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1
Zwart, Lizahn. "Investigating two AHSV non-structural proteins : tubule-forming protein NS1 and novel protein NS4". Diss., University of Pretoria, 2013. http://hdl.handle.net/2263/62198.
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African horse sickness is an equid disease caused by African horse sickness virus (AHSV). AHSV produces seven structural proteins that form the virion and four non-structural proteins with various roles during replication. The first part of this study investigated the intracellular distribution and co-localisations of NS1 with other AHSV proteins to facilitate its eventual functional characterisation. Confocal microscopy revealed that NS1 formed small cytoplasmic foci early after infection that gradually converged into large fluorescent NS1 tubule bundles. Tubule bundles were more organised in AHSV-infected cells than in cells expressing NS1 alone, suggesting that tubule bundle formation requires the presence of other AHSV proteins or regulation of NS1 expression rates. NS1 occasionally co-localised with VP7 crystalline structures, independently of other AHSV proteins. However, when NS1-eGFP, a modified NS1 protein that contains enhanced green fluorescent protein (eGFP) near the C-terminus, was co-expressed with VP7, co-localisation between these proteins occurred in most co-infected cells. It is not clear how the addition of eGFP to NS1 induces this co-localisation and further investigation will be required to determine the function of NS1 during viral replication.
The second part of the study focused on characterising the novel non-structural AHSV protein NS4. The NS4 open reading frame (ORF) occurs on segment 9, overlapping the VP6 ORF in a different reading frame. In silico analysis of segment 9 nucleotide and NS4 predicted amino acid sequences revealed a large amount of variation between serotypes, and two main types of NS4 were identified based on these analyses. These proteins differed in length and amino acid sequence and were named NS4-I and NS4-II. Immunoblotting confirmed that AHSV NS4 is translated in AHSV infected insect and mammalian cells, and also in Sf9 insect cells infected with recombinant baculoviruses that overexpress the genome segment 9 proteins, VP6 and NS4. Confocal microscopy showed that NS4 localised to both the cytoplasm and nucleus, but not the nucleolus, in AHSV-infected cells and recombinant baculovirus infected Sf9 cells. Nucleic acid protection assays using bacterially expressed purified NS4 showed that both types of NS4 bind dsDNA, but not dsRNA. This was the first study to focus on AHSV NS4. Future work will focus on determining the role of non-structural proteins in viral pathogenesis, and will involve the use of a reverse genetics system for AHSV.Dissertation (MSc)--University of Pretoria, 2013.
Genetics
MSc
Unrestricted
2
Shaw, Andrew Edward. "The Role of Non-Structural Proteins in Bluetongue Virus Replication". Thesis, University College London (University of London), 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.519459.
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Xiao, Yibei [Verfasser]. "Structural and functional studies on coronavirus non-structural proteins 7/8 and 5 / Yibei Xiao". Lübeck : Zentrale Hochschulbibliothek Lübeck, 2014. http://d-nb.info/1050905830/34.
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Horscroft, Nigel John. "Orbivirus non-structural protein NS2 : its role in virus replication". Thesis, University of Oxford, 1997. http://ora.ox.ac.uk/objects/uuid:9b550db6-dd9d-4127-941f-93eab2b6e038.
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5
Young, Natalie Jane. "Bovine Viral Diarrhoea Virus Subunit Vaccines : Contribution of Non-Structural Proteins". Thesis, Royal Veterinary College (University of London), 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.498686.
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6
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.
7
Chung, Keun-Taik. "Interactions and functions of rotavirus non-structural proteins NSP1 and NSP3". Thesis, University of Warwick, 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414314.
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8
Ponnusamy, Rajesh [Verfasser]. "Crystallographic and biochemical investigations on coronarvirus replication proteins - non-structural proteins 8 and 9 / Rajesh Ponnusamy". Lübeck : Zentrale Hochschulbibliothek Lübeck, 2010. http://d-nb.info/1004773706/34.
