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1
Sabaratnam, Keshalini. „The interaction between the Marek's Disease Virus (MDV) neurovirulence factor pp14 and the host transcription factor, CREB3“. Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:d2fc6bd4-bc3a-4a37-924b-86881096a9b5.
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Marek's Disease Virus (MDV) induces a wide range of neurological syndromes in susceptible hosts; however, the mechanisms behind the MDV-induced neuropathology are still poorly understood. The immediate-early 14kDa phosphoprotein, pp14, is associated with the neurovirulence phenotype of the virus. Yeast-two-hybrid screening identified the ER-bound transcription regulator, human CREB3 (cAMP Response Element-Binding protein), as an interacting partner of pp14, and fluorescence colocalisation between pp14 and chicken CREB3 (chCREB3) in MDV infected cells suggested an interaction between these proteins. The primary focus of this DPhil project was to further investigate this putative interaction using in vitro studies, with a view to determining if the interaction is linked to the neurovirulence of MDV. This investigation, which employed a combination of biochemical, cellular, and functional assays, found no conclusive evidence in support of the predicted interaction. In addition, this project aimed to gain structural and functional insights into the MDV neurovirulence factor pp14 and the host transcription factor, chCREB3. Biophysical characterisation of recombinant pp14B identifies pp14 as a molten globule. The results reveal the protein, while possessing substantial secondary structure, is largely disordered lacking a stable tertiary structure. Multiple lines of evidence from this study also indicate pp14 is a putative zinc-binding protein. Moreover, phosphorylation analysis of recombinant pp14B, extracted from DF1 cells, by mass spectrometry provides conclusive evidence for the presence of two phosphorylation sites in the shared C-terminal region of pp14 - serines 72 and 76 of pp14B. Structural flexibility, through a lack of a definite ordered tertiary structure, and functional features that can induce structural modifications indicate pp14 might interact with a number of binding partners and therefore could play multiple roles during MDV infection - a strong possibility due to the expression of the protein in all the different stages of virus infection. Furthermore, this thesis presents the crystal structure of the homodimeric chCREB3 bZIP. The chCREB3 bZIP possesses a structured DNA binding region even in the absence of DNA, a feature that could potentially enhance both the DNA-binding specificity and affinity of chCREB3. Significantly, chCREB3 has a covalent intermolecular disulphide bond in the hydrophobic core of the bZIP, which may play a role in promoting stability. Moreover, sequence alignment of bZIP sequences from chicken, human and mouse reveals only members of the CREB3 subfamily possess this cysteine residue, indicating it could act as a redoxsensor. These results indicate members of the CREB3 subfamily, by possessing a putative redox-sensitive cysteine with the capacity to form an intermolecular disulphide bond, may be activated in response to oxidative stress.
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Conley, Michaela Jayne. „Structural and functional characterisation of feline calicivirus entry“. Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/8920/.
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The Caliciviridae are a group of small, non-enveloped viruses with a positive sense, single stranded RNA genome. Caliciviruses include the noroviruses, responsible for winter vomiting disease, as well as several important veterinary pathogens. Feline calicivirus (FCV) is an excellent model for studying calicivirus entry, having a known protein receptor and being readily propagated in cell culture. Here we explore calicivirus entry, using FCV. Virus entry is the critical first step of infection and is therefore an important area of study. Both alpha 2-6 linked sialic acid and feline junctional adhesion molecule A (fJAM-A) have been identified as receptors for FCV. The attachment of FCV to fJAM-A, is followed by uptake via clathrin mediated endocytosis. Little is known, however, on the viral escape mechanism leading to delivery of the viral RNA into the cytoplasm. We set out to explore the nature of FCV attachment and uncoating using structural, biochemical and biophysical analyses. By cryogenic electron microscopy we have characterized the virus-receptor interaction at high-resolution. Using electron microscopy and an RNA release assay, we have investigated virion uncoating. Finally, we have explored the importance of receptor glycosylation, and oligomerisation. Our analysis has allowed us to construct an atomic model of the major capsid protein VP1. Upon binding to fJAM-A, FCV undergoes a conformational change (rotation and tilting of the capsomeres). Flexibility in the receptor decorated virion has prevented high-resolution structure analysis of the conformational change or the virus-receptor interaction. We have, however, seen that the structural changes are limited to the capsid spikes. We hypothesised that the conformational change may be a priming step that would prepare the virus for uncoating upon internalisation. We found that upon lowering the pH below 5, receptor decorated virions disassembled, supporting this hypothesis. Disassembly of the virus-receptor complex at low pH presented a tool for estimating the quantity of receptor needed to prime the capsid for uncoating. Cryo-EM studies reveal that FCV bound fJAM-A is monomeric although the receptor was found to be dimeric in solution as previously described for the human and murine homologues. Furthermore, it is hypothesised that this is the form found at tight junctions between cells. We propose that disruption of fJAM-A homodimers may be the mechanism by which induction of viral uptake by endocytosis is triggered. Finally, we have confirmed the presence of an N-linked glycosylation on fJAM-A and show that the removal of this carbohydrate moiety does not affect viral binding in vitro.
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Thompson, Catherine Isabelle. „Protein interaction studies on the rotavirus non-structural protein NSP1“. Thesis, University of Warwick, 1999. http://wrap.warwick.ac.uk/80266/.
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Rotavirus encodes six structural and six non-structural proteins. In contrast to the structural proteins, the functional roles of the non-structural proteins are not well defined beyond a realisation that they must have a role in the viral replication cycle. A fuller understanding of the replication cycle must therefore rest on determining the specific roles played by the non-structural proteins. Non-structural protein NSP1 shows high levels of sequence divergence. A generally well conserved cysteine-rich region at the amino-terminus may form a zinc finger structure. It has been shown to possess non-specific RNA-binding activity, and has been found associated with the smallest of three replication intermediates (RIs) found in infected cells, together with the viral proteins VP1, VP3 and NSP3. VP2 and VP6 are added sequentially to the pre-core RI to form the core RI and single-shelled RI respectively. The function of NSP1 in the replication cycle and the importance of its presence in early replication complexes has not been determined. The intermolecular interactions that occur between the components of the RIs have not been defined. Protein-protein interactions between NSP1 and VP1, VP2, VP3, and NSP3, from the UKtc strain of bovine rotavirus, were investigated using a variety of approaches, the first of which was the yeast two-hybrid system. In this assay a self-interaction of NSP1 was not detected. Protein-protein interactions between NSPl and VPl, VP2, VP3, and NSP3, were also not detected. Both the full-length protein and a truncated NSPl, consisting of only the amino terminal third of the protein, were tested. A direct self-interaction of NSP3 was shown and quantified. Radio-immunoprecipitation analysis of in vitro translated viral proteins using specific anti-NSP1 serum was also employed. However, it failed to detect direct protein-protein interactions between NSP1 and VPI, VP2, and VP3. Immunoprecipitation of UKtc rotavirus-infected celllysates with anti-NSP1 serum showed the co-precipitation of viral proteins VPl, VP2, VP3NP4, VP6 and NSP3, with NSP1. It was proposed that NSP1 formed a previously unrecognised complex with these proteins. Immunoprecipitation of nuclease-treated infected cell lysates showed a reduction in the co-precipitation of VP2, VP3NP4 and NSP3 with NSP1. No reduction in the co-precipitation of VP6 was seen. The association of the complex proteins may be mediated by RNA binding. Immunoprecipitation with an anti-VP6 monoclonal antibody reciprocally precipitated small amounts of NSP1, VP2, VP3/VP4, and NSP3, with VP6.
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Rezelj, Veronica Valentina. „Characterization of the non-structural (NSs) protein of tick-borne phleboviruses“. Thesis, University of Glasgow, 2017. http://theses.gla.ac.uk/8149/.
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In recent years, a number of newly discovered tick-borne viruses exhibiting a wide spectrum of diseases in humans have been ascribed to the Phlebovirus genus of the Bunyaviridae family. These viruses have a tripartite RNA genome composed of two negative-sense RNA segments (medium and large) and one ambisense segment (small), which encode four structural proteins and one non-structural protein (NSs). The NSs protein is the major virulence factor of bunyaviruses, and acts as an antagonist of a key component of the first line of defence against viral infections: the interferon (IFN) system (Bridgen et al., 2001; Weber et al., 2002). The work presented herein describes the characterization of tick-borne phlebovirus NSs proteins as IFN antagonists. The development of a reverse genetics system for the apathogenic tick-borne Uukuniemi phlebovirus (UUKV) enabled the recovery of infectious UUKV entirely from cDNA. A recombinant UUKV lacking NSs induced higher amounts of IFN in infected cells compared to wild-type UUKV, suggesting a role of NSs in modulating the IFN response. The weak IFN antagonistic activity of UUKV NSs was evident using transient transfection reporter assays in comparison to the NSs protein of either pathogenic Heartland virus (HRTV) or Severe fever with thrombocytopenia syndrome virus (SFTSV). The sensitivity of UUKV, HRTV and SFTSV to exogenous and virus-induced IFN, as well as their growth kinetics in IFN-competent cells were examined. The molecular mechanisms employed by UUKV, HRTV and SFTSV NSs proteins to evade antiviral immunity were investigated using reporter assays, immunofluorescence, and immunoprecipitation studies. Collectively, these assays showed that UUKV NSs was able to weakly inhibit IFN induction but not IFN signalling, through a novel interaction with MAVS (mitochondrial antiviral signalling protein). On the other hand, HRTV and SFTSV NSs proteins potently inhibited IFN induction through an interaction with TBK1, and type I but not type II IFN signalling via an interaction with STAT2. Finally, the development of a minigenome system for HRTV in conjunction with minigenomes developed for UUKV and SFTSV (Brennan et al., 2015) provided preliminary data to assess possible outcomes of tick-borne phlebovirus reassortment. In summary, the results described in this thesis offer insights into how tick-borne phlebovirus pathogenicity may be linked to the capacity of their NSs proteins to block the innate immune system. The data presented also illustrate the plethora of viral immune evasion strategies utilized by emerging phleboviruses, and provide an insight into the possibility of tick-borne phlebovirus reassortment.
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Howard, Susan Teresa. „Structural and functional analyses on the SalI G fragment of vaccinia virus“. Thesis, University of Cambridge, 1991. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.386088.
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Martin, Morgan Mackensie. „Functional analysis of hepatitis C virus non-structural protein (NS) 3 protease and viral cofactor NS4A“. Thesis, University of British Columbia, 2008. http://hdl.handle.net/2429/1522.
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The hepatitis C virus (HCV) was identified in 1989 as the major causative agent of transfusion-associated non-A, non-B hepatitis and today represents a worldwide health crisis with prevalence estimates of 2.2%. HCV-specific therapeutics have never been more urgently needed. One of the validated drug targets is the non-structural (NS) protein 3 (NS3) membrane-bound protease. The major aim of this thesis was characterization of NS3 allosteric activation by its viral cofactor, NS4A. We hypothesized that there would be specific residues that dominate the interaction between NS3 and NS4A, and further hypothesized that binding and activation may be separate events mediated by different residues.
This thesis details the development of novel cell-based assays for detection of NS3-4A protease activity and heterocomplex formation. The protease assay substrate was a membrane-targeted intracellular protein, which upon proteolysis released a red fluorescent protein (FP) reporter, DsRed-Express, into the cytoplasm; this change was detected by microscopy or quantified by Western blotting. The complex formation assay detected fluorescence resonance energy transfer (FRET) between yellow and cyan FP-tagged NS3 and NS4A, respectively.
Our data shows binding can be functionally separated from activation. We identified two NS4A residues (I25 and I29) important for NS3 binding and two NS4A residues (V23 and I25) important for NS3 activation. Therefore the binding-pockets of these residues are prime targets for small-molecule therapeutic development.
In addition, I have compared the NS3-4A substrate sequence cleavage efficiencies in vivo. I have been able to show that the activation-dependent NS4B/NS5A junction is processed efficiently and the NS4A/NS4B junction is not. I have also shown NS3-4A substrate specificity is not modulated by replicase components; however the specific activity of this enzyme is increased.
The strength of this thesis work stems from the novel and creative development of cell-based assays that can easily be modified to study other membrane-associated proteases. In vitro assays fall short in that they do not take into account the unique micro-environment in which these proteases are found.
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Lauder, Rebecca Pink. „Structural analysis of adenovirus bound to blood coagulation factors that influence viral tropism“. Thesis, University of Glasgow, 2011. http://theses.gla.ac.uk/2636/.
