supramolecular approach has been applied in order to synthesize a homopolymer and a heteropolymer.
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Martin, Morgan Mackensie. „Functional analysis of hepatitis C virus non-structural protein (NS) 3 protease and viral cofactor NS4A“. Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/1522.
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The hepatitis C virus (HCV) was identified in 1989 as the major causative agent of transfusion-associated non-A, non-B hepatitis and today represents a worldwide health crisis with prevalence estimates of 2.2%. HCV-specific therapeutics have never been more urgently needed. One of the validated drug targets is the non-structural (NS) protein 3 (NS3) membrane-bound protease. The major aim of this thesis was characterization of NS3 allosteric activation by its viral cofactor, NS4A. We hypothesized that there would be specific residues that dominate the interaction between NS3 and NS4A, and further hypothesized that binding and activation may be separate events mediated by different residues.
This thesis details the development of novel cell-based assays for detection of NS3-4A protease activity and heterocomplex formation. The protease assay substrate was a membrane-targeted intracellular protein, which upon proteolysis released a red fluorescent protein (FP) reporter, DsRed-Express, into the cytoplasm; this change was detected by microscopy or quantified by Western blotting. The complex formation assay detected fluorescence resonance energy transfer (FRET) between yellow and cyan FP-tagged NS3 and NS4A, respectively.
Our data shows binding can be functionally separated from activation. We identified two NS4A residues (I25 and I29) important for NS3 binding and two NS4A residues (V23 and I25) important for NS3 activation. Therefore the binding-pockets of these residues are prime targets for small-molecule therapeutic development.
In addition, I have compared the NS3-4A substrate sequence cleavage efficiencies in vivo. I have been able to show that the activation-dependent NS4B/NS5A junction is processed efficiently and the NS4A/NS4B junction is not. I have also shown NS3-4A substrate specificity is not modulated by replicase components; however the specific activity of this enzyme is increased.
The strength of this thesis work stems from the novel and creative development of cell-based assays that can easily be modified to study other membrane-associated proteases. In vitro assays fall short in that they do not take into account the unique micro-environment in which these proteases are found.