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Tai, Hung, i 戴雄. "The role of the non-structural protein, NS1, in influenza virus replication". Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2010. http://hub.hku.hk/bib/B44660303.
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10
Motari, Edwin Mwamba. "Structural Studies of Oligosaccharides Attached to Proteins Expressed in Different Organisms and PEGylation of a non-Glycosylated Protein". The Ohio State University, 2010. http://rave.ohiolink.edu/etdc/view?acc_num=osu1274803162.
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11
Barski, Michał S. "Structural studies of bunyavirus interferon antagonist proteins". Thesis, University of St Andrews, 2016. http://hdl.handle.net/10023/8408.
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Bunyaviridae is one of the biggest known viral families, and includes many viruses of clinical and economic importance. The major virulence factor of most bunyaviruses is the non-structural protein (NSs). NSs is expressed early in infection and inhibits the innate immune response of the host by blocking several steps in the interferon induction and signalling pathways. Hence, NSs significantly contributes to the establishment of a successful viral infection and replication, persistent infection and the zoonotic capacity of bunyaviruses. Although functions and structures of many viral interferon antagonists are known, no structure of a bunyavirus NSs protein has been solved to date. This strongly limits our understanding of the role and the mechanism of interferon antagonism in this large virus family. In this work the first structure for a bunyavirus interferon antagonist, the core domain crystal structure of NSs from the Rift Valley fever virus (RVFV) is presented. RVFV is one of the most clinically significant members of the Bunyaviridae family, causing recurrent epidemics in Africa and Arabia, often featuring high-mortality haemorrhagic fevers. The structure shows a novel all-helical fold. The unique molecular packing of NSs in the crystal creates stable fibrillar networks, which could correspond to the characteristic fibrillation of NSs observed in vivo in the nuclei of RVFV infected cells. This first NSs structure might be a useful template for future structure-aided design of drugs that target the RVFV interferon antagonism. Attempts at characterising other bunyavirus NSs proteins of other genera were made, but were hampered by problems with obtaining sufficient amounts of soluble and folded protein. The approaches that proved unsuccessful for the solubilisation of these NSs proteins, however, should inform future experiments aimed at obtaining recombinant NSs for structural studies.
12
Van, Niekerk Stephanie. "Association of respiratory syncytial virus glyco- and non-structural proteins with disease severity in infected children". Diss., University of Pretoria, 2012. http://hdl.handle.net/2263/31128.
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Respiratory Syncytial Virus (RSV) is a major cause of bronchiolitis and pneumonia in infants, immunocompromised and the elderly in both developed and developing countries. Re-infections are common and G protein variability is one mechanism to overcome herd immunity. This is illustrated by the appearance of the BA genotype with a 60 nucleotide duplication dominating the subtype B genotypes in epidemics worldwide. To investigate the evolution of subtype A and B in South Africa (SA) since 2002 the genetic variability in the G protein was analysed in all recent strains isolated over four years (2006-2009) in SA hospitals. Bayesian analysis revealed a replacement of all subtype B genotypes previously identified in SA with the BA genotype since 2006, while subtype A genotypes identified in previous years are still circulating. The evolutionary rate of the SA BA genotype was shown to be 2.305 x 10-3 nucleotide substitutions/site/year while subtype A was shown to have 3.382 x 10-3 substitutions/site/year and drift was evident. The most recent common ancestor (MRCA) of the SA BA viruses was determined to date back to 1997. All SA BA isolates clustered with the BA-IV sub-genotype and the appearance of new sub-genotypes within this branch may occur if drift continues. Sequencing of the complete G protein of selected BA SA strains revealed an additional 6 nucleotide deletion. When comparing G protein variability with disease severity, the dominating subtype/genotype in each season was also the subtype/genotype that was associated with increased hospitalizations. No direct association between G protein variability and disease severity was seen. Subtype/genotype switching was evident over the four years most probably because of herd immunity. G protein variability may play a role in RSV‟s ability to re-establish annual epidemics by allowing immune evasion. Certain substitutions or alterations may enhance the fitness of viruses as is evident with the BA strains that replaced all other B genotypes previously identified in SA. The G protein‟s ability to accommodate such substantial changes and facilitate immune evasion may complicate vaccine development. It remains to be seen if this BA genotype will remain dominant or if the dominance will eventually fade because of herd immunity.
