Thèses sur le sujet « NS5A inhibitor »

At the moment, several companies are studying the clinical potential of different all-oral combinations of direct-acting antivirals in ongoing studies. The most promising interferon-free combination therapies that are on the horizon include linear or cyclic NS3/4A protease inhibitors, nucleoside as well as non-nucleoside NS5B polymerase inhibitors , and NS5A inhibitors. DAAs that target NS3/4A (protease) and NS5B (RNA-dependent RNA polymerase) inhibit the enzymatic activity of these proteins. NS5A replication complex inhibitors will likely form a component of future interferon-free drug regimens but despite their remarkable potential to treat chronic hepatitis C, the detailed mechanism of action for this class of drug remains unclear. The goal of my work was to investigate the mechanism of action of different classes of antiviral agents believed to target the NS5A protein in the replication complex in order to improve the possibility to translate basic knowledge to a more meaningful clinical application. More specifically I focused my research on two classes of compounds, characterized by distinct resistance patterns in NS5A: a first class – with examples at the final stages of clinical development, represented by Daclatasvir (Lemm et al., 2011), and a second class - at earlier stages of development - represented by anilino-quinazolines such as A-831/AZD-2836 (Quinkert et al., 2008). I contributed to demonstrate that both of these inhibitor classes, by binding respectively to either HCV NS5A or to an NS5A-associated protein, PI4KIIIα, eventually interfere with the accumulation of PI4P 98 and cholesterol in the HCV replication membranous compartment, thus abrogating the ability of the virus form to replicate its RNA genome.

Introduction : Le virus de l’hépatite C (VHC), est l’une des premières causes de pathologie hépatique dans le monde. Ce virus à ARN est responsable de l’hépatite C qui aboutit au développement de la cirrhose et du cancer du foie. Selon l’Organisation Mondiale de la Santé, le VHC infecte actuellement plus de 170 millions de personnes dans le monde, soit 3% de la population. L’hépatite C chronique connait toujours en Tunisie un taux de guérison faible pour le génotype 1 car le traitement standard actuellement disponible est la bithérapie interféron pégylé associé à la ribavirine. A l’heure actuelle, le développement de différentes molécules ciblant spécifiquement le VHC, appelées les antiviraux à action directe (AAD), apparait comme une potentielle révolution dans le traitement de l’infection par le VHC.Ces AAD comprennent les inhibiteurs de protéase (IP), les inhibiteurs nucléos(t)idiques (IN) et les inhibiteurs non-nucléosidiques (INN) de la polymérase NS5B ainsi que les inhibiteurs de la protéine NS5A. La quasi-espèce virale est formée d’un mélange complexe de variants viraux parmi lesquels se trouvent des variants associés à des degrés variables à la résistance aux AAD. Ces variants peuvent donc exister naturellement en absence de toute pression médicamenteuse et sont susceptibles d’avoir un impact sur la réponse aux différents traitements par AAD. Notre objectif était de déterminer la prévalence des variants associés à la résistance dans les souches tunisiennes circulantes en préambule à l’introduction deces molécules en Tunisie. Méthodes : L’amplification et le séquençage direct de la protéase NS3, de la polymérase NS5B ainsi que la région NS5A ont été effectuées chez 149 patients tunisiens naïfs de traitement et infectés par le VHC de génotype 1 (génotype 1b = 142 ; génotype 1a = 7). Résultats : Douze séquences NS3 (12/131 ; 9,2%) ont montré des mutations connes pour conférer une résistance aux IP. Une seule séquence (1/95 ; 1,1%) a montré la mutation V321I connue pour conférer une résistance aux IN-NS5B. Trente quatre séquences (34/95 ; 35,8%) ont montré des mutations connues pour diminuer la sensibilité des INN-NS5B. Une seule séquence de génotype 1a (1/7 ; 14,3%) et 17 séquences de génotype 1b (17/112 ; 16,2%) ont montré des mutations connues pour conférer une résistance au inhibiteurs de la protéine NS5A. Conclusions : Notre étude a permis de mettre en évidence la présence de substitutions conférant une diminution de la sensibilité aux AAD chez des patients tunisiens naïfs de tout traitement anti-VHC. Des études in situ seront nécessaires pour évaluer l’impact de ces mutations sur la réponse au traitement
Introduction: Hepatitis C virus (HCV) is a major cause of liver disease worldwide. This RNA virus is responsible for hepatitis C, which leads to the development of cirrhosis and liver cancer. According to the World Health Organization, HCV infects more than 170 million people worldwide, about 3% of the population. Chronic hepatitis C still know in Tunisia low cure rates for genotype 1, because the currently standard treatment available is combination therapy of pegylated interferon plus ribavirin. At present, the development of different molecules that specifically target HCV, called direct-acting antivirals (DAA) appears as a potential revolution in the treatment of HCV infection. These DAA include protease inhibitors (PI), nucleos(t)ide (NI) and non-nucleoside inhibitors (NNI) for NS5B polymerase and NS5A inhibitors. The viral quasispecies is formed by a complex mixture of viral variants including variants associated with variable degrees of resistance to DAA. These variants may therefore exist naturally in absence of drug pressure and may affect response to different treatments by DAA. Our objective was to determine the prevalence of variants associated with resistance in circulating Tunisian strains preamble to the introduction of these molecules in Tunisia. Methods: Amplification and direct sequencing of NS3 protease, NS5B polymerase and NS5A region were performed in 149 Tunisian naïve patients infected with HCV genotype 1 (genotype 1b = 142; genotype 1a = 7) . Results: Twelve sequences NS3 (12/131; 9.2%) showed mutations known to confer resistance to PI. One sequence (1/95; 1.1%) showed the V321I mutation known to confer resistance to NS5B-IN. Thirty four sequences (34/95; 35.8%) showed mutations known to reduce the sensitivity of NS5B-INN. One genotype 1a sequence (1/7; 14.3%) and 17 genotype 1b sequences (17/112; 16.2%) showed mutations known to confer resistance to NS5A inhibitors.Conclusions: Our study highlighted the presence of substitutions conferring decreased susceptibility to DAA in naïve patients infected with HCV genotype 1. Field studies will be needed to evaluate the impact of these mutations on the treatment response