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Adenoviruses are currently the most commonly used vectors for clinical gene therapy trials. Of these, Ad5 is the most commonly used serotype. Upon intravenous delivery, the vectors are sequestered in the liver which reduces their efficacy. While the coxsackievirus and adenovirus receptor is responsible for in vitro cell entry, this pathway is not used in vivo. Blood coagulation factors have been implicated in mediating in vivo hepatic transduction, and it is therefore important to characterise the interaction between these and Ad5 in order to permit development of more efficacious and safer viral vectors. Ad35 is a rare human pathogen and presents less pre-existing immunity than Ad5 and other common serotypes. It has potential as a gene therapy vector as this may reduce side effects and toxicity linked to pre-existing immunity. Ad35 uses CD46 as a receptor instead of CAR which allows it to infect hepatocytes, however blood coagulation factor X has also been implicated in in vivo hepatic transduction. This interaction must therefore also be characterised. We used low dose cryo electron microscopy to collect images of Ad5, Ad5 bound to FX and to FIX, Ad35 and Ad35 bound to FX. These were used to generate three dimensional icosahedral reconstructions of Ad5 at 27Å, Ad5 bound to FX at 26Å and 14Å, Ad5 bound to FIX at 21Å, Ad35 at 35Å and Ad35 bound to FX at 30Å. High resolution structural data were fitted to the Ad5 and Ad5-FX reconstructions in order to model the interaction and identify the binding sites. The structural data presented show that FX binds to Ad5 hexon with a stoichiometry of 1/3. Fitting experiments showed that the Gla domain of FX corresponds with the density in the centre of the hexon trimer, with arms corresponding to the EGF domains extending from this and terminating in the globular serine protease domain. Furthermore FX binds to Ad35 hexon in a similar manner. The data also shows that FIX does not bind to Ad5 hexon, however we were unable to confirm it if instead binds to the fiber as suggested by biochemical studies, due to limitations in the reconstruction technique used.
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Leigh, Kendra Elizabeth. „Structural Studies of a Subunit of the Murine Cytomegalovirus Nuclear Egress Complex“. Thesis, Harvard University, 2015. http://nrs.harvard.edu/urn-3:HUL.InstRepos:14226065.
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The Herpesviridae family of viruses includes a number of human pathogens of clinical importance. Like other herpesviruses, cytomegaloviruses require a heterodimeric nuclear egress complex (NEC) consisting of a membrane-bound protein and a soluble nucleoplasmic protein, termed in murine cytomegalovirus (MCMV) M50 and M53, respectively. Genetic, electron microscopic, and immunocytochemical studies have revealed the importance of this complex for viral replication, most predominantly in facilitating egress of viral nucleocapsids across the nuclear membrane. Despite the significance of the NEC to the herpesvirus life cycle, there is a dearth of structural information regarding the components of the complex. We present here an NMR-determined solution-state structure of the conserved, structured, soluble portion of M50 (residues 1-168), which exhibits novel structural character. We mapped the binding site of a highly conserved minimal binding domain of the M53 homologue from human cytomegalovirus (HCMV; UL53) required for heterodimerization onto the structure and identified specific residues in a groove within the M50 protein fold that interact with the UL53 peptide. This site was verified biophysically and biologically: single amino acid substitutions of the corresponding residues of the homologous protein from HCMV (UL50) resulted in decreased UL53 binding in vitro, as measured by isothermal titration calorimetry, and substitutions that had the greatest effect on binding affinity caused disruption of UL50-UL53 co-localization and lethal defects in the context of HCMV infection. We then compared the effect of binding UL53 peptide with binding of the larger natural binding partner, M53 (residues 103-333) via NMR, with the results suggesting that conformational changes most likely occur on a fold-wide level in the context of the full complex. We suggest that these findings combined with the clinical relevance, the virus-specific aspects of nuclear egress, and the novelty of the structure make the HCMV NEC an attractive potential drug target. To this end, we used in silico screening to identify possible small molecule inhibitors and have begun validating top screen hits biophysically and biologically.
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Ruiz, Arroyo Víctor Manuel. „Structural and functional analysis of Zika Virus NS5 protein“. Doctoral thesis, Universitat de Barcelona, 2020. http://hdl.handle.net/10803/671922.
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Zika virus (ZIKV) belongs to the Flaviviridae family and constitute an important public health concern since ZIKV infection produced devastating effects in new born infants. Flaviviruses present a positive sense single stranded RNA genome flanked by highly structured untranslated regions (UTR) carrying one open reading frame that codifies for three structural proteins (C, prM, E) and five nonstructural proteins (NS1-5). At the most C-terminal end, NS5 protein carries a RNA dependent RNA polymerase (RdRP) and a methyl transferase domain (MTase) for genome copying and 5’ capping activities of the newly synthesized RNA, respectively. Given the crucial role of this enzyme for viral replication, NS5 constitutes an attractive antiviral target to inhibit viral replication. In this study, we determined the structure of the ZIKV NS5 protein using X-Ray crystallography combined with several structural biology approaches to characterize the supramolecular arrangement of the ZIKV NS5 protein. We identified the monomer-monomer and dimer-diner interactions to form fibril-like structures, and evaluated the role of oligomer formation, using in-vitro polymerization assays. We also evaluated the in-vivo effect of NS5-oligomerisation in chicken embryos, stablishing a connection between this protein and microcephaly.
One of the most important RNA structures present at the 5’UTR of flavivirus genomes is the 5SLA. This structure was identified previously to bind the NS5 protein, acting as a promoter and being essential for viral replication. We assayed and optimized the NS5-5SLA complex stability using biophysical and biochemical techniques and determined the structure of the complex by single particle cryo-EM. Comparisons between the NS5-5SLA complex and the NS5 crystallographic structure revealed for the first time in flavivirus, important conformational changes in the NS5 RdRP. We identified the residues involved in complex formation and characterized the effect of this binding on NS5 polymerization, shedding new light on the understanding of replication mechanisms in flaviviruses.El virus Zika (ZIKV) pertenece a la familia Flaviviridae y constituye una amenaza para la salud pública, especialmente debido a las malformaciones provocadas en neonatos. Los flavivirus presentan un genoma RNA de simple cadena con polaridad positiva, flanqueado por regiones no traducidas (UTR) que presentan una elevada estructura secundaria, seguido de una región codificante para una única poliproteína que por proteólisis dará lugar a tres proteínas estructurales (C, prM, E) y cinco proteinas no estructurales (NS1-5). En el extremo C-terminal se encuentra la proteina NS5 que presenta actividad ARN polimerasa dependiente de ARN (RdRP) y un dominio metil-transferasa (MTase) para copiar el genoma y añadir una caperuza al extremo 5’ del nuevo ARN sintetizado, respectivamente. Dado el papel crucial de este enzima en la replicación viral, la proteina NS5 constituye una diana antiviral muy atractiva para inhibir la replicación del virus. En este estudio, determinamos la estructura de la proteína NS5 de ZIKV, usando cristalografía de Rayos-X combinada con diferentes técnicas biofísicas para caracterizar la organización supramolecular de la proteína. Identificamos las interacciones monomero-monomero y dimero-dimero para caracterizar las estructuras fibrilares de la proteína y evaluamos los efectos de la dimerización en la actividad polimerasa in-vitro. También evaluamos los efectos de la oligomerización de NS5 in-vivo en embriones de pollo, estableciendo una conexión entre esta proteína y la aparición de microcefalia en fetos infectados. Una de las estructuras de ARN más importantes presentes en el 5’UTR del genoma de los flavivirus es el 5SLA. Previamente se describió que esta estructura se unía a NS5 y actuaba como un promotor, siendo ademas esencial para la replicación viral. Medimos y optimizamos la estabilidad del complejo NS5-5SLA mediante técnicas biofísicas y bioquímicas y determinamos la estructura del complejo mediante cryo-EM. Las comparaciones entre la estructura cristalográfica y cryo-EM de NS5 revelaron, por primera vez en flavivirus, cambios conformacionales importantes en el dominio RdRP. Identificamos los residuos implicados en la formación del complejo y caracterizamos el efecto de la unión de NS5 a 5SLA sobre su actividad polimerasa. Estos resultados arrojan nueva luz para entender los mecanismos de replicación en los flavivirus.
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Rainsford, Edward. „Functional studies on the rotavirus non-structural proteins NSP5 and NSP6“. Thesis, University of Warwick, 2005. http://wrap.warwick.ac.uk/53876/.
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The rotavirus replication cycle has not been fully characterised, one vital stage of virus replication involves large cytoplasmic occlusion bodies termed viroplasms. These are the sites of synthesis and replication of dsRNA, packaging of viral RNA into newly synthesized cores and the formation of double-shelled previrions. The detailed mechanism by which these events occur is poorly understood but is thought to be mediated by the non-structural proteins localised to these structures. Rotavirus gene segment 11 expresses two proteins NSP5 and NSP6 which are found in alternate open reading frames. NSP5 exists is several isoforms which differ on their level of phosphorylation. It has been shown to be essential for virus replication and localises to the viroplasms. The smaller NSP6 protein is the most uncharacterised of all of the rotavirus proteins. It has however been shown to interact with NSP5 and has been tentatively suggested to be localised to the viroplasms. To further investigate these two proteins the pET expression system was utilised to obtain purified protein which was subsequently used to generate mono specific polyclonal antisera. Studies into the function and localisation of these proteins found that both localised to the viroplasms and their relative distributions within these structures were defined. NSP6 was found to be expressed at a low level throughout the course of a rotavirus infection and in contrast to other non-structural proteins, to have a high rate of turnover. The RNA binding ability of both NSP5 and NSP6 was investigated using quantitative filter binding assays and these showed both have sequence independent nucleic acid binding ability. Studies were also conducted into the mechanism of NSP6 expression from the second open reading frame of gene 11, the results obtained being consistent with a leaky scanning mechanism of expression.
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Halldorsson, Steinar. „Molecular determinants of phleboviral cell entry“. Thesis, University of Oxford, 2017. http://ora.ox.ac.uk/objects/uuid:56c5ef37-b023-4a8f-bdf2-8388226dc3b3.
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Phleboviruses are emerging zoonotic pathogens which constitute a global threat to human and animal health. The mosquito-borne Rift Valley fever virus (RVFV) is a widespread problem across the African continent and causes regular deadly outbreaks in ruminants. The recently emerged severe fever with thrombocytopenia syndrome virus (SFTSV) is a serious human public health concern in China which has rapidly spread to Japan and Korea with fatality rates as high as 16-30%. Phleboviral cell entry is mediated by two viral glycoproteins: the class II fusion protein Gc and the lesser known Gn. Initial cell attachment is glycan dependent and the penetration into the cell cytoplasm is mediated by the Gc fusion protein which catalyses viral and cell membrane merger. The entry mechanism is not well understood from a structural perspective which limits mechanistic insights. The purpose of this thesis is to further our understanding of the cell entry process by filling in the missing structural information on the phleboviral glycoprotein layer. To this end, an integrated structural approach using cryo-EM and X-ray crystallography was adopted. The crystal structure of the Gn ectodomain is presented which reveals an unprecedented structural relationship with seemingly unrelated viruses. Single-particle cryo-EM and localized reconstruction reveal the glycoprotein layer of the RVFV and a pseudo-atomic model of the RVFV is presented. The assembly shows the shielding of the Gc fusion protein and suggests that the Gn functions as a fusion chaperone. The post-fusion crystal structure of the Gc protein from SFTSV further consolidates a mechanism of membrane fusion by class II fusion proteins. Finally, preliminary data on receptor binding and mechanism of antibody mediated neutralization are presented. The work presented herein provides a novel platform for studying and understanding entry and assembly of phleboviruses as well as the design of novel therapeutics.
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Arnaud, Charles-Adrien. „Structure de la queue du phage T5 et mécanisme de perforation de l’enveloppe bactérienne par les Siphoviridae“. Thesis, Université Grenoble Alpes (ComUE), 2017. http://www.theses.fr/2017GREAV086/document.
