Siga este enlace para ver otros tipos de publicaciones sobre el tema: NS4B protein.
Tesis sobre el tema "NS4B protein"
Crea una cita precisa en los estilos APA, MLA, Chicago, Harvard y otros
Consulte los 36 mejores tesis para su investigación sobre el tema "NS4B protein".
Junto a cada fuente en la lista de referencias hay un botón "Agregar a la bibliografía". Pulsa este botón, y generaremos automáticamente la referencia bibliográfica para la obra elegida en el estilo de cita que necesites: APA, MLA, Harvard, Vancouver, Chicago, etc.
También puede descargar el texto completo de la publicación académica en formato pdf y leer en línea su resumen siempre que esté disponible en los metadatos.
Explore tesis sobre una amplia variedad de disciplinas y organice su bibliografía correctamente.
1
Meyer, Aline Katharina [Verfasser] y Christoph [Akademischer Betreuer] Sarrazin. "Bedeutung eines prädizierten Leuzinzippermotivs im NS4B-Protein des Hepatitis-C-Virus für NS4B-Proteininteraktionen / Aline Katharina Meyer. Betreuer: Christoph Sarrazin". Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2013. http://d-nb.info/105290498X/34.
Texto completo
2
Lundin, Marika. "Topology and membrane rearrangements of the hepatitis C virus protein NS4B /". Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-927-0/.
Texto completo
3
Gretton, Sarah N. "Topology and biophysical characterisation of the hepatitis C virus NS4B protein". Thesis, University of Glasgow, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.433078.
Texto completo
4
Ismail, Rosmani. "Elucidation of the mechanism of action of a mutation in the dengue virus NS4B protein that confers a persistent phenotype in cell culture". Thesis, University of Bristol, 2015. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.684745.
Texto completoResumen
Dengue viruses (DENVs) are mosquito-borne flaviviruses, which are responsible for a wide range of illnesses, from a mild febrile illness to the more serious dengue haemorrhagic fever and dengue shock syndromes. The DENV NS4B is a highly hydrophobic integral ER membrane protein that has been reported to perturb type I interferon (IFN) signalling and has emerged as a major target for anti-viral strategies. The objective of this study was to determine the mechanism of action of two consecutive nucleotide mutations in the NS4B gene (nt 7020 and 7021) that result in the substitution of threonine 66 with alanine, which allow the virus to replicate persistently in mammalian and mosquito cells. Initially, a transient gene expression system was developed to analyse the effect of the mutations on the localisation of NS4B and its ability to perturb the IFN response and the unfolded protein response. The mutations were not found to significantly affect any of these processes. Novel cell lines were then produced that stably expressed the NS4B gene and its mutant counterpart in an inducible fashion. The cell lines were used to identify cellular proteins (DNAJA2, PFN2, FAF2, BAX and PMSG1) that potentially interact with the NS4B proteins using a high-throughput proteomic approach. Finally, HEK293T cell lines that were either stably persistently infected with a virus containing the NS4B mutations or contained a wild type DENV replicon or a replicon containing the NS4B mutations were established. Genome sequencing demonstrated that the NS4B mutations were more stable in a replicon than in the full viral genome. The effect of the NS4B mutations on host cell processes was investigated by transcriptomic analysis using RNAseq. The transcriptomic analysis revealed that canonical cellular pathways were affected by viral replication including IFN signaling, virus recognition, ubiquitination, apoptosis, and ER stress. Further analysis showed that the NS4B mutations affected the ability of the virus and a DENV replicon to effectively suppress IFN signaling and IFN sensitive gene expression. This study provides further insight into the role of the NS4B protein in the viral-host interaction and provides a mechanism to explain how a persistent DENV infection may occur.
5
Lindström, Hannah Kim. "Molecular studies of the hepatitis C virus : the role of IRES activity for therapy response, and the impact of the non-structural protein NS4B on the viral proliferation /". Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-875-4/.
Texto completo
6
Machmouchi, Dana. "Exploring the Pathogenic Mechanisms of West African Zika Virus : viral Replication and Host Interaction". Electronic Thesis or Diss., La Réunion, 2024. https://elgebar.univ-reunion.fr/login?url=http://thesesenligne.univ.run/24_14_D_MACHMOUCHI.pdf.
Texto completoResumen
Le virus Zika (ZIKV), historiquement limité à l'Afrique et à l'Asie, est devenu une préoccupation mondiale majeure, surtout après les épidémies récentes dans les Amériques associées à des malformations congénitales graves et des troubles neurologiques. Bien que la recherche se soit principalement concentrée sur le génotype asiatique/américain, des preuves croissantes montrent que les souches africaines du ZIKV pourraient également représenter une menace sévère, notamment en termes de pathogénicité fœtale. Cette thèse vise à améliorer notre compréhension des mécanismes moléculaires de la pathogénicité des souches contemporaines de ZIKV d'Afrique de l'Ouest, en mettant l'accent sur les protéines non structurales dans la réplication virale, l'évasion immunitaire et la réponse au stress des cellules hôtes.Pour cela, nous avons généré un clone moléculaire infectieux, GUINEA-18, à partir d'une souche de ZIKV (ZIKV-15555) isolée en Guinée en 2018, représentant une souche contemporaine de la lignée africaine. Nous avons comparé ce clone avec le clone bien caractérisé de la souche historique MR766 (MR766MC). Les propriétés de réplication des deux clones ont été examinées dans les lignées cellulaires VeroE6, A549 et HCM3. GUINEA-18 a montré un taux de réplication plus lent, une cytotoxicité réduite et une moindre capacité à activer le système immunitaire inné comparé à MR766MC, suggérant une interaction différente avec les cellules hôtes.Pour explorer ces différences, nous avons construit des virus chimériques en échangeant les régions codantes des protéines non structurales entre GUINEA-18 et MR766MC. Nos résultats ont souligné les rôles critiques des protéines NS1 à NS4B dans la réplication et la pathogénicité, avec NS4B étant clé pour GUINEA-18. Nous avons également découvert que GUINEA-18 inhibe efficacement l'assemblage des granules de stress cytoplasmiques (SGs) dans les cellules A549, un mécanisme de défense cellulaire. Cette inhibition dépend des protéines NS1 à NS4B, soulignant leur rôle dans l'évasion des défenses de l'hôte.La thèse examine aussi le rôle de NS1 dans la pathogénicité des souches contemporaines. L'alignement des séquences protéiques a révélé sept substitutions dans la protéine NS1 de GUINEA-18 par rapport à celle de MR766. Ces mutations montrent que NS1CWA est sécrétée plus efficacement et présente une localisation subcellulaire différente de NS1MR766, augmentant la réplication virale et la cytotoxicité tout en réduisant l'activation des réponses immunitaires. Un virus chimérique MR766 avec NS1CWA a montré ces traits pathogéniques renforcés, soulignant l'importance de NS1 dans la virulence des souches contemporaines du ZIKV d'Afrique de l'Ouest.En conclusion, cette thèse analyse les déterminants moléculaires de la réplication et de la pathogénicité des souches contemporaines du ZIKV d'Afrique de l'Ouest. Les recherches mettent en évidence les rôles critiques des protéines NS1 à NS4B, en particulier NS1 et NS4B. Les résultats soulèvent des questions sur les risques associés aux souches actuelles du ZIKV en Afrique subsaharienne et soulignent la nécessité de surveillance continue pour comprendre les implications pour la santé publique. Ce travail offre des perspectives pour les stratégies de gestion et de prévention des maladies associées au ZIKV, surtout dans les régions où la lignée africaine est prévalenteThe Zika virus (ZIKV), historically confined to Africa and Asia, has become a significant global health concern, especially after recent outbreaks in the Americas linked to severe congenital malformations and neurological disorders. While much research has focused on the Asian/American ZIKV genotype, evidence suggests that African ZIKV strains might also pose a serious threat to public health, particularly regarding fetal pathogenicity. This thesis aims to enhance our understanding of the molecular mechanisms underlying the pathogenicity of contemporary ZIKV strains from West Africa, focusing on nonstructural proteins involved in viral replication, immune evasion, and the host cell stress response.To achieve this, we generated an infectious molecular clone, GUINEA-18, from a ZIKV strain (ZIKV-15555) isolated in Guinea in 2018. This clone represents a contemporary African ZIKV strain. We compared it with the infectious molecular clone of the historical African