HCV infection leads to liver failure. Genotype 3 (GT3) is known to respond poorly to newly-developed direct-acting antivirals, especially inhibitors of the multifunctional NS5A protein. This work reports the establishment of efficient transient replication of the S52 GT3 sub-genomic replicon (SGR) by further culture adaptation of S52 in the context of an optimised luciferase reporter. Also documented is the development of hepatoma cells with immune-attenuating modifications and expressing the lipid binding factor hSEC14L2 to support transient replication of S52. In parallel, stable replication of S52 in SGR-harbouring cells was used to investigate the differences between early and established replication. Differences in sensitivity to the NS5A inhibitor Daclatasvir (DCV) in both transient and stable S52 were observed compared to other genotypes, and between transient and stable replication. In addition, it is shown here that the resistance-associated substitution (RAS) Y93H conferred a significant fitness cost which is not apparent for stable S52 selected with DCV, despite this RAS being detected. This thesis explores the molecular basis of such an observation and highlights a potential mechanism which warrants further research. The role of RAS in the development of resistance is still unclear though this work reports that the presence of a RAS within a mixed population greatly influenced the development of resistance to DCV in vitro. Moreover, this work identified that a cellular metabolism-regulating factor, AMP-mediated protein kinase (AMPK), may be differentially regulated during GT3 infection compared to other GTs. This thesis presents the hypothesis that AMPK regulation by HCV may contribute to hepatic steatosis as a direct consequence of viral infection, which is unique to GT3. More insight into the propensity of GT3 to develop resistance can aid further antiviral design, and an understanding of the molecular basis of steatosis offers a rationale for treating the symptoms of HCV in addition to direct targeting of the virus.

La dengue, une des maladies les plus largement émergents actuellement, avec 390 millions d'infections chaque année (OMS), est causée par le virus de la dengue contre lequel il n’existe pas de traitements. La protéine NS5 a un rôle important dans le cycle de réplication. Cette protéine se compose d'une méthionine S-transférase d’adénosyl en N-terminal et une ARN polymérase dépendante de l'ARN (RdRp) en C-terminal. Cette NS5 RdRp peut catalyser non seulement la synthèse du brin négatif de l'ARN, utilisé comme matrice pour synthétiser l'ARN brin plus-supplémentaire, mais aussi pour la synthèse d'un ARN complémentaire à partir d'une matrice court e d'ARN sans amorce (de novo). Dans ce travail de thèse, nous présentons la production et le test de l'activité de la protéine NS5, ainsi que du domaine polymérase RdRp pour les quatre sérotypes du virus de la dengue en développant un nouveau test enzymatique, en utilisant comme un réactif fluorescent. L'utilisation de ce réactif fluorescent a également contribué à la détermination des conditions optimisées pour développer un essai de criblage de l'activité polymérase pour identifier des inhibiteurs contre le virus de la dengue. En outre, quatre flavonoïdes, Hinokiflavone, apigénine, la quercétine et Amentoflavone ont montré des valeurs d’IC50 équivalentes contre toutes les constructions NS5 et les domaines polymérase des quatre sérotypes
Dengue fever, one of the most widely emerging diseases nowadays with 390 million infections each year (WHO), is caused by Dengue virus in which no official antiviral reagent or vaccine is available. The NS5 protein has an important role in the replication cycle. This protein consists of a S-adenosyl methionine transferase at N-terminal and a RNA dependent RNA polymerase (RdRp) at C-terminal. This NS5 RdRp can catalyse for not only synthesis of minus-strand RNA to be used as the template to synthesize additional plus-strand RNA but also synthesizing a complement RNA from a short RNA template without primer (de novo). In this research we present the production and activity test for NS5 protein and N-terminal extended sequence 266-900 from NS5 RdRp of all first four serotypes of Dengue virus and a construct of sequence 273-900 using a new enzymatic assay, using Picogreen as fluorescent reagent. Using this fluorescent reagent also helped determining the optimised conditions to develop a screening assay for inhibitors against dengue polymerase activity. In addition, four flavonoids, Hinokiflavone, Apigenin, Quercetin and Amentoflavone showed approximate IC50 values when testing on all NS5 and polymerase protein constructs of all four serotypes