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La grande majorité des bactériophages connus ont un virion équipé d'une queue permettant la reconnaissance de l'hôte, la perforation de l'enveloppe bactérienne et l'éjection du matériel génétique viral directement dans le cytoplasme de la bactérie. La famille des Siphoviridae représente 60% des phages caudés et est caractérisée par une queue longue et non-contractile. Le tube de la queue est formé par un empilement de protéine majeur de tube (TTP) polymérisé autour de la protéine vernier (TMP). L'extrémité distale de la queue est équipé d'un complexe de protéine dans lequel se trouve les protéines de liaison au récepteur (RBP). La séquence d'évènement permettant l'éjection de l'ADN et l'infection est encore mal décrite.Au cours de cette thèse, la structure de pb6, la TTP du phage T5 qui s'assemble de façon non-canonique en trimères, a été résolue par cristallographie à une résolution de 2,2 Å. L'analyse de cette structure confirme cependant une homologie structurale de pb6 avec les autres TTPs et avec des protéines bactériennes du système de sécrétion de type VI et des pyocines R. Une étude RMN comparant pb6 dans ses états de monomère et de tube polymérisé est en cours et permettra à terme une description très fine de cet assemblage.De plus, les structures du complexe distal de queue et du tube de la queue (tube de pb6) ont été résolue des résolutions intermédiaires avant et après interaction avec le récepteur bactérien. Ces structures obtenues par cryo-microscopie électronique révèle une absence de changements structuraux au niveau du tube, en contradiction avec le modèle jusque là proposé que la TTP transmettait l'information de fixation du récepteur à la capside.Bien qu'à un stade préliminaire, les reconstructions du complexe distal sont très informatives sur les rôles de protéines pb2 et pb4.L'ensemble de ses données ainsi que des expériences biochimique et la comparaison avec d'autres systèmes bien décrits dans la littérature permet de proposer un nouveau modèle pour les premières étapes de l'infection des Siphoviridae. Ce modèle a également un intérêt pour l'étude du mécanisme d'autres familles de virus (Myoviridae). Les différences, similarité et parenté d'éléments de la queue du phage T5 avec d'autres systèmes de perforation de membrane sont discutésThe vast majority (96%) of bacteriophages possess a tail that allows host cell recognition, cell wall perforation and safe viral DNA channelling from the capsid to the cytoplasm of the bacterium. Siphoviridae is a familly representing 60% of all tailed phages characterized by a long flexible tail. The tail tube is formed by stacks of hexamers of the tail tube protein (TTP) polymerised around the tape measure protein (TMP). At the distal end of the tail, the tail tip complex harbours the receptor binding proteins (RBP). For these phages, little is known on the mechanism that triggers DNA ejection after binding to the host.We report the crystal structure at 2.2 Å resolution of pb6, an unusual trimeric TTP, of siphophage T5. Structure analysis however confirms the homology of pb6 with all TTPs, related tube proteins of bacterial puncturing devices (type VI secretion system and R-pyocin) and procapsid proteases. We fit this structure into the cryo-electron microscopy map of the tail tube determined at 6 Å resolution. Comparing the structure of the tail tube before and after interaction with the host receptor, we show that unlike previously proposed, the host binding information is not propagated to the capsid by the tail tube, as the two structures, at that resolution, are identical. An ambitious NMR comparative study of the TTP in its monomeric and tube form is underway to further describe this assembly.Moreover, the structures of the tail tip complex prior and after interaction with the bacterial receptor were solved at intermediate resolution. These structures reveal interesting conformationnal changes triggered by the RBP binding to the bacterial receptor. Those rearrangements are the first to occur after phage irreversible binding to its host and they induce the TMP ejection, the capsid opening, the enveloppe perforation and ultimately DNA channeling to the host cytoplasm.Together with biochemical data and comparison with other known system in the litterature we are able to propose a model for Siphoviridae very first steps of infection. These findings might be of interest for the mechanism of other viral familly (notably Myoviridae) and similarity with other membrane perforating systems is discussed
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Zwart, Lizahn. „Investigating two AHSV non-structural proteins : tubule-forming protein NS1 and novel protein NS4“. Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/62198.
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African horse sickness is an equid disease caused by African horse sickness virus (AHSV). AHSV produces seven structural proteins that form the virion and four non-structural proteins with various roles during replication. The first part of this study investigated the intracellular distribution and co-localisations of NS1 with other AHSV proteins to facilitate its eventual functional characterisation. Confocal microscopy revealed that NS1 formed small cytoplasmic foci early after infection that gradually converged into large fluorescent NS1 tubule bundles. Tubule bundles were more organised in AHSV-infected cells than in cells expressing NS1 alone, suggesting that tubule bundle formation requires the presence of other AHSV proteins or regulation of NS1 expression rates. NS1 occasionally co-localised with VP7 crystalline structures, independently of other AHSV proteins. However, when NS1-eGFP, a modified NS1 protein that contains enhanced green fluorescent protein (eGFP) near the C-terminus, was co-expressed with VP7, co-localisation between these proteins occurred in most co-infected cells. It is not clear how the addition of eGFP to NS1 induces this co-localisation and further investigation will be required to determine the function of NS1 during viral replication.
The second part of the study focused on characterising the novel non-structural AHSV protein NS4. The NS4 open reading frame (ORF) occurs on segment 9, overlapping the VP6 ORF in a different reading frame. In silico analysis of segment 9 nucleotide and NS4 predicted amino acid sequences revealed a large amount of variation between serotypes, and two main types of NS4 were identified based on these analyses. These proteins differed in length and amino acid sequence and were named NS4-I and NS4-II. Immunoblotting confirmed that AHSV NS4 is translated in AHSV infected insect and mammalian cells, and also in Sf9 insect cells infected with recombinant baculoviruses that overexpress the genome segment 9 proteins, VP6 and NS4. Confocal microscopy showed that NS4 localised to both the cytoplasm and nucleus, but not the nucleolus, in AHSV-infected cells and recombinant baculovirus infected Sf9 cells. Nucleic acid protection assays using bacterially expressed purified NS4 showed that both types of NS4 bind dsDNA, but not dsRNA. This was the first study to focus on AHSV NS4. Future work will focus on determining the role of non-structural proteins in viral pathogenesis, and will involve the use of a reverse genetics system for AHSV.Dissertation (MSc)--University of Pretoria, 2013.
Genetics
MSc
Unrestricted
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Koneru, Pratibha Chowdary. „Mechanistic and Structural Investigations into the Mode of Action of Allosteric HIV-1 Integrase Inhibitors“. The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1560532618539888.
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Meek, Richard William. „Structural and functional analysis of proteins involved in the C-DI-GMP network of the predatory bacterium Bdellovibrio bacteriovirus“. Thesis, University of Birmingham, 2018. http://etheses.bham.ac.uk//id/eprint/8115/.
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Bdellovibrio bacteriovorus HD 100 is a σ-proteobacterium that predates on Gram-negative bacteria. The lifecycle of Bdellovibrio bacteriovorus is complex and regulated in part by the cyclic nucleotide, c-di-GMP. Gene knockouts of diguanylate cyclases reveal discrete phenotypes in Bdellovibrio at different time points of predation. This thesis presents the first structure of the Bdellovibrio diguanylate cyclase, Bd0742 in an inhibitory conformation. Bd0742 mediates invasion but the mechanism of its regulation was unknown. Our structure suggests that an N-terminal tail attached to the forkhead-associated domain of Bd0742 regulates diguanylate cyclase activity via self-binding. We present structural and biochemical evidence demonstrating that the Nterminal tail regulates Bd0742 activity. An active mutant of Bd0742 was produced by introduction of a disulphide bond. We also solved the structure of the glucose-6-phosphate isomerase Bd07 41. Bd07 41 likely catalyses isomerization of glucose-6-phosphate and of fructose-6-phosphate. Finally, this thesis presents a structure of a region of the degenerate diguanylate cyclase, Bd3125. Mutant strains deficient of Bd3125 invade prey more slowly. The Bd3125 structure reveals that a previously cryptic domain is a GAF (cGMP-specific phosphodiesterase, Adenylyl cyclases and FhiA) domain, with a potential role in controlling invasion speed.
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Chauché, Caroline Marie. „Molecular evolution of equine influenza virus non-structural protein 1“. Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/8877/.
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Influenza A viruses (IAVs) are common infections of certain avian reservoir species, and they periodically transfer to mammalian hosts. These cross-species jumps are usually associated with sporadic outbreaks, and on rare occasions lead to the establishment of a lineage in the new host species. The immune pressure exerted by the new host on the emergent virus forces it to evolve and adopt strategies to evade immunity in order to survive in nature. Understanding the biological mechanisms that allow successful inter-species transmission and adaptation to mammals is crucial to develop the theoretical tools required to predict and/or control emergence of new viruses in humans and animals. H3N8 equine influenza virus (EIV) represents an interesting model to study the dynamic of within-host variation of an avian-origin IAV. Indeed, this virus has emerged from birds in 1963 and has circulated in horse populations for more than fifty years despite the availability of vaccines. Evidence of evolution of EIV virulence factor non-structural protein 1 (NS1) also exists. NS1 is the main viral antagonist of the host interferon (IFN) response, and it relies on different strategies for overcoming these responses, which varies depending on the viral strain. While some NS1 proteins effectively block the induction of IFN and IFN stimulated genes (ISGs), others block general gene expression at a post-transcriptional level, and therefore reduce the synthesis of IFN and ISGs indirectly. Importantly, little is known about the contribution of these NS1 functions to EIV infection phenotype and adaptation to horses. In this work, we characterised NS1 proteins spanning the entire EIV lineage and showed that NS1s from different time periods after EIV emergence counteract the IFN response using different and mutually exclusive mechanisms. While EIVs circulating in the early 1960s blocked general gene expression by a NS1-mediated blockade of the cleavage and polyadenylation specificity factor 30 (CPSF30), NS1s from contemporary EIVs specifically inhibit the induction of ISGs by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially during EIV evolution, E186K substitution and C-terminal truncation. These changes in NS1 allowed contemporary EIVs to replicate in the presence of high levels of IFN. The results shown here with EIV indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation.
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Hill, Alison. „Emergence of simian immunodeficiency virus in rhesus macaques is characterized by changes in structural and accessory genes that enhance fitness in the new host species“. Thesis, Harvard University, 2016. http://nrs.harvard.edu/urn-3:HUL.InstRepos:33493400.
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The distribution of lentiviruses among primates reflects a history of interspecies transmission and emergence of new virus-host relationships. The degree to which viruses must adapt to the genetic environment of new host species, and how adaptations to the new host initially affect viral fitness are two understudied elements of emergence. The simian immunodeficiency virus (SIV) of rhesus macaques (SIVmac) emerged as the result of a cross-species transmission of SIV from the sooty mangabey monkey (SIVsm) into rhesus macaques, and comparing cohorts of SIVmac- and SIVsm-infected macaques provides an opportunity to examine a lentivirus evolving during the early stages of emergence. Using archived samples from four cohorts of macaques, we compared evolution of “established” macaque-adapted viruses (SIVmac239, SIVmac251) to incompletely-adapted, “emerging” viruses (SIVsmE543, SIVsmE660). Longitudinal samples included the inoculum for each cohort, as well as acute and chronic plasma samples for each animal. Samples were processed for deep sequencing, and consensus sequences of complete viral coding regions were assembled de novo. Computational and manual analysis of the sequences revealed a set of loci that diverged considerably only in the SIVsm-infected animals, suggesting that adaptations at these loci are important for emergence of SIVsm in rhesus macaques. These candidate adaptations included known adaptations to overcome restriction by macaque TRIM5α. In order to quantify the impact of these candidate adaptations on viral replication, each mutation was introduced into SIVsmE543 (forward mutations, reflecting adaptation to the macaque host) and SIVmac239 (reversions to the ancestral residue). These were then tested in a deep sequencing-based fitness assay, in which changes in the frequencies of mutant and parental sequences replicating in cell culture were used to calculate differences in relative fitness. Substitutions in the coding sequences for the Matrix, Capsid, and Vif proteins were found to enhance fitness of SIVsm in rhesus cells, confirming the hypothesis that they represent species-specific adaptations. Together, these studies represent a novel approach to the identification and functional characterization of viral determinants of cross-species transmission.Medical Sciences
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Kissel, Jay D. „Target specificity and structural characterization of single-stranded DNA aptamer RT1t49, a broad inhibitor of HIV-1 reverse transcriptases“. [Bloomington, Ind.] : Indiana University, 2007. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3274917.
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Thesis (Ph.D.)--Indiana University, Dept. of Biology, 2007.Source: Dissertation Abstracts International, Volume: 68-07, Section: B, page: 4320. Adviser: Donald H. Burke-Aguero. Title from dissertation home page (viewed Apr. 22, 2008).
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Persson, Magnus. „Structural Studies of Bacteriophage PRR1 and HIV-1 protease“. Doctoral thesis, Uppsala universitet, Strukturell molekylärbiologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-135159.
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Viruses are a diverse genera of organisms adapted to thrive in many different hosts from prokaryotic to eukaryotic. We present here the structure of bacteriophage PRR1 virus-like particle (VLP), belonging to Leviviridae family. Our structure reveals calcium ions in the VLP. Metal ions are rare in the VLP among the Leviviridae and the calcium ions were found to affect VLP stability. Gene expression in Leviviridae is controlled by a specific interaction between the viral coat protein that assembles to create the VLP, and the genomic RNA. This interaction has been thoroughly studied for the levivirus MS2 but other structural data are scarce. We have solved the structure of PRR1 VLP in complex with its RNA operator stem-loop. Binding of the stem-loop in PRR1 shows similarities to MS2 but also a different arrangement of the nucleotides, in the area of the loop that we could interpret, compared to MS2. The structures of PRR1 increase our knowledge about translational control in Leviviridae and add new information about particle stability within this family. The other virus we investigated is the more infamous human pathogen, the HIV. Because of the high mutation rate of HIV new drugs are needed on a continuous basis. We describe here the structure of two new protease inhibitors bound to the HIV-1 protease and compare them with two previously published inhibitors. Due to an extended P1´site the new compounds are able to exploit a new interaction to Phe53 in the protease structure.Felaktigt tryckt som Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 724
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Brown, Heather Piehl. „Homology-based Structural Prediction of the Binding Interface Between the Tick-Borne Encephalitis Virus Restriction Factor TRIM79 and the Flavivirus Non-structural 5 Protein“. University of Toledo Health Science Campus / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=mco1481304908426729.