ZIKV strain MR766, designated MR766MC. The replication properties of both viral clones were examined in VeroE6, A549, and HCM3 cells. GUINEA-18 exhibited a slower replication rate, reduced cytotoxicity, and a lower ability to activate the host’s innate immune system compared to MR766MC, suggesting different interactions with host cells.To dissect these differences, we created chimeric viruses by swapping nonstructural protein-coding regions between GUINEA-18 and MR766MC. Results highlighted the critical roles of NS1 to NS4B proteins in replication efficiency and pathogenicity, with NS4B being crucial for GUINEA-18’s replication properties. GUINEA-18 also developed an efficient mechanism to inhibit the assembly of cytoplasmic stress granules (SGs) in A549 cells, a defense mechanism typically triggered by viral infection. The ability of GUINEA-18 to block SG formation depended on the NS1 to NS4B proteins, underscoring their role in evading host defenses.Further investigation into the NS1 protein revealed seven amino acid substitutions in GUINEA-18 compared to MR766. Functional analyses showed that the contemporary NS1 protein (NS1CWA) is secreted more efficiently and has a different subcellular localization than NS1 from MR766 (NS1MR766). This altered behavior of NS1CWA significantly enhances viral replication and cytotoxicity while reducing the activation of innate immune responses in infected cells. A chimeric MR766 virus containing NS1CWA demonstrated these enhanced traits, emphasizing NS1’s role in the virulence of contemporary West African ZIKV strains.In conclusion, this thesis provides a comprehensive analysis of the molecular determinants of replication and pathogenicity in contemporary West African ZIKV strains. The research underscores the crucial roles of NS1 to NS4B proteins, particularly NS1 and NS4B, in these processes. The findings raise questions about risks associated with circulating ZIKV strains in sub-Saharan Africa and highlight the need for ongoing surveillance and research to understand the public health implications. This work contributes valuable insights that could inform future strategies for managing and preventing ZIKV-associated diseases, especially in regions where the African lineage of the virus is prevalent
7
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.
Texto completoResumen
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
8
Taylor, Annette Irene. "The intracellular localisation and membrane-altering properties of hepititis C virus proteins NS4B and NS5A". Thesis, University of Glasgow, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.274768.
Texto completo
9
Jin, Yi. "Characterisation of the African horse sickness virus NS4 protein". Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/8973/.
Texto completoResumen
African horse sickness is one of the most deadly infectious diseases of horses. The disease is caused by African horse sickness virus (AHSV), an arbovirus transmitted by culicoides midges. AHSV is classified within the genus Orbivirus, family Reovirdae. The AHSV genome is composed by ten segments of double stranded RNA (dsRNA) encoding seven structural and at least four non-structural (NS) proteins. AHSV shares structural and functional similarities with Bluetongue virus, the ‘prototype’ species of the genus Orbivirus. An alternative open reading frame (ORF), overlapping the main ORF encoding the VP6, has been identified in segment 9 in both AHSV and BTV. This additional ORF encodes the non-structural protein NS4. The BTV NS4 localises in the nucleoli of the infected cells. NS4 is an interferon antagonist and a determinant of virus virulence. In this thesis, I aimed to characterise the AHSV NS4. Unlike the BTV NS4, the AHSV NS4 are relatively variable mong different virus strains. I have divided these proteins into four different subtypes: NS4I, NS4-IIα, -IIβ, and IIγ based upon their sequence similarity and on the presence of N-terminal or C-terminal truncations. In contrast to BTV, all four of these NS4 types localise in the cytoplasm of either transfected or infected cells. In addition, in transient transfection assays all the NS4 types show the ability to hamper gene expression, with NS4-IIβ being the most efficient. In order to further understand the biological significance of the AHSV NS4 we used reverse genetics to generate viruses expressing the four types of NS4 (AHSV-NS4-I, AHSV-NS4-IIα, AHSV-NS4-IIβ, AHSV-NS4-IIγ) and the corresponding NS4 deletion mutants (AHSV-ΔNS4-I etc.). Deletion of NS4 did not affect virus replication kinetics in either KC cells or interferon incompetent cells such as the BSR cell line. Similarly, both AHSV-NS4-IIβ and the corresponding ΔNS4 mutant showed similar replication kinetics in the interferon competent E. Derm cell line and in primary horse endothelial cells. In contrast, AHSV-NS4-I, AHSV-NS4-IIα, and AHSV-NS4-IIγ replicated more efficiently than the corresponding ΔNS4 viruses in these horse cells. Interestingly, the defects in replication of the NS4 viruses were removed after treatment with an inhibitor of the JAK/STAT pathway. Indeed, we observed that primary horse cells infected with the NS4 mutants released higher levels of type I interferon (IFN) than cells infected with the corresponding NS4 expressing viruses. In addition, we found the NS4 to be a determinant of virus virulence in vivo in NIH-Swiss mice infected with the viruses described above. Collectively, the data described in this thesis suggest that the NS4 is one of the proteins used by AHSV to modulate the IFN response.
10
Choi, Yook-Wah. "Structural and functional characterization of human DDX5 and its interaction with NS5B of hepatitis C virus". University of the Western Cape, 2011. http://hdl.handle.net/11394/5299.
Texto completoResumen
Philosophiae Doctor - PhDHepatitis C was first recognized as a transfusion-associated liver disease not caused by hepatitis A or hepatitis B virus after serological tests were developed to screen for their presence in the blood. The infectious agent was finally identified with the cloning of the cDNA of hepatitis C virus (HCV) using random polymerase chain reaction (PCR) screening of nucleic acids extracted from plasma of a large pool of chimpanzee infected with non-A non-B hepatitis. NS5B, a membrane-associated RNA-dependent RNA polymerase essential in the replication of HCV, initiates the synthesis of a complementary negative-strand RNA from the genomic positive-strand RNA so that more positive-strand HCV RNA can then be generated from the newly synthesised negative-strand template. The crystal structure of NS5B presented typical fingers, palm and thumb sub-domains encircling the GDD active site, which is also seen in other RNA-dependent RNA polymerases, and is similar to the structure of reverse transcriptase of HIV-1 and murine Moloney leukaemia virus. The last 21 amino acids in the C-terminus of NS5B anchor the protein to the endoplasmic reticulum (ER)-derived membranous web. NS5B has been shown to interact with the core, NS3/NS4A, NS4B and NS5A proteins, either directly or indirectly. Numerous interactions with cellular proteins have also been reported. These proteins are mainly associated with genome replication, vesicular transport, protein kinase C-related kinase 2, P68 (DDX5), α-actinin, nucleolin, human eukaryotic initiation factor 4AII, and human VAMP-associated protein. Previous studies have confirmed that NS5B binds to full-length DDX5. By constructing deletion mutants of DDX5, we proceeded to characterize this interaction between DDX5 and HCV NS5B. We report here the identification of two exclusive HCV NS5B binding sites in DDX5, one in the N-terminal region of amino acids 1 to 384 and the other in the C-terminal region of amino acids 387 to 614. Proteins spanning different regions of DDX5 were expressed and purified for crystallization trials. The N-terminal region of DDX5 from amino acids 1 to 305 which contains the conserved domain I of the DEAD-box helicase was also cloned and expressed in Escherichia coli. The cloning, expression, purification and crystallization conditions are presented in this work. Subsequently, the crystal structure of DDX5 1-305 was solved and the high resolution three-dimensional structure shows that in front of domain I is the highly variable and disordered N terminal region (NTR) of which amino acids 51-78 is observable, but whose function is unknown. This region forms an extensive loop and supplements the core with an additional α-helix. Co-immunoprecipitation experiments demonstrated that the NTR of DDX5 1-305 auto-inhibit its interaction with NS5B. Interestingly, the α-helix in NTR is essential for this auto-inhibition and seems to mediate the interaction between the highly flexible 1-60 residues in NTR and NS5B binding site in DDX5 1-305, presumably located within residues 79-305. Furthermore, co-immunoprecipitation experiments revealed that DDX5 can also interact with other HCV proteins, besides NS5B.