L'une des principales cibles pour la thérapie visant le virus de l'hépatite C (VHC) est l'ARN polymérase dépendante de l'ARN NS5B indispensable à la réplication du génome virale. NS5B est l'une des enzymes clefs du cycle virale de VHC et son activation met en jeu aussi bien des interactions intramoléculaires que des interactions avec des cofacteurs viraux et cellulaires au sein du complexe de réplication. Nous avons développé une nouvelle stratégie d'inhibition de NS5B basée sur l'élaboration de peptides courts dérivés de motifs exposés à la surface de l'enzyme dans le but de cibler les nombreuses interactions impliquées dans l'activation de cette protéine. En associant une analyse fine de la structure cristallographique de NS5B avec de la modélisation moléculaire, nous avons élaboré des peptides courts mimant les motifs « hotspot » de la protéine. Ces peptides ont été évalués sur système réplicon de génotype 1b et nous avons ainsi identifié un peptide leader Moon1 de 15 résidus correspondant à un motif hautement conservé du domaine "thumb". Dans ce travail, nous avons étudié en détail la structure et le mécanisme moléculaire de ce nouvel inhibiteur de NS5B. Moon1 inhibe l'activité polymérase de la forme sauvage de NS5B ainsi que celle de mutants résistants au inhibiteurs nucléosidiques et non nucléosidiques. Nous avons démontré que la fixation de Moon1 entraine un changement de conformation de NS5B et se fait préférentiellement avec NS5B dans une conformation fermée. Ce peptide inhibe spécifiquement l'interaction entre NS5B et l'ARN double brin, indépendamment de la présence d'ions métalliques et de manière dose-dépendante. Moon1 bloque la transition entre l'étape d'initiation de novo de la synthèse d'ARN et l'extension du primer. Nous avons démontré que les résidus essentiels à l'activité de Moon1 sont hautement conservés à travers les différents génotypes et sous-types de VHC. De plus, nous avons établi une séquence minimale pour l'activité de Moon1. Nos travaux permettent de valider l'intérêt d'une stratégie interfaciale ciblant une enzyme clef du cycle du VHC et les interactions intra et intermoléculaires nécessaires à son activation
The non-structural protein RNA-dependent RNA polymerase (RdRp) NS5B plays a key role in hepatitis C virus (HCV) replication and is currently considered as one of the most relevant target to develop safe anti-HCV agents. Although many small molecules have been identified as inhibitors of NS5B, very few are active in clinic. The structure and function of NS5B have been well characterized and as other polymerases, NS5B adopts a typical “right-hand” conformation containing the characteristic fingers, palm and thumb subdomains. The activation of NS5B requires conformational changes involving intramolecular contacts as well interactions with viral proteins and host factors in the replication complex. We developed a new strategy for NS5B inhibition based on short interfacial peptides derived from NS5B surface accessible motifs that target protein-protein interfaces or essential motifs involved in NS5B-activation. Combining the NS5B crystallogaphic structure and molecular modelling, we have designed short peptides derived from NS5B surface “hotspots” that were screened using HCV genotype 1b replicon cell system. We have identified Moon1, a short 15-residu peptide, derived from a well-conserved motif located in the NS5B thumb domain that inhibits HCV replication in the low nanomolar range. Moon1 tightly binds NS5B in a conformational-dependent manner and induces NS5B conformational changes. This peptide specifically inhibits double-stranded RNA/NS5B interactions in a dose-dependent and metal ions-independent manner. Moon1 blocks the transition between RNA de novo initiation and primer-extension. We showed that residues required for Moon-1 anti-polymerase activity are well-conserved among HCV genotypes and subtypes and a minimal Moon1 active motif was established. Taken together, these results demonstrate that NS5B structural dynamics constitute an attractive target for HCV chemotherapeutics and for the design of more specific new antiviral drugs

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-résistantes
The 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