Subtyping of RSV strains over the four years identified G protein PCR amplicons significantly reduced in size in 2 out of 209 clinical specimens. Sequence analysis revealed subtype B strains lacking nearly the entire G protein ectodomain in one HIV positive and one HIV exposed child hospitalized with pneumonia. G protein deletion mutants replicate effectively in vitro but have not been detected in nature. This study suggests that RSV clinical strains that lack most of the G protein gene may occur in immunocompromised patients with lower respiratory tract infection (LRTI). The molecular mechanism whereby this occurs is not clear, however reduced immune pressure in these patients may allow these strains to utilise the F protein for binding and replication. Further characterization of such strains may elucidate the replication and pathogenic potential, however low viral load and long term storage of specimens complicated the isolation of such strains.
Acquisition of the 60 nucleotide duplication appeared to have improved the fitness of the BA viruses and more recent subtype B strains may need to be included in experimental vaccines to evaluate their efficacy in the current setting of evolved circulating strains. In addition, the association of clinical strains lacking most of the G protein with LRTI may have implications for the utilisation of certain attenuated strains in immunocompromised children.
RSV is unique among paramyxoviruses in having two non-structural (NS) proteins that play a major role in inhibiting the host‟s immune response. Sequence and quantification analysis of these proteins were performed to determine its role in disease severity. We were unable to attribute specific protein polymorphisms with differences in disease severity but identified genome heterogeneity (quasispecies) within patients which may have an influence on the NS protein function, and also have an influence on the innate immune response and thus an effect on disease severity. When comparing patients with mild and severe disease, higher expression levels of NS1 were seen in the hospitalized group compared to the out-patient group, supporting our hypothesis that increased levels of NS may lead to enhanced suppression of the interferon pathway and in effect result in more severe disease. However, the opposite was found for NS2 with higher expression levels in the mild group. Genetic variability within the gene-end and gene-start sequences were not seen thus could not explain the differences in expression levels observed, although variability within the promoter area may need to be investigated.Dissertation (MSc)--University of Pretoria, 2012.
Medical Virology
MSc
Unrestricted
13
Langerwisch, Ulrike [Verfasser]. "Characterization of the non-structural proteins 4B and 5A of the bovine viral diarrhea virus / Ulrike Langerwisch". Lübeck : Zentrale Hochschulbibliothek Lübeck, 2016. http://d-nb.info/1096210754/34.
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14
Aydin, Cihan. "Hepatitis C Virus Non-Structural Protein 3/4A: A Tale of Two Domains: A Dissertation". eScholarship@UMMS, 2012. https://escholarship.umassmed.edu/gsbs_diss/626.
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Two decades after the discovery of the Hepatitis C Virus (HCV), Hepatitis C infection still persists to be a global health problem. With the recent approval of the first set of directly acting antivirals (DAAs), the rate of sustained viral response for HCV-infected patients increased significantly. However, a complete cure has not been found yet. Drug development efforts primarily target NS3/4A protease, bifunctional serine protease-RNA helicase of HCV. HCV NS3/4A is critical in viral function; protease domain processes the viral polyprotein and helicase domain aids replication of HCV genome by unwinding double stranded RNA transcripts produced by NS5B, RNA-dependent RNA polymerase of HCV. Protease and helicase domains can be isolated, expressed and purified separately while retaining function. Isolated domains of HCV NS3/4A have been extensively used in biochemical and biophysical studies for scientific and therapeutic purposes to evaluate functional capability and mechanism. However, these domains are highly interdependent and modulate the activities of each other bidirectionally. Interdomain dependence was demonstrated in comparative studies where activities of isolated domains versus the full length protein were evaluated. Nevertheless, specific factors affecting interdependence have not been thoroughly studied.