11
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.
Texto completoResumen
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.
12
Meguellati, Amel. "Synthèse de biomolécules agissant comme inhibiteurs de l'ARN polymérase ARN dépendante du virus de l'hépatite C et développement de nouveaux surfactants comme stabilisants des protéines membranaires par réseaux de ponts salins". Thesis, Université Grenoble Alpes (ComUE), 2015. http://www.theses.fr/2015GRENV001.
Texto completoResumen
Le projet de thèse se focalise sur la synthèse de biomolécules et se subdivise en deux parties. La première partie concerne la conception et la synthèse de dérivés de produits naturels d'intérêt thérapeutique nommés aurones en vue de mettre au point de nouvelles molécules à activité antivirale. Récemment, les aurones ont été identifiées comme étant des inhibiteurs de l'ARN-polymérase ARN-dépendante (NS5B) du virus de l'hépatite C (VHC). Cette enzyme, présente chez le virus mais absente chez l'homme, joue un rôle central dans la réplication virale. Suite à ces résultats antérieurs, les efforts ont été poursuivis et, dans le cadre de cette thèse, nous avons entrepris,d'une part, la synthèse d'analogues originaux dont le cycle B des aurones a été remplacé par des hétérocycles et, d'autre part, la synthèse depseudodimères d'aurones dans le but d'affiner les exigences structurales pour améliorer l'effet inhibiteur.L'activité a été évaluée selon des tests enzymatiques et cellulaires et a permis d'identifier quelques candidats doués d'une bonne activité inhibitrice et d'une faible toxicité. La deuxième partie du projet de thèse, sans lien avec la première partie,concerne des aspects plus fondamentaux et porte sur la synthèse de nouveaux surfactants agissant comme agents stabilisants lors des procédures d'extraction et de cristallisation des protéines membranaires. Les surfactants sont des composants clés dans le domaine de la biologie structurale et de la biochimie des protéines membranaire. Ils sont nécessaires pour maintenir les protéines membranaires dans leur état fonctionnel après extraction. La grande majorité des protéines membranaires est riche en résidus basiques à l'interface. Sur la base de cette caractéristique, une nouvelle famille de surfactants est développée et testée sur des protéines membranaires appartenant aux pompes d'efflux ABC multi-résistantesThe PhD project focuses on biomolecules and is divided into two parts. The first part concerns the design and synthesis of natural product derivatives with therapeutic interest in order to develop new molecules with antiviral activity. Recently, aurones were identified as new inhibitors of hepatitis C virus (HCV) NS5B polymerase. Following these results, efforts were continuedand we undertook, on the one hand,the synthesis of original analogues in which the aurone B-ring was replaced by a heterocyclic rings and, on the other hand, the synthesis of aurone pseudodimers in order to refine the structural requirements to improve the inhibitory effect. The potent NS5B inhibitory activity combined with their low toxicity make aurones attractive drug candidates against HCV infection. The second part of the PhD thesis is unrelated to the first part and concerns more fundamental aspects. It focused on the synthesis of new surfactants acting as stabilizing agents during extraction of membrane proteins (PM). Surfactants are required for maintaining PM in their functional state after extraction from membrane lipid matrix. The vast majority of PM shares a net enrichment in basic residues at the interface between membrane and cytoplasm, a property known as the positive inside rule. Based on this feature, a new family of surfactants is developed and tested on membrane proteins belonging to the multidrug ABC efflux pumps family
13
Hung, Yu Fu Verfasser] y Dieter [Akademischer Betreuer] [Willbold. "Biophysical characterization of the N-terminal region of Dengue virus NS4A protein / Yu -Fu Hung. Gutachter: Dieter Willbold". Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2014. http://d-nb.info/1059855232/34.
Texto completo
14
Mamigonian, Bessa Luíza. "Investigation of the hepatitis C virus RNA polymerase NS5B in solution by nuclear magnetic resonance and its interaction with intrinsically disordered domain 2 of the NS5A protein". Thesis, Lille 1, 2017. http://www.theses.fr/2017LIL10117/document.
Texto completoResumen
NS5B est l’ARN polymérase du virus de l’hépatite C (VHC). Sa structure a beaucoup été étudiée par radiocristallographie, elle contient trois sous-domaines appelés doigts, paume et pouce. Cependant, les études structurales de cette protéine en solution sont très limitées. La résonance magnétique nucléaire (RMN) a été utilisée pour étudier NS5B en solution ainsi que son interaction avec différents partenaires. L’emploi d’un échantillon de NS5B (65kDa) perdeuterée et sélectivement enrichie au niveau des méthyles 1 des résidus d’isoleucines a permis d’obtenir un spectre simplifié et de bonne qualité. Cette étude a confirmé la présence d’une dynamique particulière dans le pouce et a permis de mettre en évidence des effets à longues distances que se transmettent aux autres sous-domaines. Cette approche a alors été utilisée pour étudier l’interaction entre NS5B et le domaine 2 de la protéine NS5A (NS5A-D2) du VHC. Celui-ci est un domaine intrinsèquement désordonné qui interagit directement avec NS5B in vitro. Nous avons identifié que NS5A-D2 se lie via deux sites d’interaction sur le sous-domaine du pouce. Puisqu’un de ces sites est le site de liaison de l’inhibiteur allostérique filibuvir, nous avons étudié la liaison de cette molécule à la polymérase. Sa liaison cause des effets à longues distances tout au long de NS5B. Enfin, nous avons caractérisé la liaison d’un ARN simple brin à NS5B et nous avons identifié que NS5A-D2 et filibuvir réduisent mais ne suppriment pas l’interaction de NS5B avec l’ARN. L’analyse de NS5B par RMN en solution a permis d’étudier des interactions et d’accéder à des paramètres dynamiques très complémentaires des études cristallographiquesNS5B is the hepatitis C virus (HCV) RNA-dependent RNA polymerase. This protein has been extensively studied by X-ray crystallography and shows an organization in three subdomains called fingers, palm and thumb. Whereas static crystallographic data are abundant, structural studies of this protein in solution are limited. Nuclear magnetic resonance (NMR) spectroscopy was used to study the 65 kDa NS5B in solution as well as its interaction with binding partners. It was characterized using selective isotopic labeling of isoleucine side-chain methyl groups, which gives rise to a simplified NMR spectrum with an improved signal-to-noise ratio. This characterization confirmed the presence of particular dynamics in the subdomains, especially in the thumb, as well as long-range effects that are transmitted through to other subdomains. Furthermore, this system was used to investigate the binding of the domain 2 of NS5A (NS5A-D2), a disordered domain of another HCV protein that has been shown to directly interact with NS5B in vitro. With paramagnetic relaxation enhancement experiments we showed that NS5A-D2 binds to NS5B via, at least, two binding sites on the thumb subdomain. As one of these sites was the binding site of allosteric inhibitor filibuvir, we characterized the binding of this small molecule to NS5B by NMR and found long-range effects of its binding throughout the polymerase. Finally, we studied the binding of a small RNA template strand to NS5B and found that both NS5A-D2 and filibuvir reduce but do not abolish the interaction between the polymerase and RNA. In sum, NMR spectroscopy was used to study dynamic properties of NS5B and its interactions with binding partners
15
Dünnes, Nadia [Verfasser]. "Analyse der Interaktion von microRNA-122-Protein-Komplexen mit der NS5B-kodierenden Region und der 3´-untranslatierten Region der Hepatitis C Virus-RNA / Nadia Dünnes". Gießen : Universitätsbibliothek, 2016. http://d-nb.info/1118289773/34.