The hepatitis C virus polymerase NS5b is required for replication of the viral genome, making it an attractive target for antiviral development. The polymerase contains no proof-reading activity and generates viral variants during replication with a high degree of genetic heterogeneity, complicating the development of effective antiviral therapies since resistance mutations are readily selected under drug pressure. A successful treatment regimen will likely require a combination therapy that can suppress the emergence of resistance. Here, we have described the mechanism of action of a novel class of polymerase active site inhibitors, pyrophosphate analogues. We studied interactions between these compounds and NS5b in the presence of the resistance mutations G152E and P156L and have identified interactions leading to resistance. Additionally, we have combined the pyrophosphate analogues with a second class of polymerase active site inhibitors, nucleoside analogue inhibitors (NIs). We found that the combination can interfere with excision, a potential mechanism of resistance to NIs. We have also examined fidelity of the polymerase to better understand its contribution to variability in the viral genome. Our biochemical findings suggest that the efficiency of nucleotide mismatch formation during replication influences the prevalence of resistance mutations within the viral quasispecies population. This is supported by deep-sequencing data from an HCV-infected patient cohort. Based on these findings, we have developed a mathematical model showing that combining inhibitors selecting for resistance mutations generated through difficult-to-form nucleotide mismatches could delay the onset of resistance.We extended this study by performing transient kinetic assays to characterize incorporation of NIs by NS5b and compared this to the efficiency of mismatched nucleotide incorporation. These studies demonstrate that current NIs incorporate more efficiently than mismatched nucleotides. The incorporation efficiency of the guanosine analogue ribavirin was low as compared to other NIs tested and also as compared to G:U and U:G mismatches examined in our fidelity study, suggesting its incorporation during RNA synthesis does not cause error catastrophe. Overall, these studies provide a greater understanding of the mechanism of action of polymerase inhibitors, and of the role of the polymerase in the development of antiviral resistance.
La polymérase NS5b du virus de l'hépatite C est nécessaire pour la réplication du génome viral et représente donc une cible importante pour la découverte et le développement de nouveaux médicaments. La polymérase contient aucune activité de relecture et génère des variantes du virus avec un haut degré d'hétérogénéité génétique lors de sa réplication. Ceci nuit au développement de traitements antiviraux efficaces puisque les mutations de résistance sont facilement sélectionnées sous pression de médicaments. Un traitement efficace exigera probablement une combinaison thérapeutique qui pourrait empêcher la résistance. Ici, nous avons décrit le mécanisme d'action d'une nouvelle classe d'inhibiteurs du site actif de la polymérase, les analogues du pyrophosphate. Nous avons étudié les interactions entre ces inhibiteurs et NS5b, en présence des mutations de résistance G152E et P156L en plus d'identifier des interactions conduisant à la résistance. De plus, nous avons combiné les analogues du pyrophosphate avec une deuxième classe d'inhibiteurs du site actif de la polymérase, les inhibiteurs nucléotidiques (INs). Nous avons constaté que la combinaison peut interférer avec l'excision, un mécanisme potentiel de résistance aux INs. Nous avons également examiné la fidélité de la polymérase pour mieux comprendre sa contribution à la variabilité du génome viral. Nos résultats biochimiques suggèrent que l'efficacité de la formation de décalage lors de la réplication influence la prévalence des mutations de résistance au sein de la population virale quasi-espèces. Ceci est soutenu par les données obtenues suite au séquençage à très haut débit d'une cohorte de patients infectés par le VHC. Basé sur ces résultats, nous avons développé un modèle mathématique démontrant que la combinaison d'inhibiteurs qui sélectionnent des mutations de résistance générées par des mésappariements nucléotidiques difficiles à former pourrait retarder l'apparition de la résistance. Nous avons poursuivi cette étude en caractérisant l'incorporation des INs par NS5b et en comparant cela à l'efficacité de l'incorporation de nucléotides dépareillés. Ces études démontrent que les INs actuelles sont incorporées avec plus d'efficacité que les nucléotides dépareillés. L'efficacité d'incorporation de l'analogue ribavirine était faible par rapport aux autres INs testés et aussi par rapport aux mésappariements G: U et U: G examinés dans notre étude de fidélité. Ceci suggère que l'incorporation de la ribavirine lors de la synthèse d'ARN ne provoque pas d'erreur catastrophique. Globalement, ces études nous mènent à une meilleure compréhension du mécanisme d'action des inhibiteurs de la polymérase NS5b, et du rôle de la polymérase dans le développement de la résistance aux antiviraux.

Au cours de ma thèse je me suis intéressée aux interactions protéine-protéine (PPI’s). Les PPI’s jouent un rôle majeur dans une grande diversité de processus cellulaires et sont maintenant considérées comme une cible majeure dans le but de développer de nouveaux médicaments. Cependant, cibler ce type d’interactions requiert le développement de chimiothèques dédiées, permettant d’accélérer la découverte de molécules « touches ». Pour surmonter ce problème, une chimiothèque orientée PPI (2P2I3D) a été conçu au laboratoire. Dans un premier temps, j’ai donc évalué cette chimiothèque sur différents complexes possédant des interfaces variées. Les résultats obtenus ont révélé des taux de touches supérieurs à ceux obtenus avec des chimiothèques non orientées, de 0,2 à 1,6% contre 0,01 à 0,1%, respectivement. Cette étude a permis d’établir une preuve de concept de la faisabilité de créer une chimiothèque orientée PPI, permettant ainsi une accélération de la découverte de composés biologiquement actifs.Dans un deuxième temps, je me suis intéressée à l’interaction entre deux protéines majeures du virus de la dengue : les protéines NS3 et NS5. J’ai tout d’abord identifié et caractérisé un nouveau site d’interaction, ce qui m’a permis de mettre en évidence que cette interaction avait pour conséquence d’augmenter l’activité enzymatique du domaine hélicase. J’ai par la suite recherché et identifié des petites molécules chimiques capable d’inhiber cette interaction. Les différentes caractérisations effectuées ont permis de mettre en évidence un effet antiviral. Ces inhibiteurs constituent un excellent point de départ afin d’étudier plus en détail le rôle biologique de ce complexe
In my thesis I became interested in protein-protein interactions (PPI's). PPI's play a major role in a variety of cellular processes and are now considered a major target in order to develop new drugs. However, targeting such interactions requires the development of dedicated libraries, to accelerate the discovery of “hits”molecules .To overcome this issue, a focused chemical library PPI (2P2I3D) was designed in the laboratory.At first, I evaluated this chemical library on different complexes with diverse interfaces. The results showed higher hit rate to those obtained with non-oriented libraries, from 0.2 to 1.6% against 0.01 to 0.1%, respectively. This study has established a proof of concept of the feasibility of creating a focused chemical library PPI, thus accelerating the discovery of biologically active compounds.Secondly, I am interested in the interaction between two major proteins of dengue virus: the NS3 and NS5 proteins. I initially identified and characterized a novel interaction site, which allowed me to demonstrate that this interaction had the effect of increasing the enzymatic activity of the helicase domain. I searched and identified small molecules able to inhibit this interaction. The different characterizations helped to highlight an antiviral effect. These inhibitors are an excellent starting point to further explore the biological role of this complex