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Stone, Nicholas P. „Elucidating the structural mechanisms of capsid stability and assembly using a hyperthermophilic bacteriophage“. eScholarship@UMMS, 2019. https://escholarship.umassmed.edu/gsbs_diss/1042.
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Nearly all viruses encapsulate their genomes in protective protein shells known as capsids. Capsids self-assemble from repeating protein subunits, which surround the viral genome. Many viruses use a powerful biomotor to pump DNA into preformed capsid shells. Therefore, not only does the capsid protect the genome from environmental stress, it additionally stabilizes against high internal pressure caused by the tightly-packaged genome inside. To understand how capsids remain stable despite extreme conditions, I use thermophilic bacteriophage P74-26 as a model to probe the structural mechanisms that govern capsid assembly and stability. P74-26 capsids have a similar architecture to capsids of mesophilic tailed bacteriophages, allowing direct comparison to elucidate the structural basis of enhanced thermostability. Here I determine the structure of the P74-26 capsid decoration protein, which contains a core beta-barrel domain termed the ‘beta-tulip’ domain. The beta-tulip domain is conserved in structural proteins from both Herpesviruses and phage, as well as a broad-spectrum Cas9 inhibitor, providing evidence of shared evolutionary ancestry. Additionally, my high-resolution structure of the P74-26 virion capsid reveals unique interdigitated architectural features that contribute to enhanced stability in the thermophile. P74-26 has a significantly larger capsid than related mesophiles yet retains the same icosahedral geometry, demonstrating a novel mechanism for increasing capsid capacity. Furthermore, my thesis work explores capsid assembly and maturation mechanisms in vitro, establishing P74-26 as a platform for future development of novel nanoparticles and therapeutic delivery systems. Taken together, this work illuminates the incredible stability of a thermophilic virus and illustrates its utility as a powerful tool for studying viral maturation.
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Zeng, Yingying. „Modeling and structural studies of single-stranded RNA viruses“. Diss., Georgia Institute of Technology, 2013. http://hdl.handle.net/1853/47630.
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My research focuses on structures of the genomes of single-stranded RNA viruses. The first project is concerned with the sequence and secondary structure of HIV-1 RNA. Based on the secondary structure that Watts et al. determined, I performed a series of analysis and the results suggested that the abundance of Adenosines at the wobble position of the codons leads to an unusual structure with numerous unpaired nucleotides. The findings indicated how the virus balances evolutionary pressures on the genomic RNA secondary structure against pressures on the sequence of the viral proteins.
The second project is the modeling of satellite tobacco mosaic virus (STMV). STMV is a T=1 icosahedral virus with a single piece of RNA that has 1058 nucleotides. X-ray crystallography studies of this RNA have revealed a structure containing 30 helices. The linkers between the helices, the possible structures at the interior of the icosahedron, and the sequence of the RNA were all missing in the crystal structure. To explore how the genome is organized within the protein capsid, I built a 3D model based on the RNA secondary structure predicted by Susan Schroeder. Being the first all-atom model of any virus, this model is highly correlated with the crystal structure; and the comparison with the in vitro structure of the same RNA supports the hypothesis that capsid protein plays an important role in RNA folding during assembly.
The third project includes the modeling of bacteriophage MS2 (MS2) and the examination of the compactness of RNA in different viruses. MS2 is a T=3 RNA virus, and the cryo-EM studies have revealed a double-shell conformation of the genome. My final model of MS2 recaptures the double-shell structure of the RNA presented in the cryo-EM density. In addition, the predicted secondary structure that I used for the construction of the model shares a strong similarity with the in vitro structure determined in 1980s. This similarity contrasts with the striking difference between in vivo and in vitro RNA structures observed in STMV. Inspired by this finding, I examined the compactness of the RNA of several different viruses. The results strongly suggest that the RNAs of viruses requiring packaging signals have evolved to be structurally compact, which facilitates post-replicational RNA packaging. In contrast, viruses that do not depend on packaging signals probably adopt co-replicational RNA packaging.
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Whelan, Jillian Nicole. „Investigation of Respiratory Syncytial Virus Structural Determinants and Exploitation of the Host Ubiquitin System“. Scholar Commons, 2016. http://scholarcommons.usf.edu/etd/6431.
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Respiratory syncytial virus (RSV) is a globally circulating, non-segmented, negative sense (NNS) RNA virus that causes severe lower respiratory infections. This study explored several avenues to ultimately expand upon our understanding of RSV pathogenesis at the protein level. Evaluation of RSV intrinsic protein disorder increased the relatively limited description of the RSV structure-function relationship. Global proteomics analysis provided direction for further hypothesis-driven investigation of host pathways altered by RSV infection, specifically the interaction between the RSV NS2 protein and the host ubiquitin system. NS2 primarily acts to antagonize the innate immune system by targeting STAT2 for proteasomal degradation. The goal was to identify NS2 residues important for interaction with the host ubiquitin system, as well as describe the mechanism by which NS2 induces host protein ubiquitination. Bioinformatics analysis provided a platform for development of loss-of-ubiquitin-function NS2 mutants. Combining critical mutations as double or triple NS2 ubiquitin mutants displayed an additive effect on reducing NS2-induced ubiquitination. Recombinant RSV (rRSV) containing NS2 ubiquitin mutations maintained their effect on ubiquitin expression during infection in addition to limiting STAT2 degradation activity. NS2 ubiquitin mutants decreased rRSV growth and increased levels of innate immune responses, indicating a correlation between NS2’s ubiquitin function and antagonism of type I IFN to enhance viral replication. Finally, several proteomics strategies were employed to identify specific cellular proteins ubiquitinated by NS2 to further define host-pathogen interactions during RSV infection. This study demonstrates an effective approach for limiting viral protein function to enhance immune responses during infection.
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Fadda, Valeria. „Structural studies on a hepatitis C virus-related immunological complex and on Ebola virus polymerase cofactor VP35“. Thesis, University of St Andrews, 2015. http://hdl.handle.net/10023/7703.
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Hepatitis C virus (HCV) is one of the leading causes of hepatocellular carcinoma worldwide. HCV-neutralizing antibody AP33 recognizes a linear, highly conserved epitope on the viral entry protein E2, disrupting the interaction with the cellular receptor CD81 that leads to viral entry. AP33-related anti-idiotypes (Ab₂s) have the potential to carry the internal image of the antigen E2, eliciting the production of AP33-like antibodies in humans. This study reports the mid-resolution structure of the Fab fragment of anti-idiotype A164.3 and the high-resolution structure of the Fab fragment of AP33 in complex with the Fv fragment of anti-idiotype B2.1A. Analysis of the structures and comparison with the previously published structure of AP33 in complex with a peptide corresponding to the E2 epitope, suggests that while A164.3 does not mimic the antigen E2, B2.1A is characterized by high surface complementarity with AP33 and functional antigen mimicry. Thus, B2.1A can be classified as an Ab₂-β, a subgroup of anti-idiotypes carrying the internal image of the antigen. Preliminary binding studies show that AP33 binds B2.1A with nanomolar affinity, supporting the role of B2.1A as an idiotypic vaccine candidate. Zaire ebola virus causes severe, often lethal hemorrhagic fever in humans. Ebola virus polymerase cofactor VP35 is a multifunctional protein involved in, among other functions, dsRNA binding and inhibition of the host's interferon pathways. VP35 contains an N-terminal oligomerization domain and a C-terminal dsRNA-binding domain (RBD). Preliminary results on the oligomerization domain of VP35 suggest that this region contains a coiled-coil motif, as previously reported. In order to validate a recently-discovered dsRNA end-capping pocket as a drug target, the structure of VP35 RBD I278A mutant was solved at high resolution, showing that even a small perturbation in the binding pocket can cause dramatic binding impairment due to loss of contacts with dsRNA.
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Flatt, Justin Wayne. „STRUCTURAL INSIGHTS INTO RECOGNITION OF ADENOVIRUS BY IMMUNOLOGIC AND SERUM FACTORS“. Case Western Reserve University School of Graduate Studies / OhioLINK, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=case1387451692.
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Hengrung, Narin. „Structure of the RNA-dependent RNA polymerase from influenza C virus“. Thesis, University of Oxford, 2014. http://ora.ox.ac.uk/objects/uuid:694e16a6-f94e-4375-a1f9-7e250aea7343.
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The influenza virus causes a disease that kills approximately 500,000 people worldwide each year. Influenza is a negative-sense RNA virus that encodes its own RNA-dependent RNA polymerase. This protein (FluPol) carries out both genome replication and viral transcription. Therefore, like the L-proteins of non-segmented negative-sense RNA (nsRNA) viruses, FluPol also contains mRNA capping and polyadenylation functionality. In FluPol, capping is achieved by snatching cap structures from cellular mRNAs, so requiring cap-binding and endonuclease activities. This makes FluPol a substantial machine. It is a heterotrimeric complex, composed of PB1, PB2 and PA/P3 subunits, with a total molecular weight of 255 kDa. PB1 houses the polymerase active site, whereas PB2 and PA contain, respectively, cap-binding and endonuclease domains. Currently, we only have high resolution structural information for isolated fragments of FluPol. This severely hampers our understanding of influenza replication and consequently inhibits the development of therapies against the virus. In this DPhil project, I have determined a preliminary structure for the heterotrimeric FluPol of influenza C/Johannesburg/1/66, solved by x-ray crystallography to 3.6 Å. Overall, FluPol has an elongated structure with a conspicuous deep groove. PB1 displays the canonical right-hand-like polymerase fold. It sits at the centre of the particle, sandwiched between the two domains of P3, and with PB2 stacked against one side of this dimer. In the structure, the polymerase and endonuclease catalytic sites are both ~40 Å away from the cap-binding pocket. This pocket also faces a tunnel leading to the polymerase core. This suggests a mechanism for how capped cellular mRNAs are cleaved and then fed into the polymerase active site to prime transcription. The structure also hints at a unique trajectory for template RNA, in which the RNA exits at an angle ~180° from which it came in. This provides an explanation for how the polymerases of influenza, and other nsRNA viruses, can copy templates that are packaged into ribonucleoprotein complexes. My work reveals the first molecular structure of any polymerase from an nsRNA virus. It uncovers the arrangement of functional domains within FluPol, illuminating the mechanisms of this and related viral polymerases. This work will help focus future experiments into FluPol biology, and should hopefully spur the development of novel antiviral drugs.
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Minoves, Marie. „Etude fonctionnelle et structurale de la glycoprotéine du virus de la Stomatite Vésiculaire et des Lyssavirus“. Electronic Thesis or Diss., université Paris-Saclay, 2024. http://www.theses.fr/2024UPASL068.