Texto completo
16
Kuo, Yi-Chen y 郭逸楨. "The NTPase Activity of Hepatitis C Virus NS4B protein". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/47366072821472642267.
Texto completoResumen
碩士國立臺灣大學
生物化學暨分子生物學研究所
93
Hepatitis C virus (HCV) is a major cause of liver disease worldwide. HCV contains a positive-stranded RNA genome of 9.6 kilobases encodes a polyprotein of about 3000 amino acid residues. The polyprotein undergoes cellular and viral protease processing to generate structural and nonstructural proteins. Nonstructural protein 4B (NS4B) is a relatively hydrophobic 27 kDa protein of unknown function. NS4B has four transmembrane segments and has the ability to induce a tight structure formation as membranous web. It is proposed that HCV NS5B RNA polymerase forms replication complex with other nonstructural proteins at the membranous web. NS4B possesses GTPase activity. It contains a nucleotide binding motif (NBM) that mediates binding and hydrolysis of GTP previously demonstrated to be important for HCV replication. Substitutions at the conserved K-1846 with serine or arginine in the A motif (1840-GX1X2X3X4GK-1846) of NBM impaired GTP binding and hydrolysis, and resulted in inhibition of HCV RNA replication. Nevertheless, a cell culture adaptive mutant K1846T in which K-1846 was mutated to threonine enhances HCV RNA replication to 30 fold. In this study, NS4B and NS4B(K1846T) were detected in the cytoplasm of transfected cells and preferentially localized at the perinuclear region by immunofluorescence staining assay. To examine the NTPase activities of NS4B and NS4B(K1846T), GST-NS4B and GST-NS4B(K1846T) were expressed in E. coli, purified by glutathione Sepharose 4B, and cleaved to NS4B and NS4B(K1846T) by PreScission protease. HCV NS4B protein possesses both GTPase and ATPase activities. K1846T mutation decreased the ATPase activity of NS4B to 40%, but had little effect on GTPase activity. The results indicate that the lysine-1846 in the nucleotide binding motif is not absolutely required for the GTPase activity of NS4B. Different from previous studies, the results suggest that the adaptive mutant K1846T may promote HCV replication by mechanisms other than the GTPase activity of NS4B protein.
17
楊馥嘉. "Functional Expression of Nonstructural Proteins NS2A, NS2B, NS4A, and NS4B of Dengue Virus Type 2 PL046 Strain". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/92535778180784776173.
Texto completo
18
Racine, Marie-Eve. "Étude du réseau d'interactions entre les protéines du Virus de l'Hépatite C". Thèse, 2007. http://hdl.handle.net/1866/7243.
Texto completo
19
Lin, Hsu Jye y 徐婕琳. "Expression of Nonstructural Proteins: NS2A, NS2B, NS4A, and NS4B of Dengue Virus Type 2 PL046 in E. coli, Mammalian, and Pichia pastoris Expression Systems". Thesis, 2003. http://ndltd.ncl.edu.tw/handle/19256682776855580136.
Texto completoResumen
碩士國立交通大學
生物科技研究所
91
Dengue virus is a member of flaviviridas, which cause many infectious diseases of human. Dengue virus encodes 11 proteins in a single open reading frame. Among them, NS2A, 2B, 4A and 4B are small non-structural proteins exhibiting conserved hydrophobicity profiles among flaviviuses, suggesting their roles as membrane-associated proteins. Only NS2B has been suggested to be involved in protease activity. Specific biological functions for NS2A, 4A and 4B have not been identified. To delineate the structure and function of these four proteins, I ttempted to establish cloning and expression of these four genes. They were initially expressed as His-HA (polyhistidine-influenza hemagglutinin protein) fusion proteins in E. coli, Pichia pastoris, and mammalian cells. For expression of these proteins in different expression systems, the coding region of NS2A, 2B, 4A and 4B were RT-PCR amplified from dengue virus type 2 PL-046 strain and were successfully cloned into pcDNA3, an E. coli/mammalian cells expression shuttle vector, and pPIC3.5, a Pichia pastoris expression vector. Recombinant proteins of NS2A, 2B, 4A and 4B fusing with HA-His tag can be detected in soluble fraction of E. coli expression system by Western blotting analysis and purified with nickel column. Protein expression of NS2A, 2B and 4A in mammalian cell and NS2B and NS4A in Pichia pastoris, KM71 was also detected by Western analysis.
20
(8623374), Shishir Poudyal. "A COMBINED GENETIC AND CHIMERIC ANALYSIS OF THE FLAVIVIRAL NON-STRUCTURAL PROTEINS". Thesis, 2020.
Buscar texto completoResumen
A successful flaviviral life cycle involves several coordinated events between viral proteins and host factors. The polyprotein processing at the surface of the ER membrane results in the formation of several replication proteins that bring about changes in the ER membrane making it permissive for viral genome amplification. Non-structural proteins 4A (NS4A) and non-structural protein 4B (NS4B) are two of the most important integral membrane proteins of DENV that are essential part of the viral replicase complex. The cleavage at NS4A-2K-NS4B is temporally and spatially regulated. The cleavage at the N-terminal of 2K is carried out by viral NS2B/3 protease while host signalase cleaves on the C-terminal side at the ER lumen to give rise to a mature NS4B protein. This thesis primarily focuses on demonstrating the function of 2K as an independent peptide rather than simply a signal sequence, and the role 2K plays, when present as 2K-NS4B vs NS4B. Moreover, this thesis has attempted to explore the function of transmembrane domains (TMDs) in replication separating them from their membrane anchor function. This thesis will also describe the development of a ZIKV replicon and its use in screening small molecule inhibitors in the last chapter.
In Chapter 2 of the thesis, we established 2K as an independent, information carrying peptide rather than just a signal peptide. A strategy involving chimeric virus generation and mutational analysis supported the notion that 2K is rather unique and important for viral replication and infectious particle production. Using an interserotypic 2K chimeric virus, it was established that the 2Ks of DENV are serotype specific, however, they are interchangeable with a huge fitness cost in infectious particle production. We further showed that individual amino acid residues towards then end of h-region and C-terminus of the 2K peptide affect viral replication and infectious particle production. Moreover, it was shown that the 2K peptide consists of a highly conserved ‘DNQL’ region at its N-terminal that plays an important role in viral replication.
Chapter 3 details the mechanistic aspect of the effects observed in interserotypic 2K chimeric viruses. The interserotypic chimeric viruses were comparable to wild type in replication, however, they were deficient in infectious particle production early in the life cycle. The major change to be noted in the chimeric viruses was the absence of signalase cleavage at the 2K-NS4B junction. We demonstrated that in a virus infected system, 2K-NS4B and NS4B populations are always present which led us to look for any specific functions of the cleaved vs uncleaved 2K-NS4B protein. Using a transcomplementation system where NS4B was presented in the absence of 2K, we showed that particle production can be rescued in the interserotypic 2K chimeric viruses. It was further concluded using NS4B truncations that the property of NS4B to rescue particle production was concentrated in the ER luminal loop. Further, alanine scanning mutagenesis of the conserved residues of ER loop resulted in pinpointing T198 and its involvement in the early stages of viral packaging.