The aim of these studies was to increase our understanding of the non-structural proteins 3 and 5B (NS3 and NS5B) from the hepatitis C virus (HCV), and thereby contribute to the development of new and better drugs against HCV. By studying NS3 with substitutions identified to be associated with resistance to NS3 inhibitors in clinical trials (R155Q, A156T and D168V) it was found that not all inhibitors were affected, indicating that cross-resistance can be avoided. Substitutions at position 526 and 528 in the helicase domain of this bifunctional enzyme were introduced and the effect on the protease was investigated. These substitutions affected protease inhibition, showing that the helicase can influence the protease. This interplay between the two domains is also involved in the discovered activation of the enzyme at low inhibitor concentrations. Being a case of "enzyme memory", the phenomenon stresses the importance of using full-length NS3 for enzymatic assays. Inhibitors with novel designs, with presumed increased stability in vivo, were developed and, even though they were found to be of low potency, provide alternative ideas of how to design an inhibitor. Detailed information about the interaction between NS3 and its protein cofactor NS4A or several protease inhibitors were determined using a direct binding assay. The rate constants of the inhibitor interactions were affected by NS4A and it was also possible to visualize time-dependent binding inhibitors. A good correlation between interaction data (Kd or koff) and inhibition data (Ki) or replicon data (EC50) was also seen. The same approach was used for studying the interactions between NS5B and several non-nucleoside inhibitors, providing information of the chemodynamics and giving insights into inhibitor design.   Taken together, all these studies have resulted in new information about, and new tools with which to study, NS3 and NS5B. This is of great importance in the struggle to find new and potent drugs, leading to a cure for HCV infection.

Les travaux présentés dans ce manuscrit relatent les recherches les plus significatives de mon parcours professionnel au cours des onze dernières années. Dans un premier temps, nous montrons comment il est possible de prospecter de nouvelles molécules actives contre l’hépatite C en développant de nouveaux acides aminés non naturels innovants. Cette étude a été assistée par le logiciel de docking GenMol pour le design des divers composés. Nous y détaillons l’évolution des structures produites en fonctions des propriétés recherchées et des dernières avancées du domaine. Les synthèses énantio convergeantes, appuyées par la chimie des peptides, ont pu aboutir à cinq molécules finales qui ont montré différents potentiels antiviraux. Ensuite, la synthèse et l’étude structurale du composé BHH-BTT sont abordées. Nous y décrivons les propriétés physico-chimiques de l’empilement des molécules et comment celles-ci finissent par influer les propriétés de transport des charges. Nous avons ainsi élaboré des OFETs performants qui montrent de bonnes perspectives en termes de futurs détecteurs. Enfin nous rapportons la première intégration de matériaux semi-conducteurs organiques (BTBT) au sein de matériaux hybrides organique-silice. Cette démarche impliquait la réalisation d’un précurseur sol-gel optimisé bis(triéthoxysilane) qui nous permettait de contrôler l’organisation des cœurs π conjugués lors de la création des matériaux. Nous avons, grâce à des suivis spectroscopiques détaillés, démontré une agrégation de type J lors de la formation des films minces et une réorganisation des cœurs π-conjuguées en agrégation de type H lors l’application du procédé sol-gel. Après une mise au point au niveau de l’interface semi-conducteur/électrodes, nous avons alors réalisé des transistors hybrides organique-silice à effet de champs entièrement réticulés dont la robustesse face à une grande diversité de solvants organique a été démontrée
The different works presented in this manuscript relate the most relevant investigations representing my career evolution along this last eleven years. First, we show how it is possible to explore new active molecules by developing innovating non-natural amino-acids. This investigation was supported by the docking software GenMol. We show here the structures evolution of the produced molecules according to desired properties and to last advances in the field. The enantio convergent synthesis supported by peptide chemistry allowed us to reach five final molecules with diverse antiviral potentials. Then, synthesis and structural investigation of the compound BHH-BTBT was broached. We describe here physicochemical properties of the packing and how they affect charge transport properties. So then, we achieved OFET with good performance that show interesting perspective for future sensors. Finally we reported the first integration of a semi-conducting material (BTBT) in a hybrid organo-silica material. This approach involved the conception of an informed sol-gel bis(triethoxysilane) precursor that allowed us to control the π-conjugated cores organisation during the creation of the material. With detailed spectroscopic monitoring we demonstrated a J-aggregation during the thin film formation and a reorganisation to a H-aggregation during the sol-gel process. After optimisation of the semiconductor/electrode interface, we obtained hybrid organo-silica field effect transistors entirely reticulated showing a high resilience to a large diversity of organic solvent