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Le virus de la stomatite vésiculaire (VSV) est un virus enveloppé appartenant au genre Vésiculovirus et à la famille des Rhabdoviridae. Son unique glycoprotéine G reconnait un récepteur à la surface de la cellule hôte puis, après endocytose du virion, déclenche la fusion membranaire grâce à une transition structurale induite à faible pH depuis la forme pré-fusion de G vers sa forme post-fusion. Par ailleurs, G est la cible des anticorps neutralisant le virus. Les structures cristallographiques des formes pré- et post-fusion de l'ectodomaine soluble de G (i.e. sans sa partie transmembranaire) ont été déterminées par radiocristallographie. Ces structures ont installé G comme étant le prototype des glycoprotéines de fusion de classe III. L'organisation de l'extrémité carboxyterminale de l'ectodomaine et du domaine transmembranaire de G, qui jouent un rôle important durant le processus de fusion, n'est cependant pas connue. Nous avons donc réalisé une étude par cryo-microscopie électronique sur la glycoprotéine complète, purifiée à partir de particules virales, seule ou en complexe avec un anticorps monoclonal. Cette étude nous a permis de compléter les structures de l'ectodomaine dans ses conformations pré et post-fusion. Elle suggère que les domaines transmembranaires sont mobiles au sein de la membrane. Par ailleurs, nous avons résolu deux structures de G en complexe avec un FAb dérivé d'un anticorps neutralisant, reconnaissant à la fois les formes pré- et post-fusion de plusieurs souches de Vésiculovirus. Cette première structure d'un complexe entre G et un anticorps nous a permis de caractériser finement l'épitope, d'identifier les résidus de G clefs dans l'interaction et de proposer un mécanisme de neutralisation. Ce travail augmente de façon significative nos connaissances sur la structure de G qui est la glycoprotéine la plus utilisée en biotechnologie pour délivrer un cargo et en thérapie génique pour le pseudotypage de vecteurs lentiviraux. Nous avons également débuté une étude visant à caractériser les glycoprotéines des Lyssavirus, genre appartenant également à la famille des Rhabdoviridae, et dont le virus de la rage est le prototype. Nous avons produit et purifié les ectodomaines de plusieurs Lyssavirus, et nous avons pu obtenir une structure cristallographique de l'ectodomaine du virus Ikoma (IKOV G) qui correspondrait à un intermédiaire monomérique tardif. Afin de poursuivre le travail de caractérisation de cette structure, plusieurs approches sont en cours. Nous avons notamment réalisé une sélection par phage display de ligands alphaReps dirigés contre IKOV G. Les alphareps sont des protéines artificielles constituées par des répétitions hélicoïdales. 6 des 11 alphareps sélectionnées sont capables de lier IKOV G. La caractérisation des complexes IKOV G est en cours. Nous envisageons i) d'utiliser ces alphareps pour piéger des conformations différentes de G que celle obtenues pour en obtenir la structure par cristallographie ou cryo-EM ii) tester l'activité antivirale des apharep sélectionnéesVesicular stomatitis virus (VSV), an enveloped virus, is the prototype species of the genus Vesiculovirus within the family Rhabdoviridae. Its G glycoprotein is responsible for receptor recognition, on the host cell surface, that triggers clathrin-mediated endocytosis of VSV. Then, within the acidic environment of the endosome, VSV G undergoes a fusogenic conformational change from the pre-fusion form of G to its post-fusion form, leading the fusion of both membranes. G is also the target of virus-neutralizing antibodies. Both structures of the pre- and post-fusion forms of the soluble ectodomain of G (i.e. without its transmembrane part) were determined by radiocrystallography. These structures established G as the prototype of class III fusion glycoproteins. However, the organization of the carboxyterminal part of the ectodomain and the transmembrane domain of G, which play an important role during the fusion process, remains unknown. Therefore, we carried out a cryo-electron microscopy study on the complete glycoprotein, directly purified from viral particles, alone or in complex with a monoclonal antibody. This study led to complete the structures of the ectodomain in its pre- and post-fusion conformations. It also revealed that the transmembrane domains are mobile within the membrane. We have also solved two structures of G in complex with a FAb derived from a neutralizing antibody, recognizing both pre- and post-fusion forms of G from several strains of Vesiculovirus. Based on these first structures of a complex between G and an antibody, we could characterize the epitope, identify the key G residues in the interaction and propose a neutralization mechanism. This work significantly increases our knowledge of the structure of G, which is the most widely used glycoprotein in biotechnology for cargo delivery and in gene therapy (by lentivirus pseudotyping).We also initiated a study aimed at characterizing the glycoproteins of Lyssaviruses, a genus also part of the Rhabdoviridae family, and for which rabies virus is the prototype. We produced and purified the ectodomains of several Lyssaviruses, and we were able to obtain a crystallographic structure of the ectodomain of Ikoma virus (IKOV G), which corresponds to a late monomeric intermediate. Several approaches are underway to further characterize this structure. We also carried out a phage display selection of alphaReps directed against IKOV G. Alphareps are artificial proteins binders consisting of helical repeats. 6 out of 11 alphareps are able to bind IKOVG. Complexes of IKOV G with alphareps are currently being characterized. We plan to i) use these tools as crystallization helpers to trap different conformations of G in crystallography or cryo-EM ii) evaluate the potential antiviral activity of these alphareps
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Shandilya, Shivender. „Structural Studies of the Anti-HIV Human Protein APOBEC3G Catalytic Domain: A Dissertation“. eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/562.
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HIV/AIDS is a disease of grave global importance with over 33 million people infected world-wide and nearly 2 million deaths each year. The rapid emergence of drug resistance, due to viral mutation, renders anti-retroviral drug candidates ineffective with alarming speed and regularity. Instead of targeting mutation prone viral proteins, an alternative approach is to target host proteins that interact with viral proteins and are critical for the HIV life-cycle. APOBEC3G is a host anti-HIV restriction factor that can exert tremendous negative pressure by hypermutating the viral genome and has the potential to be a promising candidate for anti-retroviral therapeutic research.
The work presented in this thesis is focused on investigating the A3G catalytic domain structure and implications of various observed structural features for biological function. High-resolution crystal structures of the A3G catalytic domain were solved using data from macromolecular X-ray crystallographic experiments, revealing a novel intermolecular zinc coordinating motif unique to A3G. Major intermolecular interfaces observed in the crystal structure were investigated for relevance to biochemical activity and biological function.
Co-crystallization with a small-molecule A3G inhibitor, discovered using high-throughput screening assays, revealed a cysteine residue near the active site that is critical for inhibition of catalytic activity by catechol moieties. The serendipitous discovery of covalent interactions between this inhibitor and a surface cysteine residue led to further biochemical experiments that revealed the other cysteine, near the active site, to be critical for inhibition.
Computational modeling was used to propose a steric-hinderance based mechanism of action that was supported by mutational experiments. Structures of other human APOBEC3 homologs were modeled using in-silico methods examined for similarities and differences with A3G catalytic domain crystal structures. Comparisons based on these homology models suggest putative structural features that may endow substrate specificity and other characteristics to the APOBEC3 family members.
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Luque, Santolaria Antoni. „Structure, Mechanical Properties, and Self-Assembly of Viral Capsids“. Doctoral thesis, Universitat de Barcelona, 2011. http://hdl.handle.net/10803/31993.
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Viruses are submicroscopic biological entities that need to infect a host cell in order to replicate. In their simplest form viruses are constituted by an infective genetic material and a protein shell (the capsid) that protects the viral genome. In this thesis we try to elucidate the general physical principles playing a major role in the morphology, stability, and assembly of viral capsids.
Therefore, in the first part of the thesis, we develop a general theory that characterizes spherical and bacilliform (or prolate) capsids based on icosahedral symmetry under the same geometrical framework. In addition, we demonstrate that the structures derived in this geometrical study are obtained spontaneously from the free energy minimization of a very generic interaction among the viral capsomers of the capsid.
In the second part of the thesis, we analyze the role of the discrete nature of capsids and the organization of capsomers in the actual mechanical properties of shells. We show that the icosahedral class P influences the stability and mechanical response of the quasi-spherical capsids. We also determine that under expansion, spherical shells tend to produce polyhedral structures (buckling), which are more resistant, and it is in consonance with the maturation process observed in some viruses. We also unveil the existence of built-in stress
in the empty procapsids of the elongated bacteriophage φ29. This phenomenon is intimately related to the discrete nature of the structure, and reinforces the mechanical properties of the shell inverting the classical anisotropic response expected from continuum elasticity theory.
In the last part of the thesis, we show that viral assembly and disassembly are activated processes controlled by nucleation barrier, which can be explained adapting classical nucleation theory (CNT). We focus on the case of spherical shells and confirm that the underlying assumptions of CNT are surprisingly good in characterizing the assembly of discrete shells, using the physical model introduced in the previous parts. Finally, we also unveil an interesting closure mechanism during the assembly of capsids.
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Soumana, Djade I. „Hepatitis C Virus: Structural Insights into Protease Inhibitor Efficacy and Drug Resistance: A Dissertation“. eScholarship@UMMS, 2015. http://escholarship.umassmed.edu/gsbs_diss/803.
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The Hepatitis C Virus (HCV) is a global health problem as it afflicts an estimated 170 million people worldwide and is the major cause of viral hepatitis, cirrhosis and liver cancer. HCV is a rapidly evolving virus, with 6 major genotypes and multiple subtypes. Over the past 20 years, HCV therapeutic efforts have focused on identifying the best-in-class direct acting antiviral (DAA) targeting crucial components of the viral lifecycle, The NS3/4A protease is responsible for processing the viral polyprotein, a crucial step in viral maturation, and for cleaving host factors involved in activating immunity. Thus targeting the NS3/4A constitutes a dual strategy of restoring the immune response and halting viral maturation. This high priority target has 4 FDA approved inhibitors as well as several others in clinical development. Unfortunately, the heterogeneity of the virus causes seriously therapeutic challenges, particularly the NS3/4A protease inhibitors (PIs), which suffer from both the rapid emergence of drug resistant mutants as well as a lack of pan-genotypic activity.
My thesis research focused on filling two critical gaps in our structural understanding of inhibitor binding modes. The first gap in knowledge is the molecular basis by which macrocyclization of PIs improves antiviral activity. Macrocycles are hydrophobic chains used to link neighboring chemical moieties within an inhibitor and create a structurally pre-organized ligand. In HCV PIs, macrocycle come in two forms: a P1 - P3 and P2 - P4 strategy. I investigated the structural and thermodynamic basis of the role of macrocyclization in reducing resistance susceptibility. For a rigorous comparison, we designed and synthesized both a P1 - P3 and a linear analog of grazoprevir, a P2 - P4 inhibitor. I found that, while the P2 - P4 strategy is more favorable for achieving potency, it does not allow the inhibitor sufficient flexibility to accommodate resistance mutations. On the other hand, the P1 - P3 strategy strikes a better balance between potency and resistance barrier.
The second gap my thesis addresses is elucidating the structural basis by which highly potent protease inhibitors function in genotype 1 but not in genotype 3, despite having an 87% sequence similarity. After mapping the amino acids responsible for this differential efficacy in genotypes 1 and 3, I engineered a 1a3a chimeric protease for crystallographic studies. My structural characterization of three PIs in complex with both the 1a3a and genotype 1 protease revealed that the loss of inhibitor efficacy in the 1a3a and GT-3 proteases is a consequence of disrupted electrostatic interactions between amino acids 168 and 155, which is critical for potent binding of quinoline and isoindoline based PIs. Here, I have revealed details of molecular and structural basis for the lack of PI efficacy against GT-3, which are needed for design of pan-genotypic inhibitors.
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Koopman, Tammy L. „Production of Porcine Single Chain Variable Fragment (SCFV) selected against a recombinant fragment of Porcine Reproductive and Respiratory Syndrome virus non structural protein 2“. Thesis, Kansas State University, 2011. http://hdl.handle.net/2097/13189.
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Master of ScienceDepartment of Diagnostic Medicine/Pathobiology
Richard 'Dick' Hesse
Carol Wyatt
Over the last two decades molecular laboratory techniques have enabled researchers to investigate the infection, replication and pathogenesis of viral disease. In the early eighties, Dr. George Smith developed a unique system of molecular selection. He showed that the fd bacteriophage genome could be manipulated to carry a sequence of DNA coding for a protein not contained in the phage genome. Infection of the recombinant bacteriophage or phagemid into a specific strain of the bacterium, Escherichia coli, produced progeny phage with the coded protein displayed as a fusion with the phage's coat protein. Antibody phage display utilizes the same technology with the DNA encoding an antibody fragment. The DNA insert can carry the information to produce either a single chain variable fragment (scFv) producing the heavy chain variable and light chain variable (VH-VL) portion or a Fab fragment which also contains the heavy chain constant 1 with the light chain constant (CH and CL) portion of an antibody. Screening an antibody phage display library has the possibility of producing an antibody not produced in the normal course of immune selection. This decade also saw the emergence of a viral disease affecting the porcine population. The Porcine Reproductive and Respiratory Syndrome virus (PRRSV) has been one of the most costly diseases affecting the pig producer. Molecular investigations found that PRRSV is a single, positive-stranded RNA virus which codes for five structural and 12-13 nonstructural proteins producing an enveloped, icosahedral virus. An interesting characteristic of PRRSV is the ability to produce infective progeny with genomic deletions, insertions and mutations within the nonstructural protein 2 (nsp2). With this knowledge, many researchers have produced marker vaccines containing fluorescent tags with the hope of developing a DIVA (Differentiate Infected from Vaccinated Animals) vaccine. In my Master‟s studies, I studied the techniques of antibody phage display technology and how to apply these methods to producing scFvs which recognize a recombinant PRRSV nsp2 fragment protein and the native protein during infection of MARC-145 cells.
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Arista, Romero Maria. „Unveiling viral structures by single-molecule localization microscopy“. Doctoral thesis, Universitat de Barcelona, 2021. http://hdl.handle.net/10803/672262.
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Influenza A virus is one of the most outstanding human viruses.
The major treatments against influenza are small analogues, monoclonal antibodies and vaccines, however, due to the fast mutation of the seasonal influenza strain, these methods are easily outdated, so vaccine production and antiviral development
need to be in continuous growth and study to improve immunity and fight against influenza disease. The characterization of the viral structure and the identification of the mechanisms of action of newly synthesized antivirals are crucial to develop fast and powerful treatments, nonetheless, due to the small size of viruses, conventional fluorescence techniques are lacking the resolution power to resolve individual viral structures. In this context, super-resolution microscopy has positioned in the last decade as a powerful technique to characterize viral constructs by achieving resolutions up to 10 nm.