Chapter 4 examined the role of TMDs of NS4A and NS4B and attempted to define their roles separately from their membrane anchoring functions. Several interserotypic TMD chimeric viruses were generated to address the function of these domains. We concluded that TMD1 and TMD3 of NS4A could be replaced with partial success across the DENV serotypes, whereas, TMD2 was serotype specific. The specificity of TMD2 of NS4A is not contributed by a single amino acid and should be a function of the secondary structure formed by TMD2 as it sits on the inner leaflet of the ER membrane. We demonstrated the variable roles different TMDs of NS4B play in viral replication using a similar strategy of reverse genetics of chimeric viruses. TMD1 of NS4B was replaceable with no to minimal effect, whereas, the remaining four showed variable effect upon substitution. More importantly, we demonstrated how the reorientation of TMD5 of NS4B post NS2B/3 cleavage might vary in different serotypes of DENV using revertant virus obtained from the TMD5 interserotypic chimera. Analysis of interserotypic cytosolic and ER luminal loop chimeras of NS4B pointed to functional conservation of the cytosolic loop between DENV-2 and DENV-3, whereas, the remaining cytosolic loops and the ER loops showed variable level of defects upon substitution, suggesting their functions in serotype-dependent manner.
Chapter 5 describes the construction and characterization of a ZIKV replicon system and use of it to screen several small molecule inhibitors of the flaviviruses MTase. Several small molecule inhibitors of flavivirus N-7-MTase were designed/synthesized in Dr. Arun K Ghosh’s lab which would target the extra pocket unique to the flavivirus SAM-binding site. We analyzed the docking of a set of these compounds into MTase domain of NS5 of ZIKV, DENV and YFV and screened them for their ability to inhibit replication of ZIKV, DENV and YFV. A huge variation in the activity profile of these compounds were observed against different flaviviruses even though these compounds were targeted against the highly conserved MTase domain of flavivirus NS5. GRL-002- and GRL-004-16-MT specifically inhibited ZIKV replication with low micromolar IC50 value, while these compounds showed little to no effect on DENV and YFV. On the other hand, compounds GRL-007-, GRL-0012- and GRL-0015-16-MT demonstrated a dual inhibitory effect against DENV and YFV albeit the CC50 values of the GRL-012 and GRL-015 were concerning. Compounds GRL-007-16-MT showed broad spectrum activity against ZIKV, DENV and YFV even though it was slightly cytotoxic to Vero cells. Moreover, GRL-002-16 was inhibitory to YFV while ineffective against DENV, whereas, GRL-016-16 had the opposite effect. Our results reveal the differential efficacies of the small molecule inhibitors targeting N-7-MTase. The experimental data suggests these compounds have different cytotoxicities in different cell lines and the compounds act in a virus-specific way. Nonetheless, we were able to shortlist some potent compounds for future modifications.
21
Chen, Ming-Yuan y 陳明圓. "Hepatitis C Virus Nonstructural Protein 5B (NS5B) Interacts with Akt". Thesis, 2008. http://ndltd.ncl.edu.tw/handle/87849171233555725407.
Texto completoResumen
碩士國立陽明大學
藥理學研究所
96
Hepatitis C virus (HCV) is recognized as a major causative agent of chronic hepatitis, cirrhosis, and hepatocellular carcinoma. The phosphatidylinositol 3-kinase (PI3K)–Akt pathway is utilized by many viruses, including HBV and HCV, for inhibition of apoptosis of infected cells. Akt, a serine/threonine kinase, is a substrate of PI3K and acts as an important signal mediator of cell survival following activation. The HCV NS5B, a putative serine phosphoprotein, is the viral RNA-dependent RNA polymerase (RdRP) required for replication of the HCV RNA genome. The phosphorylated form of HCV NS5B was observed when expressed in insect cells using a recombinant baculovirus. Though protein kinase C-related kinase 2 was implicated to be responsible for HCV NS5B phosphorylation, phosphorylation of HCV NS5B in human liver cells has never been demonstrated. Previously, we noticed that there is a putative Akt-phosphorylation motif, (RXRXXS/T), containing the potential phosphorylated serine residue at amino acid 506 and two arginine residues at amino acid 501 and 503 in the catalytic core region of HCV NS5B. Most importantly, this motif is highly conserved in HCV genotype 1 but not in non-1 genotype. Therefore, we constructed several mammalian vectors expressing NS5B, including wild type, S506A, S506D, from HCV genotype 1b and genotype 2b (A506). By using co-immunoprecipitation, we demonstrated that NS5B of HCV genotype 1b interacts with Akt in both non-hepatocytes-derived 293T and hepatocyte-derived Huh7 cell lines. NS5B-1b mutant (S506A) and NS5B-2b also bound Akt but with weaker affinity than NS5B-1b (S506). The in vivo interaction of NS5B-Akt was further verified in confocal microscopy study. By using GST pull down assay, the in vitro interaction of NS5B and Akt was also demonstrated. The Akt binding site on NS5B could be mapped to the thumb domain of NS5B (372-591), the region containing the putative Akt phosphorylation motif. Most importantly, both full length (1-591) and the thumb domain of NS5B (372-591) bind the activated Akt with its serine phosphorylated form, implicating that NS5B might be a substrate of Akt. Further study aiming to elucidate the biological significance of the interplay between NS5B and Akt and its effect on HCV replication and host signaling pathways is ongoing. Hopefully, understanding of the interplay of HCV NS5B and Akt may also facilitate new therapeutic strategy development for HCV infection.
22
Henningson, Jamie N. "Influence of BVDV nonstructural proteins N(pro) and NS4B on virulence in experimental acutely infected calves". 2008. http://proquest.umi.com/pqdweb?did=1584072581&sid=3&Fmt=2&clientId=14215&RQT=309&VName=PQD.
Texto completoResumen
Thesis (Ph.D.)--University of Nebraska-Lincoln, 2008.Title from title screen (site viewed Jan. 13, 2009). PDF text: vii, 268 p. : col. ill. ; 5 Mb. UMI publication number: AAT 3321565. Includes bibliographical references. Also available in microfilm and microfiche formats.
23
Wang, Yi-Ming y 王怡敏. "The Mechanisms of Hepatitis C Virus NS4A Protein on the Inhibition of Protein Synthesis and theInternal Cleavage of NS3 Protein". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/23492695413222383664.
Texto completoResumen
碩士國立臺灣大學
微生物學研究所
93
Hepatitis C virus (HCV) is a positive, single-stranded RNA virus. Previous studies have demonstrated that the viral nonstructural protein NS4A interacts with NS3 and is a cofactor of NS3 serine protease essential for the proteolytic processing of the viral polyprotein. NS4A protein was also demonstrated to inhibit cellular and viral protein synthesis. By performing GST pull down assay, our laboratory has previously identified eEF1A that specifically interacted with NS4A. The purpose of this study was to examine whether NS4A inhibits protein translation via interacting with eEF1A. By performing luciferase assay, the inhibition of translation by NS4A protein was confirmed in this study. The eEF1A C-terminal domain, responsible for binding with eEF1B and tRNA was identified to be involved in the interaction. Furthermore, mutations at Val-23 [NS4A(V23A)] and at Ile-25 and Val-26 [NS4A(I25AV26A)] disrupted the interaction between NS4A and eEF1A. The effect of NS4A on translation inhibition was also decreased with V23 and I25AV26A mutations. In contrast, a mutation at Val-24 [NS4A(V24A)] remained the ability of NS4A to interact with eEF1A and the inhibition effect on luciferase activity. These results suggest NS4A may interfere with protein synthesis by forming complex with eEF1A. This may be involved in HCV infection and helps its survival in host cells. It was reported the NS3 protein is internally cleaved in the presence of NS4A when expressed in HepG2, COS-7, and NIH3T3 cells. The internal cleavage of NS3 protein required not only NS4A as a cofactor but also the activity of NS3 serine protease. However, in this study, we found that NS4A(I25AV26A) that retains the function to act as a cofactor of NS3 was unable to induce the internal cleavage of NS3. It suggests that the serine protease activity of NS3 is not sufficient for its internal cleavage. In addition, the internal cleavage products of NS3 appeared to have higher oncogenic potential than the intact NS3. In this study, soft agar analysis was performed to compare the transformation activity of NS3 in the presence of NS4A or NS4A(I25AV26A). Preliminary data did indicate a higher transformation activity of NS3 in the presence of wild type NS4A. Nevertheless, the numbers of transforming colonies were low and may need to be further confirmed. The possible roles of the interactions among the viral nonstructural proteins, on the oncogenesis of HCV infection remain to be elucidated.