INTRODUÇÃO: A hepatite C crônica é uma das principais causas de hepatopatia em todo mundo. A coinfecção pelo vírus C (VHC) e o HIV não é incomum, pois ambos compartilham vias similares de transmissão. Recentemente, a terapêutica da hepatite C crônica foi radicalmente modificada com o advento das drogas antivirais de ação direta (DAAs), elevando as taxas de RVS mesmo na população coinfectada. O VHC é caracterizado pela sua alta taxa replicativa e por grande diversidade populacional. Substituições de ocorrência natural na protease viral associadas a resistência podem comprometer a terapêutica em alguns regimes baseados no uso de inibidores de protease (IPs). OBJETIVOS: Estimar a prevalência de polimorfismos e mutações de ocorrência natural associadas a resistência aos IPs em pacientes monoinfectados e coinfectados pelo VHC e HIV e identificar fatores clínicos e virológicos associados a presença de tais substituições. MATERIAIS E MÉTODOS: Dados epidemiológicos e clínicos foram obtidos de 247 pacientes (135 monoinfectados e 112 coinfectados pelo VHC e HIV). VHC RNA foi extraído do plasma dos indivíduos participantes e um fragmento de 765 pares de base da região NS3 foi amplificado e sequenciado por metodologia populacional (técnica de Sanger). O estadiamento da fibrose hepática foi realizado pelo escore não invasivo FIB- 4. RESULTADOS: 54 indivíduos (21,9%) apresentaram pelo menos uma substituição na região NS3/NS4A do VHC. Somente 14 pacientes (5,7%) apresentaram pelo menos uma mutação de resistência aos IPs (T54S, V55A ou Q80R). A Q80K não foi identificada em nenhuma das amostras. Não houve diferença entre monoinfectados e coinfectados quanto à ocorrência de polimorfismos ou mutações associadas a resistência. As variáveis independentemente associadas com substituições na região da protease foram infecção pelo VHC genótipo 1b, bilirrubinas totais > 1,5 vezes o LSN e níveis de albumina < 3,5 g/dL. Fibrose hepática avançada (FIB-4 > 3.25) não esteve associada a presença de substituições. A análise de diversidade nucleotídica na protease viral revelou maior heterogeneidade do VHC genótipo 1b em relação ao 1a. Contudo, a análise de pressão seletiva não demonstrou maior variabilidade de quasiespécies no grupo de hepatopatia avançada, achado este compatível com uma sequência genômica relativamente conservada. CONCLUSÕES: As substituições na região NS3/NS4 do VHC consistiram majoritariamente por polimorfismos naturais sem impacto clínico num eventual tratamento que envolva o uso de IPs. A prevalência de substituições associadas a resistência foi baixa e compatível com os valores informados pela maioria dos estudos nacionais e internacionais. A coinfecção pelo HIV não parece elevar a frequência de substituições na protease do VHC. A região NS3 do genótipo 1b foi altamente variável em relação ao genótipo 1a, reforçando o conceito de possíveis diferenças geográficas em relação ao perfil genético deste vírus
INTRODUCTION: Chronic hepatitis C is a major cause of liver disease worldwide. Hepatitis C vírus (HCV) and HIV coinfection is not uncommon due to similar transmission routes. Recently developed direct-acting antivirals drugs (DAAs) have increased the rate of SVR even in coinfected patients. HCV has a high replication rate and a lack of proofreading activity, leading to a greatly diverse viral population. Baseline spontaneously occurring resistance substitutions in the protease region may impair the rate of success in some protease inhibitors (PI) based regimens. OBJECTIVE: to determine the prevalence of naturally occurring polymorphisms and resistance associated variants to HCV PIs in mono and coinfected HCV HIV patients and to evaluate potential associations between amino acid substitutions in protease domain and clinical / virological features of those patients. METHODS: Clinical and epidemiological data were retrieved from medical records of 247 subjects in Brazil (135 HCV monoinfected and 112 HIV HCV coinfected patients). HCV-RNA was extracted from plasma and a fragment of 765 base pairs from the NS3 region was amplified and sequenced with Sanger-based technology. Fibrosis staging was assessed by non invasive score (FIB-4). RESULTS: Overall, 54 patients (21.9%) had at least one amino acid substitution in the NS3 region; only 14 patients (5.7%) harboured at least one resistance mutation (T54S, V55A, Q80R). Q80K mutation was not found in any sample. There was no difference between monoinfected and coinfected patients regarding the frequency of natural polymorphisms and resistance mutations. Variables independently associated with amino acid substitution were HCV subtype 1b, total bilirubin level > 1.5 ULN and albumin level < 3.5 g/dL. Advanced liver fibrosis (FIB-4 > 3.25) was not related to NS3 polymorphisms nor resistance associated variants. Examination of HCV protease nucleotide diversity revealed greater heterogeneity in subtype 1b than subtype 1a. Analysis of selective pressure did not reveal a greater quasispecies variability in advanced liver fibrosis group, being such finding consistent with a relatively conserved gene in this setting. CONCLUSION: Baseline HCV NS3 amino acid substitutions depicted herein were considered mostly natural polymorphisms with no clinical impact in a PI based therapy. The prevalence of resistance-associated substitutions was low and compatible with values reported by most national and international studies. HIV coinfection was not associated with a greater frequency of such substitutions in the studied sample. The NS3 region of genotype 1b was highly variable in relation to genotype 1a, highlighting geographic differences concerning HCV genetic profile