Here, we have optimized and established a type of super-resolution microscopy technique entitled single-molecule localization microscopy (SMLM) to study viral structures at single-particle level by characterizing several viral structures, antivirals and vaccines.
Firstly, we could characterize the filament formation of influenza virus and described how monoclonal antibodies disturbed the development of those filaments, deforming them. Further, we could relate this malformation with an inhibition of the infectivity, suggesting the crucial role of filament formation in the infectivity of influenza. Moreover, we optimized and implemented a novel SMLM called DNA-PAINT in the study of the target distribution and quantification in the nanoscale, validating this method using commercial nanoparticles for its further implementation in the study of the expression of recombinant proteins of influenza and the corresponding virus-like particle produced.
In addition, we studied and identified several other viral structures and antivirals interactions using SMLM such as the distribution of Hepatitis B and Hepatitis Delta on paraffin tissues, the interaction of analogues of sialic acid on four strains of influenza and the uptake of nanovaccines from antigen-presenting cells, obtaining features on how these viruses and antivirals interact in order to produce a smart design of antivirals and vaccines, corroborating how SMLM could increase the knowledge of the mechanism of action of viruses.
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Wall, Erin A. „ELUCIDATION OF A NOVEL PATHWAY IN STAPHYLOCOCCUS AUREUS: THE ESSENTIAL SITE-SPECIFIC PROCESSING OF RIBOSOMAL PROTEIN L27“. VCU Scholars Compass, 2015. http://scholarscompass.vcu.edu/etd/3747.
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Ribosomal protein L27 is a component of the eubacterial large ribosomal subunit that has been shown to play a critical role in substrate stabilization during protein synthesis. This function is mediated by the L27 N-terminus, which protrudes into the peptidyl transferase center where it interacts with both A-site and P-site tRNAs as well as with 23S rRNA. We observed that L27 in S. aureus and other Firmicutes is encoded with a short N-terminal extension that is not present in most Gram-negative organisms, and is absent from mature ribosomes. The extension contains a conserved cleavage motif; nine N-terminal amino acids are post-translationally removed from L27 by a site-specific protease so that conserved residues important for tRNA stabilization at the peptidyl transferase center are exposed. We have identified a novel cysteine protease in S. aureus that performs this cleavage. This protease, which we have named Prp, is conserved in all bacteria containing the L27 N-terminal extension. L27 cleavage was shown to be essential in S. aureus; un-cleavable L27 did not complement an L27 deletion. Cleavage appears to play an essential regulatory role, as a variant of L27 lacking the cleavage motif could not complement. Ribosomal biology in eubacteria has largely been studied in E. coli; our findings indicate that there are aspects of the basic biology of the ribosome in S. aureus and other related bacteria that differ substantially from that of E. coli. This research lays the foundation for the development of new therapeutic approaches that target this novel, essential pathway.
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Dhar, Jayeeta. „Suppression of Pulmonary Innate Immunity by Pneumoviruses“. Cleveland State University / OhioLINK, 2016. http://rave.ohiolink.edu/etdc/view?acc_num=csu1479673989904175.
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Benachenhou, Farid. „Retroviral long Terminal Repeats; Structure, Detection and Phylogeny“. Doctoral thesis, Uppsala universitet, Klinisk virologi, 2010. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-120028.
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Long terminal repeats (LTRs) are non-coding repeats flanking the protein-coding genes of LTR retrotransposons. The variability of LTRs poses a challenge in studying them. Hidden Markov models (HMMs), probabilistic models widely used in pattern recognition, are useful in dealing with this variability. The aim of this work was mainly to study LTRs of retroviruses and LTR retrotransposons using HMMs. Paper I describes the methodology of HMM modelling applied to different groups of LTRs from exogenous retroviruses (XRVs) and endogenous retroviruses (ERVs). The detection capabilities of HMMs were assessed and were found to be high for homogeneous groups of LTRs. The alignments generated by the HMMs displayed conserved motifs some of which could be related to known functions of XRVs. The common features of the different groups of retroviral LTRs were investigated by combining them into a single alignment. They were the short inverted terminal repeats TG and CA and three AT-rich stretches which provide retroviruses with TATA boxes and AATAAA polyadenylation signals. In Paper II, phylogenetic trees of three groups of retroviral LTRs were constructed by using HMM-based alignments. The LTR trees were consistent with trees based on other retroviral genes suggesting co-evolution between LTRs and these genes. In Paper III, the methods in Paper I and II were extended to LTRs from other retrotransposon groups, covering much of the diversity of all known LTRs. For the first time an LTR phylogeny could be achieved. There were no major disagreement between the LTR tree and trees based on three different domains of the Pol gene. The conserved LTR structure of paper I was found to apply to all LTRs. Putative Integrase recognition motifs extended up to 12 bp beyond the short inverted repeats TG/CA. Paper IV is a review article describing the use of sequence similarity and structural markers for the taxonomy of ERVs. ERVs were originally classified into three classes according to the length of the target site duplication. While this classification is useful it does not include all ERVs. A naming convention based on previous ERV and XRV nomenclature but taking into account newer information is advocated in order to provide a practical yet coherent scheme in dealing with new unclassified ERV sequences. Paper V gives an overview of bioinformatics tools for studies of ERVs and of retroviral evolution before and after endogenization. It gives some examples of recent integrations in vertebrate genomes and discusses pathogenicity of human ERVs including their possible relation to cancers. In conclusion, HMMs were able to successfully detect and align LTRs. Progress was made in understanding their conserved structure and phylogeny. The methods developed in this thesis could be applied to different kinds of non-coding DNA sequence element.
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Coutard, Bruno. „Contribution de la biotechnologie à la virologie structurale et fonctionnelle“. Aix-Marseille 2, 2007. http://theses.univ-amu.fr.lama.univ-amu.fr/2007AIX22046.pdf.
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Ozen, Aysegul. „Structure and Dynamics of Viral Substrate Recognition and Drug Resistance: A Dissertation“. eScholarship@UMMS, 2013. https://escholarship.umassmed.edu/gsbs_diss/677.
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Drug resistance is a major problem in quickly evolving diseases, including the human immunodeficiency (HIV) and hepatitis C viral (HCV) infections. The viral proteases (HIV protease and HCV NS3/4A protease) are primary drug targets. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition; the drug resistant protease variants are no longer effectively inhibited by the competitive drug molecules but can process the natural substrates with enough efficiency for viral survival. Therefore, the inhibitors that better mimic the natural substrate binding features should result in more robust inhibitors with flat drug resistance profiles. The native substrates adopt a consensus volume when bound to the enzyme, the substrate envelope. The most severe resistance mutations occur at protease residues that are contacted by the inhibitors outside the substrate envelope. To guide the design of robust inhibitors, we investigate the shared and varied properties of substrates with the protein dynamics taken into account to define the dynamic substrate envelope of both viral proteases. The NS3/4A dynamic substrate envelope is compared with inhibitors to detect the structural and dynamic basis of resistance mutation patterns. Comparative analyses of substrates and inhibitors result in a solid list of structural and dynamic features of substrates that are not shared by inhibitors. This study can help guiding the development of novel inhibitors by paying attention to the subtle differences between the binding properties of substrates versus inhibitors.
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Ozen, Aysegul. „Structure and Dynamics of Viral Substrate Recognition and Drug Resistance: A Dissertation“. eScholarship@UMMS, 2005. http://escholarship.umassmed.edu/gsbs_diss/677.
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Drug resistance is a major problem in quickly evolving diseases, including the human immunodeficiency (HIV) and hepatitis C viral (HCV) infections. The viral proteases (HIV protease and HCV NS3/4A protease) are primary drug targets. At the molecular level, drug resistance reflects a subtle change in the balance of molecular recognition; the drug resistant protease variants are no longer effectively inhibited by the competitive drug molecules but can process the natural substrates with enough efficiency for viral survival. Therefore, the inhibitors that better mimic the natural substrate binding features should result in more robust inhibitors with flat drug resistance profiles. The native substrates adopt a consensus volume when bound to the enzyme, the substrate envelope. The most severe resistance mutations occur at protease residues that are contacted by the inhibitors outside the substrate envelope. To guide the design of robust inhibitors, we investigate the shared and varied properties of substrates with the protein dynamics taken into account to define the dynamic substrate envelope of both viral proteases. The NS3/4A dynamic substrate envelope is compared with inhibitors to detect the structural and dynamic basis of resistance mutation patterns. Comparative analyses of substrates and inhibitors result in a solid list of structural and dynamic features of substrates that are not shared by inhibitors. This study can help guiding the development of novel inhibitors by paying attention to the subtle differences between the binding properties of substrates versus inhibitors.
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Feng, Yuqin. „Molecular epidemiological analysis of rabies viruses associated with population structure of bat hosts“. Thesis, University of Ottawa (Canada), 2006. http://hdl.handle.net/10393/27243.
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To explore whether rabies viral variants co-localize with discrete bat host populations (sub-populations), both the host genome and rabies virus of Eptesicus fuscus (big brown bats), Myotis lucifugus (little brown bats) and other Myotis specimens, collected during 1989 to 2004 from diagnostic submissions from across the country, were genetically characterized.
Bat species population analysis was performed by nuclear DNA genotyping, scored by variation of several microsatellite loci, and through phylogenetic analysis of Cox-1 (cytochrome oxidase subunit I) gene sequences located on mitochondrial DNA. Microsatellites are relatively short DNA stretches consisting of tandem repeats of one to five nucleotides which exhibit high levels of allelic variation. Cox-1 gene sequence analysis provides accurate species level of identification for Myotis lucifugus specimens. Two hundred and ninety five DNA samples of Eptesicus fuscus were examined at 9 microsatellite loci, and 126 DNA samples of Myotis lucifugus were examined at 7 microsatellite loci, both datasets were analyzed by a series of genetic population analysis softwares. Phylogeny of Cox-1 gene sequences with 552 nucleotides by using 106 DNA samples of bats Myotis lucifugus was analyzed as an alternative strategy of the microsatellite gene marker for population structure determination of Myotis lucifugus . Consequently, two populations---East (group I) and West (group II) were determined for Eptesicus fuscus bats in Canada. No population structure was identified for Myotis lucifugus bats.
Rabies viral variants were identified by nucleotide sequencing of the central divergent portion of the P (phosphoprotein) gene---a region previously proved to be a sensitive target for molecular epidemiology analysis. Viral RNA, isolated from 231 samples of Eptesicus fuscus and Myotis species was sequenced over a 597 by region. Phylogenetic analysis of these data identified six rabies viral variants circulating in Eptesicus fuscus and one rabies viral variant circulating in Myotis species.
Based on the results of genetic characterization, the spatial distribution of the bat host subpopulations and their associated rabies virus variants were determined. Rabies variants I and II circulate in Eptesicus fuscus population I (East); rabies variants III, IV and V circulate in Eptesicus fuscus population II (West); and rabies variant VI circulates in both populations. A distinct rabies virus variant VII was associated with Myotis species. No population substructure would be identified for Myotis lucifugus bats.
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Roznowski, Aaron. „A Structure-Function Analysis of the phiX174 DNA Piloting Protein“. Thesis, The University of Arizona, 2019. http://pqdtopen.proquest.com/#viewpdf?dispub=13812936.
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In order to initiate an infection, bacteriophages must deliver their large, hydrophilic genomes across their host’s hydrophobic cell wall. Bacteriophage ϕX174 accomplishes this task with a set of identical DNA piloting proteins. The structure of the piloting protein’s central domain was solved to 2.4 Å resolution. In it, ten proteins are oligomerized into an α-helical barrel, or tube, that is long enough to span the host’s cell wall and wide enough for the circular, ssDNA to pass through. This structure was used as a guide to explore the mechanics of ϕX174 genome delivery. In the first study, the H-tube’s highly repetitive primary and quaternary structure made it amenable to a genetic analysis using in-frame insertions and deletions. Length-altered proteins were characterized for the ability to perform the protein’s three known functions: participation in particle assembly, genome translocation, and stimulation of viral protein synthesis.
The tube’s inner surface was altered in the second study. The surface is primarily lined with amide and guanidinium containing amino acid side chains with the exception of four sites near the tube’s C-terminal end. The four sites are conserved across microvirus clades, suggesting that they may play an important role during genome delivery. To test this hypothesis and explore the general role of the amide and guanidinium containing side chains, the amino acids at these sites were changed to glutamine. The resulting mutants had a cold-sensitive phenotype at 22 °C. Viral lifecycle steps were assayed in order to determine which step was disrupted by the mutant glutamine residues. The results support a model in which a balance of forces governs genome delivery: potential energy provided by the densely packaged viral genome and/or an osmotic gradient push the genome into the cell, while the tube’s inward facing residues exert a frictional force on the genome as it passes.