24
Tseng, Fan-Wei y 曾繁偉. "Hepatitis C Virus Nonstructural Protein 5B (NS5B) is a Substrate of Akt". Thesis, 2008. http://ndltd.ncl.edu.tw/handle/89612733234015073934.
Texto completoResumen
碩士國立陽明大學
藥理學研究所
96
Hepatitis C virus (HCV) is a positive, single-stranded RNA virus. It belongs to the family of Flaviviridae. There are six major genotypes of HCV and the most common in Taiwan is 1b. HCV can generate more than ten proteins. The non-structural protein NS5B, identified as a RNA dependent RNA polymerase (RdRp), plays an important role in HCV replication. We noticed that the NS5B of HCV genotype 1 has the consensus sequence of the Akt/PKB substrate. Akt/PKB is a Ser/Thr-protein kinase which plays important roles in the cell such as cell metabolism, glucose uptake, and cell survival. In this study, we aimed to prove that the NS5B is a substrate of Akt. We constructed the 6xHis and GST tagged NS5B recombinant protein in E. coli expression system. We also used the baculovirus expression system to generate recombinant NS5B with genotype 1b and 2b in insect cells. After generation of recombinant NS5B protein from E. coli system and baculovirus expression system, we used the ÄKTAprimeTM system coupled with Q and heparin column to purify these two kinds of NS5B and the anti-FLAG M2 resin also used to capture these proteins. These purified NS5B proteins were subjected to the Akt kinase assay and immuno-complex Akt kinase assay to determine whether HCV NS5B could be phosphorylated by Akt. We were unable to induce sufficient amount of recombinant NS5B in soluble form from E. coli, though the bacteria could be induced with 0.4 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 30 ℃ for 4 hours. We succeed in generating recombinant NS5B of both genotype 1b and 2b from baculovirus expression system followed by purification by ÄKTAprimeTM system coupled with columns. The purified recombinant NS5B was incubated with the activated recombinant Akt-1 protein or BSA in the kinase buffer containing γ-32P ATP or excess cold ATP. A signal appeared only in reaction containing NS5B-2b but disappeared after thrombin digestion, suggesting the signal might come form nonspecific phosphorylation on protein kinase A site which is at upstream of NS5B. Unequal amount of NS5B-1b and NS5B-2b might be another reason for this false-positive result. To eliminate this concern, we used the M2 resin to capture the recombinant NS5B proteins and quantitated the captured NS5B proteins by western blot before thrombin digestion. Equal mount of NS5B-1b and NS5B-2b protein, with or without thrombin digestion, were applied for Akt kinase assay. By using the immuno-complex Akt kinase assay, specific phosphorylation signal appeared in reaction containing NS5B-1b but not NS5B-2b. The result clearly demonstrated that NS5B could be phosphorylated by Akt, indicating that HCV NS5B is a novel substrate of Akt. The result may have implication in pathogenesis of HCV replication and novel treatment development for HCV infection.
25
曹昌煇. "Study of Membrance Permeability Modified by Japanese Encephalitis Virus Nonstructural Protein NS2b". Thesis, 1997. http://ndltd.ncl.edu.tw/handle/01394049417075570382.
Texto completoResumen
碩士國防醫學院
微生物暨免疫學研究所
85
Membrane permeability was reported to be modified by various animal virus during infection and the viral proteins responsible for this modification has been determined for poliovirus, influenza virus and HIV, etc. by an E. coli inducible system. In this study, the ability of the membrane permeability modification by Japanese encephalitis virus (JEV) nonstructural protein NS2b~NS3 was determined by the inducible system. Overexpression of NS2b plus various lengths of NS3 in E. coli causd membrane permeability change demonstrated by the entry of protein translation inhibitor) hygromycin B. The viral protein responsible for membrane modification was further mapped to NS2b since NS2b alone but not the N-terminus of NS3 retained this membrane modification ability. The region of NS2b required for this effect was localized to almost its full-length since the N-terminal 24-amino-acids deleted and the C-terminal 6-amino-acids deleted clones failed to demonstrate membrane modification ability. The NS2b's ability to modify membrane permeability was further proved by demonstrating the decrease of bacteria growth rate after protein induction, as well as the inpairment of eukaryotic cells' protein synthesis by a luciferase reporter system. In conclusion, JEV NS2b but not NS3 could modify membrane permeability.
26
Huang, Jing-Tang y 黃景堂. "PROTEOMIC APPROACHES TO IDENTIFY HEPATITIS C VIRUS NS5B RNA POLYMERASE-INTERACTING PROTEINS". Thesis, 2005. http://ndltd.ncl.edu.tw/handle/64850437781298980141.
Texto completoResumen
碩士中國醫藥大學
醫學研究所
93
Abstract Hepatitis C virus (HCV) nonstructure protein 5B (NS5B) is a RNA-dependent RNA polymerase that acts as a key player in the HCV replication complex. Some viral and cellular factors were reported to involve in HCV replication. However, the components of the HCV replication complex are still not yet completely understood. In this study, the HCV NS5B was used as the bait in a pull down assay to screen for NS5B-interacting proteins present in Huh 7 hepatoma cells. After mass spectrophotometric analysis, a putative lipogenic enzyme, fatty acid synthase (FAS), was identified to interact with NS5B. Co-immunoprecipitation analysis further confirmed the direct binding between the cellular protein and HCV NS5B. In addition, the expression of viral proteins and RNA in HCV subgenome replicon cells was decreased by treatment with C75, a specific FAS inhibitor. Together, these results suggest a critical role of FAS in the regulation of HCV infection through the modulation of HCV replication complex.
27
Huang, Yu-Hsu y 黃裕煦. "Hepatitis C virus NS4A protein regulates clusterin expression and caspase-3 activity of host cells". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/06270102090872298157.
Texto completoResumen
碩士國立臺灣大學
微生物學研究所
95
Hepatitis C virus (HCV) NS4A is a nonstructural protein of about 6 kDa that consists of 54 amino acids. The well-known function of NS4A is the cofactor of NS3 serine protease that promotes the HCV polyprotein processing. NS4A also uses its central domain to interact with NS3 serine protease and to promote NS3 internal cleavage. A recent study demonstrated that NS4A accumulates on mitochondria membrane and induces mitochondria-mediated cell apoptosis and caspase-3 activation. cDNA microarray analysis from our laboratory revealed an increase of clusterin (CLU) mRNA in HCV subgenomic replicon cell line. CLU is a glycoprotein that expresses in virtually all tissue and fluid. CLU is associated with DNA repair, cell apoptosis and cell survival. It has two distinct isoforms: the secreted CLU (sCLU) and nuclear CLU (nCLU). sCLU and nCLU are translated from full-length CLU mRNA and alternatively spliced CLU mRNA, respectively. In this study, HCV nonstructural protein expression plasmids were transfected into 293 cells. RNA was isolated for quantitative PCR analysis. Results showed that expression of NS4A or NS5B alone specifically increased cellular steady-state CLU mRNA. NS4A could increase cellular sCLU mRNA but it had little influence on nCLU expression. NS4A expression plasmid was co-transfected with pCLU-luc reporter plasmid containing 801 bp of CLU promoter into 293 cells. RNA was extracted for RT-PCR analysis. The synthesis of luciferase mRNA in NS4A-transfected cells was not significantly different from that in vector-transfected cells. These data suggest that NS4A has no influence on the promoter activity of clusterin or NS4A-responsive elements are beyond the cloned 801 bp region. These results indicate that the mechanism of NS4A-mediated upregulation of CLU mRNA may be through inhibition of CLU mRNA degradation. The level of sCLU protein in cell lysates was increased, but sCLU protein in medium was decreased in NS4A-transfected 293 cells. These results suggest that NS4A may decrease sCLU stability in 293 cells. Furthermore, sCLU protein was slightly increased both in the cell lysates and the culture medium of doxycycline-induced HepG22-NS3-4A stable cells. On the other hand, important sequences of NS4A involved in the enhancement of caspase-3 activity were analyzed. Deletion mutant NS4A(1-34) lost the ability to activate caspase-3 activity, indicating that NS4A(35-54) domain is important for caspase-3 activation. Understanding the NS4A-mediated CLU regulation and the important domain of NS4A involved in caspase-3 activation will help us to elucidate the interaction between host and virus during HCV infection.