碩士
慈濟大學
醫學生物技術碩士班
104
Hepatitis C virus (HCV) NS5A protein could be cleaved at residue 154 under certain circumstances, e.g., apoptosis. NS5A without its N-terminal 154 amino acid possesses transcriptional activity. The function of the N-terminal 154 a.a. of NS5A (NS5A 1-154) is unknown. Over-expressed NS5A could enhance HCV replication while NS5A 1-154 would repress viral replication in the replicon system. Exosomes, nano-particles (30 -100 nm diameter) derived from cells, are formed by the inward budding of the multivesicular body (MVB) membrane. It is reported that exosomes could facilitate HCV transmission. We have also found that HCV NS5A protein or NS5A 1-154 could be detected in exosomes of Huh7 cells over-expressing these two proteins. Moreover, exosomes with NS5A enhanced HCV replication while exosomes with NS5A 1-154 repressed viral replication in HCV replicon cells. Artificial vesicles with NS5A protein or NS5A 1-154 through the sonication of Huh7 cells over-expressing these two proteins were also developed. Effects of these artificial vesicles on HCV replication are under investigations.

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.
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博士
國立臺灣大學
藥學研究所
100
Abstract The chronic hepatitis C virus (HCV) infection progressively leads to major liver disorders such as cirrhosis, end stage liver disease and hepatocellular carcinoma. While almost 3% of world’s population is infected by HCV, the lone treatment option of ribavirin in combination with pegylated interferon is insufficient to address this global epidemic. Therefore, there is an unmet clinical need for more effective and safe anti-HCV drugs. NS5B polymerase is currently pursued as the most popular target to develop safer anti-HCV agents because it is not expressed in uninfected cells. In chapter 1 lead identification and structure activity relationship (SAR) studies of more than 50 scaffolds leading to the potent NS5B inhibitors is reviewed. Even though, allosteric site inhibitors have gained much attention due to their nanomolar potency in enzyme as well as replicon assays, emergence of resistant mutants and lack of specificity against all HCV subtypes have limited their transition to the clinic. Active site inhibitors of NS5B are noncompetitive with respect to both the RNA template and to the nucleotides. Existence of a strong sequence homology within the active site of NS5B from all the nine subtypes makes active site a potential target for safe drug development against all subtypes of HCV. Scaffolds such as alpha, gamma-diketo acid (DKA) and its bioisosters such as monoethyl ester of meconic acid and dihydroxypyrimidine carboxylic acid are the only active site inhibitors of NS5B in the literature. Therefore, in chapter 2 a novel series of 2-hydroxy-1-oxo-1,2-dihydroisoquinoline-3-carboxylic acid derivatives with the built-in β-N-hydroxy-γ-keto-acid as DKA bioisoster was designed. Compounds 16a-p were synthesized via isocoumarin intermediates 18a-l. 2-Hydroxy-1-oxo-4-phenyl-1,2-dihydroisoquinoline-3-carboxylic acid (16c) was the most active compound in this series. Compound 16c inhibited NS5B polymerase in assays based on the inorganic pyrophosphate generation (IC50 = 9.5 µM) and NTP incorporation by NS5B enzyme (IC50 = 5.9 µM). Consequently, it demonstrated moderate antiviral activity (EC50 = 15.7 µM) and good selectivity in HCV genotype 1b replicon Ava5 cells. Job plot, NMR spectroscopy and docking studies showed that compound chelated to two Mg2+ ions and also deteriorated the interaction of NTP to NS5B polymerase in BIAcore assay. Enhancement of druggability is a major challenge for active site inhibitors as they possess functional groups such as hydroxyl and carboxylic acid that add to their polar nature. Change in isoquinoline core to quinazoline and carboxylic acid to carboxamide generated β-N-hydroxy-γ-keto-carboxamide pharmacophore that would act as a DKA bioisoster. Thus, in chapter 3 a series of N-substituted-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamides (8a-k) was synthesized. N-Benzyl-3-hydroxy-4-oxo-3,4-dihydroquinazolin-2-carboxamide (8a) was defined as a minimum structure to inhibit NS5B polymerase. Predicted physicochemical properties such as MolLogP, cLogP, MolPSA, and number of hydrogen bond donors (HDB) and hydrogen bond acceptors (HBA) of N-hydroxyquinazolinone analog 8a were in acceptable range and possessed higher drug likeness score than the N-hydroxyisoquinolinones. Structure activity relationship studies have led to identification of more flexible and lipophilic N-hydroxyquinazolinone 8k (IC50 = 8.77 µM and EC50 = 15.7 µM) as the most potent analog of this series. Moreover, it was nontoxic to parent Huh7 cells rendering it a safe candidate for further development. Binding predictions by molecular docking suggested that compound 8k forms chelating interactions with two magnesium ions present in the active site of NS5B polymerase. In addition to metal chelation, backbone atom of Phe224 and Asp225 form hydrogen bonds with compound, while alkyl linker and aromatic ring also have a hydrophobic interaction with Val52 and Leu159 of NS5B polymerase. A pharmacophore wherein 1,3-diketo moiety integrated with 2-N-hydroxyl group is proposed as DKA bioisoster in chapter 4. A small fragment like molecule N-hydroxysuccinimide weakly inhibited NS5B polymerase, and was subjected to structural change to generate an equipotent lead 2-hydroxyisoindole-1,3-dione (13a). A series of isoindole-1,3-diones (13a-n) was synthesized from respective phthalic anhydrides. 4-Nitro analog (13b) demonstrated moderate enzyme inhibition with an IC50 of 9.5 µM. Alternatively, a modified 1,3-diketo scaffold 2-aroylisoindoline-1,3-dione (14a-g) was developed, of which 2-benzoyl-isoindole-1,3-dione (14a) inhibited generation of pyrophosphate by NS5B polymerase with IC50 of 6.8 µM, which is more than 10 fold enhancement as compared to parent 2-hydroxyisoindole-1,3-dione. 5,6-Dichloro analog 14d was found to be selectively cytotoxic to Ava5 cells with an IC50 of 18.0 µM. Chapter 5 describes the overall conclusion of this thesis and perspectives thereof. Literature reported NS5B inhibitors are classified. The summary of three different classes of NS5B polymerase inhibitors namely, isoquinolinone, quinazolinone and isoindoledione with hydroxamate inbuilt DKA bioisosteric pharmacophores have been discussed. Though investigated for their NS5B polymerase inhibitory activity and anti-HCV activity, these classes of DKA bioisosters offer an opportunity for medicinal chemists to provide lead structures for targeting other biological targets that are metal dependent.