Bacteriophage must first identify a susceptible host prior to genome delivery. In the final study, biochemical and genetic analyses were conducted with two closely related bacteriophages, α3 and ST-1. Despite ~90% amino acid identity, the natural host of α3 is Escherichia coli C, whereas ST-1 is a K-12-specific phage. To determine which structural proteins conferred host range specificity, chimeric virions were generated by individually interchanging the coat, spike, or DNA pilot proteins. Interchanging the coat protein switched host range. However, host range expansion could be conferred by single point mutations in the coat protein. The expansion phenotype was recessive: mutant progeny from co-infected cells did not display the phenotype. Novel virus propagation and selection protocols were developed to isolate host range expansion mutants. The resulting genetic and structural data were consistent enough that host range expansion could be predicted, broadening the classical definition of antireceptors to include interfaces between protein complexes within the capsid.
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O'Hara, Maureen. „Relating the structure of the HSV-1 UL25 DNA packaging protein to its function“. Thesis, University of Glasgow, 2009. http://theses.gla.ac.uk/1326/.
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The herpes simplex virus type 1 (HSV-1) UL25 protein (pUL25) is a minor capsid protein that is essential for packaging the full-length viral genome into preformed precursor capsid. It is also important in virus entry and recently has been implicated in the egress of the virus from the cell (Coller et al., 2007, Preston et al., 2008). The crystallographic structure of an N-terminally truncated form of pUL25 (residues 134-580) has been determined to 2.1 Å, revealing a protein with a novel fold that consists mostly of a-helices and a few minor b-sheets (Bowman et al., 2006). An unusual feature of the protein is the presence of numerous flexible loops extending out from the stable core and its distinctive electrostatic distribution. Five of the extended loops contain unstructured regions, L1-L5, with three additional unstructured amino acids, L6, located at the carboxyl terminus of the protein (Bowman et al., 2006). Four potentially functional clusters of residues, C1-C4, were identified on the surface of the protein using evolutionary trace analysis (Lichtartge & Sowa, 2002). To examine the function of the protein in relation to its structure, site-directed mutations were engineered into the UL25 gene in a protein expression plasmid. A series of mutant proteins was generated, each protein containing a deletion of the unstructured residues in one of the six regions, L1-L6. Another set of mutant proteins were constructed with each member containing substitutions of selected amino acids within one of the four potentially functional clusters, C1-C4, or substitutions of the three disordered amino acids in L6. The amino acid substitutions were generally to alanine, but in one case where the SIFT program predicted alanine would not affect the function of the protein an alternative residue was substituted. To determine the functional significance of the uncrystallised part of pUL25, residues 1-133, three deletion mutant proteins that spanned this region (pUL25D1-45, pUL25D1-59 and pUL25D1-133) were included in the study. Although an existing UL25 null mutant, KUL25NS, was available at the beginning of the project for analysis of the mutant proteins, it had been made by the insertion of multiple stop codons in the UL25 ORF and as a result some UL25 sequences were still present within the virus genome. Consequently, during complementation assays recombination between the UL25 sequences in the KUL25NS genome and the transfected expression plasmid generated low levels of wild-type (wt) progeny virus. To improve the sensitivity of the assay, a new deletion mutant, ΔUL25MO, was created that lacked the entire UL25 gene. This mutant failed to form plaques in non-permissive Vero cells and grew well in the complementing cell line, 8-1. However, contrary to previously published work, electron microscopic (EM) analysis revealed that DNA-containing capsids as well as A- and B-capsids were present in the nuclei of both ΔUL25MO- and KUL25NS-infected cells. As expected, none of the progeny from ΔUL25MO-infected Vero cells expressing the wt pUL25 formed plaques on non-permissive cells. Of the 17 mutant UL25 proteins screened in the complementation assay, nine failed to complement the growth of ΔUL25MO in Vero cells. Three of the non-complementing mutant proteins examined altered the phenotype of ΔUL25MO in a transient DNA packaging assay, allowing the mutant virus to package full-length genomes in U2OS cells co-infected with ΔUL25MO and a mammalian baculovirus vector containing the mutant UL25 gene. These results indicate that viral assembly was disrupted in these cells following DNA packaging. However, five of the mutant proteins did not change the pattern of DNA encapsidation of ΔUL25MO in this system, suggesting that the wild-type residues mutated in these proteins are critical for packaging virus DNA. To determine at which point in the virus growth cycle the post-packaging blocks occurred, EM was used to investigate the pattern of virus assembly in ΔUL25MO-infected cells expressing either of the three packaging-competent mutant proteins. In addition, fluorescent in-situ hybridisation (FISH) analysis was performed to establish the distribution of virus DNA in these cells. The results showed that in ΔUL25MO-infected cells expressing two of the mutant pUL25s the C-capsids failed to exit the nucleus, whereas in cells expressing the third post-packaging mutant protein C-capsids were seen in both the nucleus and the cytoplasm. The FISH data confirmed the EM observations. These studies show that two regions of pUL25 are important for egress of the C-capsids from the nuclei. Since these two regions lie in close proximity to each other on the surface of the molecule they may represent a single functional interface of the protein. In addition, another region of pUL25 was identified that was essential for the interactions required for virus assembly after the C-capsids are released into the cytoplasm. The 62 carboxyl-terminal region of the UL36 gene product (pUL36) has previously been shown to contain a capsid-binding domain (CBD) that interacts with pUL25 (Coller et al., 2007). A GST-pull down assay was used to determine whether the mutations in the post-packaging mutant proteins disrupted the interaction of pUL25 with the CBD of pUL36. However, all of these mutant proteins and the wt pUL25 bound to the pUL36 CBD GST fusion protein. In summary, three different classes of pUL25 mutants, each of which affect a different essential function of pUL25, have been identified, revealing that pUL25 is indeed a versatile viral protein. These mutants provide the first evidence that this DNA packaging protein is crucial for virus assembly at two different stages after DNA encapsidation, one in nuclear egress of C-capsids and the other in the assembly of the virus in the cytoplasm.
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Romano, Keith P. „Mechanisms of Substrate Recognition by HCV NS3/4A Protease Provide Insights Into Drug Resistance: A Dissertation“. eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/554.
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HCV afflicts many millions of people globally, and antiviral therapies are often ineffective and intolerable. The Food and Drug Administration approved the HCV protease inhibitors telaprevir and boceprevir in May 2011, marking an important milestone in anti-HCV research over the past two decades. Nevertheless, severe drug side effects of combination therapy – flu-like symptoms, depression and anemia – limit patient adherence to treatment regimens. The acquisition of resistance challenges the long-term efficacy of antiviral therapies, including protease inhibitors, as suboptimal dosing allows for the selection of drug resistant viral variants. A better understanding of the molecular basis of drug resistance is therefore central to developing future generation protease inhibitors that retain potency against a broader spectrum of HCV strains.
To this end, my research characterizes the molecular basis of drug resistance against HCV protease inhibitors. Chapter II defines the mode of substrate recognition by the common volume shared by NS3/4A substrate products – the substrate envelope. Chapter III then correlates patterns of drug resistance to regions where drugs protrude from the substrate envelope. Lastly, Chapter IV elucidates the molecular underpinnings of resistance against four leading protease inhibitors – telaprevir, danoprevir, vaniprevir and MK-5172 – and provides practical approaches to designing novel drugs that are less susceptible to resistance. I ultimately hope my work appeals to the broader biomedical community of virologists, medicinal chemists and clinicians, who struggle to understand HCV and other human pathogens in the face of rapid disease evolution.
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Fan, Wan Ho. „Investigating the structure of herpes simplex virus - 1 at the interface between the capsid and tegument“. Thesis, University of Glasgow, 2015. http://theses.gla.ac.uk/6876/.
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The structure of the herpesviruses particle is characterised by an icosahedral capsid surrounded by a proteinaceous tegument layer and is enclosed by a lipid envelope. The understanding of the structure of the capsid, primarily through the use of cyro-electron microscopy, is greater to than of the tegument, due to the typically amorphous nature of the tegument. The interaction between the capsid and tegument has been well studied, unveiling interactions limited to the capsid vertices involving two minor capsid proteins, pUL17 and pUL25, and the large tegument protein pUL36. In herpes simplex virus – 1 (HSV-1), pUL17 and pUL25 form the capsid vertex-specific component (CVSC), a heterodimeric structure which resides in top of triplexes between peripentonal hexons. pUL36 has been suggested to connect the CVSC to the penton and to the rest of the tegument proteins. Recent studies on the gammaherpesvirus Karposi’s sarcoma-associated herpesvirus (KSHV) and the alphaherpesvirus pseudorabies virus (PrV) have questioned both the protein content of the CVSC and the organization of pUL17 and pUL25. As well as the composition of the CVSC, these studies have provided further insight as to the location of tegument assembly to the capsid, a subject that remains a highly contested. In order to clarify the content of the CVSC, virus mutants with deletions of the large inner tegument protein pUL36, and a second inner tegument protein, pUL37, were analysed using cryo-electron microscopy and icosahedral reconstructions. The examination and comparisons of these mutants with wild-type HSV-1 revealed that the CVSC is not only formed by pUL17 and pUL25 as originally reported, but also pUL36, as suggested in the most recent studies. In addition, comparisons of the capsid structure of the pUL36 deletion mutant with a mutant with a full deletion of pUL34, a protein implicated in nuclear egress as part of the nuclear envelopment complex (NEC) and therefore cannot exit the nucleus, suggest that at least part of pUL36 is present on nuclear capsids. Further analyses of the mutant virus with pUL36 deleted using immunofluorescence and Western blotting, along with the capsid reconstruction of a pUL36 deletion mutant which retains only the N-terminal 361 codons suggest that the C-terminal end of pUL36 is present in the nucleus. The work presented here offers additional evidence to clarify the roles of pUL17, pUL25 and pUL36 in tegument assembly. In particular, structural analysis has implied that the contributions of pUL36 to the nuclear capsid stabilizes the CVSC structure from a structural stand-point, emphasizing it’s importance as a multifunctioning protein which acts as a bridge between the capsid and tegument.
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Coulibaly, Fasséli. „Etude structurale des birnavirus : identification des déterminants d'antigénicité, de virulence et d'assemblage : mise en évidence d'un lien évolutif entre virus à ARN(+) et à ARN double brin“. Paris 11, 2003. http://www.theses.fr/2003PA112236.
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Les birnavirus occupent une place particulière parmi les virus icosaédriques non enveloppés. Ils sont à la fois proches des Reoviridae par leur organisation génomique et leur capside T=13 et proches de virus à ARN positif par leurs stratégies réplicatives et de morphogenèse. Ce travail présente la structure de particules subvirales à symétrie icosaédrique T=l de l'IBDV. Ces particules de 260 A de diamètre se composent de 60 sous-unités de la protéine d'attachement du virus, VP2. Celle-ci se replie sous la forme de deux domaines jelly rolls perpendiculaires reposant sur une base riche en hélices α. Le jelly roll radial forme des projections trimériques (domaine P) sur la particule et apparaît comme un domaine priviligié d'interaction virus-hôte: il porte l'ensemble des épitopes de neutralisation ainsi que les déterminants de virulence et de tropisme cellulaire. Le jelly roll tangentiel assure la surface (domaine S) icosaédrique continue de la particule. Enfin, le domaine riche en hélices α tapisse l'intérieur de la particule (domaine B) offrant ainsi une surface d'interaction interne avec d'autres constituants viraux tels que l'ARN ou VP3. En particulier, ce domaine forme un tonneau d'hélices α susceptible de participer à la sortie des ARN viraux par l'axe 5. De plus, la comparaison structurale de VP2 avec d'autres capsides de virus à ARN suggère que les birnavirus pourraient constituer un chaînon évolutif reliant les virus à ARN(+) assez simples aux virus à ARN double brin plus complexes. Enfin, la transposition des résultats obtenus sur VP2 à la particule virale complète montre la présence d'une différence conformationnelle importante. Nous proposons donc un modèle de la morphogenèse virale où le rôle régulateur de VP3 passe par une interaction VP3-VP2 induisant une trans-conformation de VP2Birnaviruses appear to be atypical among icosahedral RNA viruses. Their genomic and structural organization lead to comparisons with members of the Reoviridae family. However, birnaviruses seem to bear more functional similarities to positive-strand RNA viruses such as Nodaviridae or Picornaviridae. We have determined the structures of T=l icosahedral subviral particles of two birnaviruses. Twenty copies of the attachment protein VP2 trimers make up these 260A-wide particles. VP2 folds as two orthogonal jelly rolls on top of a helical domain. The radial jelly roll is the trimeric spike (domain P) projecting outward of a continuous icosahedral shell formed by the other jelly roll (domain S). Domain P is the major site of virus-host interactions as it bears all the neutralizing epitopes as well as the determinants of viral tropism and virulence. Helical domain B coats the inner surface of the particle providing a domain of interaction to other viral constituents such as RNA or VP3. In particular, this domain forms a helical barrel at five fold axes which might be involved in viral RNA exit. Our structural comparison of VP2 structure to other capsid proteins points to an evolutionary link between RNA(+) (Nodaviridae and Tetraviridae) and double-stranded RNA (Reoviridae) viruses embodied by Birnaviruses. Finally, a fit of VP2 in the viral particle requires a major conformational change. We propose a model for morphogenesis in which VP3 binding to VP2 would act as a molecular switch triggering assembly thereafter driven by VP2 self association capacities
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DORE, PETIT-MAIRE ISABELLE. „Utilisation des anticorps monoclonaux en virologie vegetale : diagnostic et etudes structurales de quelques tobamovirus“. Université Louis Pasteur (Strasbourg) (1971-2008), 1987. http://www.theses.fr/1987STR13097.