28
Lin, Yu Chieh y 林雨潔. "Effects of Different Domains of the HCV NS3- NS4A Protein on Its RNA Helicase Activity". Thesis, 2002. http://ndltd.ncl.edu.tw/handle/15861932597428236170.
Texto completoResumen
碩士國立臺灣大學
微生物學研究所
90
Hepatitis C virus (HCV) is the major etiological agent of post- transfusion- associated non- A, non- B hepatitis worldwide. An estimated 3 % of the world’s population is infected with HCV, according to the World Health Organization. HCV infection most commonly results in chronic hepatitis that eventually develops into cirrhosis and hepatocellular carcinoma. The current available therapy, using interferon- α and combination with ribavirin, has worked on less than 50 % of the patients. Therefore, there is an urgent need for new therapies. The non-structure protein 3 (NS3) of HCV ranges from a. a. 1027 to 1657 of the poly-protein and is a well- designed and multi- functional protein. The N terminal one-third (from a. a. 1027 to 1207) of NS3 protein contains a serine protease activity, which catalyzes the cleavage between NS3/ NS4A, NS4A/ NS4B, NS4B/ NS5A and NS5A/ NS5B. The NS4A protein is its functional cofactor. The C terminal two-thirds region of NS3 protein contains an ATPase activity and an RNA helicase activity. There is no evidence to suggest that the two domains of NS3 are separated by proteolytic procession in vivo. The thesis investigates the inter-molecular effects on RNA helicase activity between NS3 and NS4A and the intra-molecular influences between the N terminal domain and the C terminal domain of the NS3 protein. We used baculovirus expression system to produce NS3- NS4A, full-length NS3 protein, and a truncated NS3 protein that only contains the C terminal helicase domain, and assayed their helicase activity. Our data showed that NS4A up-regulated the RNA helicase activity, RNA binding ability, and the ATPase activity. Unlike the NS4A protein, the N terminal protease domain of the HCV NS3 protein down-regulated the RNA helicase activity, but enhanced the ATPase activity. Besides, the effects of the potassium ion to the helicase activities of three proteins were different. When the concentration of potassium ion was above 75 mM, the helicase activity of the NS3- NS4A protein began to decline. The helicase activity of the NS3 protein reached maximum at 75 mM KCl. But, any the helicase activity of NS3 helicase domain was greatly inhibited by K+, even at low concentration. The preliminary results should provide valuable information to further investigate the mechanisms of intermolecular and intramolecular interactions of the HCV NS3 protein. This may provide a hope to develop new therapeutic agents for hepatitis C.
29
Kou, Yi-Hen y 寇怡衡. "Molecular mechanisms of NS3 and NS4A proteins involved in the pathogenesis of hepatitis C virus". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/36322031036572804712.
Texto completoResumen
博士國立臺灣大學
微生物學研究所
95
The genomic RNA of hepatitis C virus (HCV) encodes the viral polyprotein precursor that undergoes proteolytic cleavage into structural and nonstructural proteins by cellular and the viral NS2-3 and NS3 proteases. NS4A is a cofactor of the NS3 protease. The NS3 protein was previously found to be internally cleaved but the mechanism is unclear. In this study, internal cleavages were demonstrated with the NS3 protein of genotype 1b in the presence of NS4A. Three potential cleavage sites were detected in the NS3 protein (genotype 1b) with IPT402|S being the major one. The internal cleavages required the polyprotein processing activity of NS3 protease that can be supplemented in trans. Mutational analysis demonstrated differential requirements of NS4A for the polyprotein processing and the internal cleavages of NS3, indicating that the mechanisms of NS4A involved in these two proteolytic cleavages are different. Furthermore, Ile-25, Val-26, and Ile-29 of the NS4A protein that were demonstrated to be involved in the interaction between NS3 and NS4A in culture cells are also important for the NS4A-dependent internal NS3 cleavages. These residues were also shown to be critical for the transforming activity of NS3, while mutations at these critical residues increased only slightly the efficiency of HCV replication. The internal cleavage-associated enhancement of the transforming activity of NS3 was significantly reduced when T402A substitution at the major internal cleavage site was introduced. Internal cleavage activity of the NS3-4A proteins of HCV genotypes 2a, 4a, 5a, and 6a were also examined. On the other hand, NS4A has been demonstrated to inhibit protein synthesis. In this study, a specific interaction between NS4A and eEF1A was demonstrated. The central domain from residues 21 to 34 of NS4A is the key player involved in the NS4A-mediated translation inhibition of host and the viral proteins. The translation inhibitory effect of NS4A(21-34) was relieved by the addition of purified recombinant eEF1A in an in vitro translation system. The study indicates a possible role of eEF1A in regulating HCV multiplication during a chronic infection. Taken together, the multiple roles of NS3 and NS4A involved in the viral multiplication and pathogenesis make them ideal molecular targets for HCV therapy.
30
Kou, Yi-Hen. "Molecular mechanisms of NS3 and NS4A proteins involved in the pathogenesis of hepatitis C virus". 2007. http://www.cetd.com.tw/ec/thesisdetail.aspx?etdun=U0001-2507200714162700.
Texto completo
31
Xia, Shuangluo. "High throughput screening of inhibitors for influenza protein NS1". 2009. http://hdl.handle.net/2152/14126.
Texto completoResumen
Influenza virus A and B are common pathogens that cause respiratory disease in humans. Recently, a highly virulent H5N1 subtype avian influenza virus caused disease outbreaks in poultry around the world. Drug resistant type A viruses rapidly emerged, and the recent H5N1 viruses were reported to be resistant to all current antiviral drugs. There is an urgent need for the development of new antiviral drugs target against both influenza A and B viruses. This dissertation describes work to identify small molecule inhibitors of influenza protein NS1 by a high throughput fluorescence polarization assay. The N-terminal GST fusion of NS1A (residue 1-215) and NS1B (residue 1-145) were chosen to be the NS1A and NS1B targets respectively for HT screening. In developing the assay, the concentrations of fluorophore and protein, and chemical additives were optimized. A total of 17,969 single chemicals from four compound libraries were screened using the optimized assay. Six true hits with dose-response activity were identified. Four of them show an IC₅₀ less than 1 [micromolar]. In addition, one compound, EGCG, has proven to reduce influenza virus replication in a cell based assay, presumably by interacting with the RNA binding domain of NS1. High throughput, computer based, virtual screenings were also performed using four docking programs. In terms of enrichment rate, ICM was the best program for virtual screening inhibitors against NS1-RBD. The compound ZINC0096886 was identified as an inhibitor showing an IC₅₀ around 19 [micromolars] against NS1A, and 13.8 [micromolars] against NS1B. In addition, the crystallographic structures of the NS1A effector domain (wild type, W187A, and W187Y mutants) of influenza A/Udorn/72 virus are presented. A hypothetical model of the intact NS1 dimer is also presented. Unlike the wild type dimer, the W187Y mutant behaved as a monomer in solution, but still was able to binding its target protein, CPSF30, with wild type binding affinity. This mutant may be a better target for the development of new antiviral drugs, as the CPSF30 binding pocket is more accessible to potential inhibitors. The structural information of those proteins would be very helpful for virtual screening and rational lead optimization.text
32
Cheng, Ju-Chien y 鄭如茜. "Molecular Mechanisms of the nonstructural proteins NS5B and NS3 Involved in teh Replication of Hepatitis C Virus". Thesis, 2000. http://ndltd.ncl.edu.tw/handle/78376461821549977686.