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.

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.

碩士
中國醫藥大學
醫學研究所碩士班
94
Japanese encephalitis virus (JEV) is a member of Flavivirus. JEV contains a single-stranded positive-sense RNA genome. JEV genome encodes for 3 structural proteins (C, prM, and E) and 7 nonstructural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, and NS5). JEV has been demonstrated the inhibitory effect on the IFN-α/β response by blocking Jak-Stat singling pathway. In addition, dengue virus type 2 NS2A, NS4A, and NS4B, and West Nile virus NS4B were associated with the inhibition of IFN-α/β signaling. In this study, we intend to investigate the effect of JEV NS4A and NS4B proteins on IFN-α/β signaling, and to identify the interaction of NS4A and NS4B proteins with neuron cellular factors. Our results indicated that JEV NS4A and NS4B inhibited IFN-α/β signaling using interferon stimulating response element (ISRE)-luciferase cis-acting reporter gene, and reduced stat2 protein phosphorylation with western blotting. Moreover, expression of NS4A in BHK-21 cells inhibited about 100% and 60% luciferase activity of ISRE-luciferase cis-acting reporter by INF-α and INF-β, respectively. Expression of NS4B in BHK-21 cells inhibited 15% luciferase activity of ISRE-luciferase cis-acting reporter by INF-α/β. Expression of NS4A or NS4B in TE671 cells also inhibit around 35~45% luciferase activity of ISRE-luciferase cis-acting reporter by INF-α/β. Dead box42 protein that interacted with the NS4A protein has been identified from a phage display human brain cDNA library. Co-immunoprecipitation showed the binding interaction of recombinant NS4A protein with C-terminus of Dead box42 protein. In addition, confocal imaging indicated the co-localization of NS4A with DDX42 in BHK-21 and TE671 cells. Overexpression of DDX42 in BHK-21 cell significantly blocked the inhibitory affect of INF-β by IFN-α/β signaling. This study could be helpful for understanding the JEV pathogenesis and developing antiviral drugs.

碩士
國立東華大學
生物技術研究所
98
Japanese encephalitis virus (JEV) is a single stranded positive-sense RNA virus that contains 10,976 nucleotides in length. Previous study in our lab reported an abundant accumulation of small RNA (522 nts) in JEV infected cells. This small RNA derived from the 3’-untranslated region (UTR) highly conserved region. Function of this small RNA remains unclear. In this study, the effect of small RNA in regulating the viral NS5 protein and host interfering regulatory factor-3 (IRF-3) were sought using transfecting of small RNA at various time points. Down regulation of NS5 and inhibition IRF-3 phosphorylation was found when transfecting small RNA 6 hour prior to infection or simultaneous with infection but not in the mock transfected cells. In contrast, transfecting small RNA at 24 h postinfection has no effect on NS5 expression or IRF-3 phosphorylation. Furthermore, Northern analysis revealed same pattern as well as western blot indicating the effect was on RNA level. The inhibition of IRF-3 phosphorylation by Small RNA was further confirmed using poly IC induction. Subcellular localization of IRF-3 was monitored using IRF-3 fused with green fluorescence protein and the result showed that overexpression of small RNA partially inhibited the translocation of IRF-3 to the nucleus. Taken together, these results indicated that small RNA interferes with IRF-3 activation by down regulating JEV replication in a time-dependent manner.