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Etude des epitopes reconnus par 18 anticorps monoclonaux diriges contre la proteine de l'enveloppe du virus de la mosaique du tabac, en mesurant leur reactivite vis-a-vis de differents mutants, virus et peptides synthetiques. Visualisation de la liaison entre anticorps monoclonaux et virus par microscopie electronique, dans differents tests elisa. Preparation d'anticorps monoclonaux permettant la detection du virus des taches annulaires de l'odontoglossum et du virus de la mosaique de la tomate
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Dore, Isabelle. „Utilisation des anticorps monoclonaux en virologie végétale diagnostic et études structurales de quelques tobamovirus /“. Grenoble 2 : ANRT, 1987. http://catalogue.bnf.fr/ark:/12148/cb37604651w.
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Hornsey, Crystal A. „The function of extensive structured RNA in the evasion of host anti-virus responses“. Thesis, University of Warwick, 2012. http://wrap.warwick.ac.uk/56672/.
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Genome scale ordered RNA structure (GORS) is found throughout the genome of many single stranded positive sense RNA viruses, including plant viruses. It was hypothesised that GORS may function to help evade RNAi either by preventing the generation of siRNAs or by stopping RNAi-mediated cleavage of the target. This project used Potato Leafroll Virus (PLRV) to investigate the function of GORS in plant viruses. The RNA structure of a 750nt region of the genome was modified to have less, more or the same energy as the WT sequence. The physical structure of these sequences was shown to be different using two distinct methods. Viral infectivity was tested and although all four viruses were able to replicate and spread to distal leaves, the WT virus was always able to outcompete the variant viruses in competition assays. This suggests GORS provides a distinct selective advantage to the WT virus. The effect on the siRNA response was tested using a dedicated siRNA assay. In plants, this showed that the WT sequence was more resistant to degradation by siRNAs than the variant sequences in the presence of their specific inducers. The WT inducer was also not able to cause suppression of the other targets indicating that this inducer failed to produce siRNAs or that they were not effective. The siRNA populations generated during infections were sequenced and the profiles compared. This showed that all four viruses stimulated the production of siRNAs but the location of siRNA hotspots differed. It is therefore hypothesised that GORS may function to evade the RNAi response by directing the generation of less effective siRNAs. The data presented in this thesis not only informs current work on GORS and RNA structure in viral genomes but also has wider implications for research on siRNAs and food biosecurity.
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Mittal, Seema. „Role of Protein Flexibility in Function, Resistance Pathways and Substrate Recognition Specificity in HIV-1 Protease: A Dissertation“. eScholarship@UMMS, 2011. https://escholarship.umassmed.edu/gsbs_diss/573.
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In the 30 years since the Center for Disease Control's Morbidity and Mortality Weekly Report published the first mention of what later was determined to be AIDS (Acquired immunodeficiency syndrome) and HIV (Human immunodeficiency virus) recognized as the causative pathogen, much has been done to understand this disease’s pathogenesis, development of drugs and emergence of drug resistance under selective drug therapy. Highly Active Antiretroviral Therapy (HAART), a combination of drugs that includes HIV-1 reverse transcriptase, protease, and more recently, integrase and entry inhibitors, have helped stabilize the HIV prevalence at extraordinarily high levels. Despite the recent stabilization of this global epidemic, its dimensions remain staggering with estimated (33-36 million) people living with HIV-AIDS in 2007 alone. This is because the available drugs against AIDS provide treatment for infected individuals, but HIV evolves rapidly under drug pressure and develops resistant strains, rendering the therapy ineffective. Therefore, a better understanding underlying the molecular mechanisms of viral infection and evolution is required to tackle drug resistance and develop improved drugs and treatment regimens.
HIV-1 protease is an important target for developing anti-HIV drugs. However, resistant mutations rapidly emerge within the active site of the protease and greatly reduce its affinity for the protease inhibitors. Frequently, these active site drug resistant mutations co-occur with secondary/ non-active site/ associated or compensatory mutations distal to the active site. The role of these accessory mutations is often suggested to be in maintaining viral fitness and stability of protease. Many of the non-active site drug resistant mutations are clustered in the hydrophobic core in each monomer of the protease. Molecular dynamic simulation studies suggest that the hydrophobic core residues facilitate the conformational changes that occur in protease upon ligand binding. There is a complex interdependence and interplay between the inherent adaptability, drug resistant mutations and substrate recognition by the protease. Protease is inherently dynamic and has wide substrate specificity. The PI (protease inhibitor) resistant mutations, perhaps, modulate this dynamics and bring about changes in molecular recognition, such that, in resistant proteases, the substrates are recognized specifically over the PIs for the same binding site. In this thesis research, I have investigated these three complementary phenomena in concert.
Chapter II examines the importance of hydrophobic core dynamics in modulating protease function. The hydrophobic core in the WT protease is intrinsically flexible and undergoes conformational changes required for protease to bind its substrates. This study investigated if dynamics is important for protease function by engineering restricted vs. flexible hydrophobic core region in each monomer of the protease, using disulfide chemistry. Under oxidizing conditions, disulfide bond established cross-link at the interface of putative moving domains in each monomer, thereby, restricting motion in this region. Upon reduction of the disulfide bond, the constraining influence was reversed and flexibility returned to near WT. The disulfide cross-linked protease showed significant loss of function when tested in functional cleavage assay. Two protease variants (G16C/L38C) and (R14C/E65C) were engineered and examined for changes in structure and enzymatic activity under oxidizing and reducing conditions. (R14C/E65C) was engineered as an internal control variant, such that cysteines were engineered between putative non-moving domains. Structurally, both the variants were very similar with no structural perturbations under oxidizing or reducing conditions. While significant loss in function was observed for (G16C/L38C) only under oxidizing conditions, (R14C/E65C) did not show any loss of function under oxidizing or reduced conditions, as expected. Successful regain of function for cross-linked (G16C/L38C) was obtained upon reversible reduction of the disulfide bond. Taken together, these data demonstrate that the hydrophobic core dynamics modulates protease function and support the hypothesis that the distal drug resistant mutations, possibly causing drug resistance by modulating hydrophobic core dynamics via long range structural perturbations. Since protease recognizes and cleaves more than 10 substrates at different rates, our further interest is to investigate if there is a differential loss of activity for some specific substrates over the others, and whether the order of polypeptide cleavage is somehow affected by restricted core mobility. In order to better answer these questions it is essential to understand: what determines the substrate binding specificity in protease? A two-pronged approach was applied to address this question as described in chapter III and IV respectively.
In chapter III, I investigated the determinants of substrate specificity in HIV-1 protease by using computational positive design and engineered specificity-designed asymmetric protease (Pr3, A28S/D30F/G48R) that would preferentially bind to one of its natural substrates, RT-RH over two other substrates, p2-NC and CA-p2, respectively. The designed protease was expressed, purified and analyzed for changes in structure and function relative to WT. Kinetic studies on Pr3 showed that the specificity of Pr3 for RT-RH was increased significantly compared to the wild-type (WT), as predicted by the positive design. ITC (Isothermal Titration Calorimetry) studies confirmed the kinetic data on RT-RH. Crystal structural of substrate complexes of WT protease and Pr3 variant with RT-RH, CA-p2 and p2-NC were further obtained and analyzed. The structural analysis, however, only partially confirmed to the positive design due to the inherent structural pliability of the protease. Overall, this study supports the positive computational design approach as an invaluable tool in facilitating our understanding of complex proteins such as HIV 1 protease and also proposes the integration of internal protein flexibility in the design algorithms to make the in-silico designs more robust and dependable.
Chapter IV probed the substrate specificity determining factors in HIV-1protease system by focusing on the substrate sequences. Previous studies have demonstrated that three N-terminal residues immediate to the scissile bond (P1-P3) are important in determining recognition specificity. This work investigated the structural basis of substrate binding to the protease. Catalytically active WT protease was crystallized with decameric polypeptides corresponding to five of the natural cleavage sites of protease. The structural analyses of these complexes revealed distinct P side product bound in all the structures, demonstrating the higher binding affinity of N terminal substrate for protease.
This thesis research successfully establishes that intrinsic hydrophobic core flexibility modulates function in HIV-1 protease and proposes a potential mechanism to explain the role of non-active site mutations in conferring drug resistance in protease. Additionally, the work on specificity designed and N terminal product bound protease complexes advances our understanding of substrate recognition in HIV protease.
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Eno-Ibanga, Cheryl K. „The analysis of a conserved RNA structure in the 3D polymerase encoding region of human parechovirus 1“. Thesis, University of Essex, 2016. http://repository.essex.ac.uk/19097/.
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Picornaviruses are important causes of human illness and it is necessary to understand more about how these viruses function. Human parechoviruses (HPeV) are common pathogens and studies have shown that 95% of people become infected with HPeV at a very early age, usually with symptoms such as mild diarrhoea and fever. However, one virus type HPeV3, is implicated in much more serious cases of neonatal disease and so it is important to understand HPeVs to increase the opportunity to develop drugs or vaccines against the infection. The HPeV1 genome encodes a single polyprotein that is cleaved into structural and non-structural proteins. Analysis of one region of the genome (encoding the polymerase, 3Dpol) shows that some codons are perfectly conserved, suggesting functions in addition to protein coding. This region seems to fold into an RNA secondary structure made up of three stem-loops and a tertiary structure “kissing” interaction. The structure was validated by comparing all the available HPeV sequences and found to be highly conserved. To investigate if the structure has a role in RNA stability, an EGFP fluorescent assay was used. Sequences containing the structure was added to the 3’ UTR of the EGFP gene. A mutant with 21 mutations which completely destroys the RNA structure was also used. A FACS-based method was used to measure expression levels of EGFP. The results showed that there was a significant reduction in fluorescence from the mutant construct. The effect of the structure was also investigated in infected cells and in cells exposed to different stresses which could mimic virus infection. The results suggest that the structure can positively affect RNA stability/translation. Further investigation on other possible roles such as RNA replication and translation should be performed to improve the understanding of the biology of the structure in HPeVs and a Renilla Luciferase reporter gene system was assembled to facilitate the studies in the future.
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Lin, Kuan-Hung. „Viral Proteases as Drug Targets and the Mechanisms of Drug Resistance: A Dissertation“. eScholarship@UMMS, 2016. https://escholarship.umassmed.edu/gsbs_diss/841.
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Viral proteases have been shown to be effective targets of anti-viral therapies for human immunodeficiency virus (HIV) and hepatitis C virus (HCV). However, under the pressure of therapy including protease inhibitors, the virus evolves to select drug resistance mutations both in the protease and substrates. In my thesis study, I aimed to understand the mechanisms of how this protease−substrate co-evolution contributes to drug resistance. Currently, there are no approved drugs against dengue virus (DENV); I investigated substrate recognition by DENV protease and designed cyclic peptides as inhibitors targeting the prime site of dengue protease.
First, I used X-ray crystallography and subsequent structural analysis to investigate the molecular basis of HIV-1 protease and p1-p6 substrate coevolution. I found that co-evolved p1-p6 substrates rescue the HIV-1 I50V protease’s binding activity by forming more van der Waals contacts and hydrogen bonds, and that co-evolution restores the dynamics at the active site for all three mutant substrates.
Next, I used aprotinin as a platform to investigate DENV protease–substrate recognizing pattern, which revealed that the prime side residues significantly modulate substrate affinity to protease and the optimal interactions at each residue position. Based on these results, I designed cyclic peptide inhibitors that target the prime site pocket of DENV protease. Through optimizing the length and sequence, the best inhibitor achieved a 2.9 micromolar Ki value against DENV3 protease. Since dengue protease does not share substrate sequence with human serine proteases, these cyclic peptides can be used as scaffolds for inhibitor design with higher specificity.