Texto completoResumen
博士國立臺灣大學
微生物學研究所
88
Hepatitis C virus (HCV) is the major etiology agent of posttransfusion and sporadic, community acquired non-A, non-B hepatitis. It plays a major role in the development of chronic hepatitis and hepatocellular carcinoma. The viral particle possesses a positive-sense RNA genome that contains a large open reading frame encoding a polyprotein about 3000 amino acid residues. Generation of mature proteins from the polyprotein precursor is mediated by cellular and viral proteases. Amino acid sequence analysis revealed highly conserved function motifs within the NS5 and NS3 proteins of HCV. To elucidate biological functions of NS5 and NS3 proteins, the cDNA fragments of NS5B, NS5A and NS3 were generated by reverse transcriptase-nested polymerase chain reaction from sera of HCV patients and expressed in both prokaryotic and eukaryotic systems. HCV NS5B protein possesses the conserved -GDD- motif and the viral RNA-dependent RNA polymerase. It is generally believed that the NS5B protein binds to the 3'' terminus of the viral genome to initiate viral replication. In this study, electrophoretic mobility shift assay was performed to analyze the RNA binding activity of the NS5B protein. The results demonstrated that HCV NS5B protein was capable of interacting with a 3'' viral genomic RNA with or without the 98-nucleotide at sequences extreme 3'' terminus. Both of the 3'' viral RNAs contain 295 nucleotides of the 3''coding region of the HCV polyprotein. The NS5B-RNA complexes were specifically competed away by the unlabeled homologous RNA, but not by the viral 5'' noncoding region and very poorly by the 3*''conserved 98-nucleotide tail. A 3'' coding region with conserved stem-loop structures rather than the 3'' noncoding region of the HCV genome is critical for the specific binding of NS5B. In addition, the binding activity was not observed with the HCV NS5A protein. Furthermore, two independent RNA-binding domains (RBDs) of NS5B were identified, RBD1 from amino acid residues 83 to 194 and RBD2 from 196 to 298. Interestingly, the conserved motifs of RNA dependent RNA polymerase for putative RNA-binding (220-DxxxxD-225) and template/primer position (282-S/TGxxxTxxxNS/T-292) are present in the RBD2. HCV NS3 protein possesses NTPase and helicase activities. Although the enzymatic activities have been extensively studied, the ATP- and RNA-binding domains of the NS3 helicase were not well-characterized. In this study, NS3 proteins with point mutations were analyzed for their activities on ATP binding, RNA binding, ATP hydrolysis, and RNA unwinding. UV crosslinking experiments demonstrated that the lysine residue in the 1230-AX4GKS-1237 motif was directly involved in ATP binding, whereas an NS3 mutant in which the arginine-rich motif (1486-QRRGRTGR-1493) was changed to QRRDTTGR bound ATP as well as the wild type. The binding activity of HCV NS3 helicase to the viral RNA was drastically reduced with the mutations in the arginine-rich motif and was also affected by a substitution of the lysine residue in the AX4GKS motif. Previously, Arg-1490 in the arginine-rich motif of the HCV NS3 was suggested, based on the crystal structure of an NS3-deoxyuridine octamer complex, to directly interact with the g-phosphate group of ATP. Nevertheless, our functional analysis demonstrated the critical roles of Arg-1490 in binding to the viral RNA, ATP hydrolysis, and RNA unwinding, but not in ATP binding. To further elucidate possible interactions among the nonstructural proteins of HCV to form a replicative complex, co-immunoprecipitation assay was performed. The results indicated that the HCV NS3, NS5A and NS5B proteins interacted with each other, either directly or indirectly. Although the roles of cellular factors involved in the complex formation and HCV replication remained to be analyzed, the present studies have provided the information on the molecular mechanisms of the nonstructural proteins involved in the replication of HCV.
33
Sridharan, Haripriya. "The interaction between NS1B protein of influenza B virus and the ubiquitin-like modifier ISG15 : insights into a unique species specific property of the virus". Thesis, 2009. http://hdl.handle.net/2152/21917.
Texto completoResumen
Influenza B virus causes a respiratory disease in people with a compromised immune system. The NS1B protein of influenza B virus is essential for virus growth and plays a crucial role in inhibiting the anti-viral responses mounted by the infected host cell. The N terminal 104 amino acids of NS1B bind a cellular protein called ISG15. ISG15 is an interferon induced 'ubiquitin-like' protein, and upon interferon induction, is conjugated to hundreds of targets. It has been found that both ISG15 and its conjugation inhibit many viruses. The focus of the current study was to characterize the interaction between NS1B and ISG15. Study of a recombinant influenza B virus which encoded a mutant NS1B protein that is unable to bind ISG15 revealed that ISG15 is mis-localized in cells infected with wild type but not the mutant influenza B virus. Further, such a mutant virus is attenuated in growth as compared to wild type virus in human cell lines but is not attenuated in canine cell lines. This result led to the discovery of the species specific nature of the interaction between NS1B and ISG15. Specifically, NS1B was found to bind ISG15 homologs from human and old world monkeys like Rhesus macaques and African green monkeys but not those from mouse or canines. These findings were extended by identifying the hinge between the N and C terminal domains of ISG15 as one of the major determinants of species specificity. These results highlight the importance of using human or primate cell culture models to study the effect of ISG15 on influenza B virus, and raises new possibilities on differences in the function of the ISG15 system in different species.text
34
Liu, Chun-Lin y 劉俊麟. "DENV NS2B/NS3 Structural Protein Cleavage Insufficiency Is Not the Cause of NS3 N369 Mutation-induced Virus Production Defect". Thesis, 2017. http://ndltd.ncl.edu.tw/handle/70029427814005613331.
Texto completo
35
Lan, Min-Shu y 藍敏書. "Expression of Dengue Virus Proteins NS1, NS2, NS4 and E in E.coli C41 and C43 Expression Strains". Thesis, 2009. http://ndltd.ncl.edu.tw/handle/35955526977058269023.
Texto completoResumen
碩士國立交通大學
生物醫學研究所
97
Dengue virus is a member of family Flaviviridae, genus of Flavivirus. Dengue viruses cause dengue fever, dengue hemorrhagic fever and dengue shock syndrome. Dengue virus encodes 10 proteins in a single open reading frame, including 3 structural proteins and 7 non-structural proteins. Envelope protein is one of the structural proteins locates outside of the virus particle, which can bind to receptors on host cells and causes infection. NS2A, 2B, 4A and 4B are small non-structural proteins which are membrane-associated proteins exhibiting hydrophobic profiles. NS2B has been suggested to involve in protease activity. NS1, NS2A, NS4A and NS4B have been suggested to involve in virus replication. Individual expression plasmids were constructed and expressed in E.coli C41（DE3）and C43（DE3） strains due to their hydrophobic profiles. These genes were cloned individually into a modified pET-30(+) vector, pET5T-HAHis. Their proteins were expressed as HA-His (influenza Hemagglutinin- Hexahistidine protein) fusion proteins for antibodies detection. Recombinant proteins fused with HA-His tag could be detected by Western blotting analysis, but not by Comassie blue staining.
36
Stocks, Christine Edith. "A Study of the mechanism and role of coordinated cleavages by signal peptidase and viral NS2B-3 protease at the C-prM junction in the structural protein region of the flavivirus polyprotein". Phd thesis, 2001. http://hdl.handle.net/1885/145717.
Texto completo