Chapter II investigates the domain-domain interface formed between protease and helicase domains as a determinant in interdependence. Molecular dynamics simulations performed on single chain NS3/4A constructs demonstrated the importance of interface in the coupled dynamics of the two domains. The role of the interface in interdomain communication was experimentally probed by disrupting the domain-domain interface through Ala-scanning mutations in selected residues in the interface with significant buried surface areas. These interface mutants were assayed for both helicase and protease related activities. Instead of downregulating the activities of either domain, interface mutants caused enhancement of protease and helicase activities. In addition, the interface had minimal effect in RNA unwinding activity of the helicase domain, the mere presence of the protease domain was the main protagonist in elevated RNA unwinding activity. In conclusion, I suspect that the interface formed between the domains is transient in nature and plays a regulatory role more than a functional role. In addition, I found results supporting the suggestion that an alternate domain-domain arrangement other than what is observed in crystal structures is the active, biologically relevant conformation for both the helicase and the protease.
Chapter III investigates structural features of HCV NS3/4A protease inhibitors in relation to effects on inhibitor potency, susceptibility to drug resistance and modulation of potency by the helicase domain. Nearly all NS3/4A protease inhibitors share common features, with major differences only in bulky P2 extension groups and macrocyclization statuses. Enzymatic inhibition profiles of different drugs were analyzed for wildtype isolated protease domain and single chain NS3/4A helicase-protease construct, their multi drug resistant variants, and additional helicase mutants. Inhibitor potency was mainly influenced by macrocyclization, where macrocyclic drugs were significantly more potent compared to acyclic variants. Potency loss with respect to resistance mutations primarily depended on the P2 extension, while macrocyclization had minimal effect except for P2-P4 macrocyclic compounds which were up to an order of magnitude more susceptible to mutations A156T and, in lesser extent, D168A. Modulation by helicase domain was also dependent on P2 extension, although opposite trends were observed for danoprevir analogs versus others. In conclusion, this study provides a basis for future inhibitor development in both avoiding drug resistance and exploitation of the helicase domain for additional efficacy.
In this thesis, I have provided evidence further supporting and revealing the details of domain-domain dependency in HCV NS3/4A. Lessons learned here will aid future research for dissecting the interdependency to gain a better understanding of HCV NS3/4A function, which can possibly be extended to all Flaviviridae NS3 protease-helicase complexes. In addition, interdomain dependence can be exploited in future drug development efforts to create better drugs that will pave the way to an effective cure.
15
Lindström, Hannah Kim. "Molecular studies of the hepatitis C virus : the role of IRES activity for therapy response, and the impact of the non-structural protein NS4B on the viral proliferation /". Stockholm, 2006. http://diss.kib.ki.se/2006/91-7140-875-4/.
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Miller, Cathy Lea. "Investigation of the role of minute virus of mice (MVM) small non-structural protein NS2 interactions with host cell proteins during MVM infection". free to MU campus, to others for purchase, 2001. http://wwwlib.umi.com/cr/mo/fullcit?p3025638.
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Abdullah, Syed Umer. "Structural determinants of stability to proteolysis, processing and impact on allergenic potential of non-specific lipid transfer proteins". Thesis, University of East Anglia, 2012. https://ueaeprints.uea.ac.uk/41974/.
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Lipid transfer proteins (LTPs) are a class of low molecular weight hydrophobic conserved proteins comprising four intramolecular disulphide bonds making the structure very resistant to proteolysis and harsh food processing conditions. These proteins are identified as strong allergens sensitizing through the gut and share epitopes with LTPs from closely related species. Peach LTP, Pru p 3 is the primary sensitizer in the Mediterranean area being the most frequent food allergen. Wheat LTP, Tri a 14 is a relatively weak allergen with a very low prevalence. The study here compares the structural properties of these proteins and their resistance to various digestive and processing processes. Ligand binding experiments showed that Pru p 3 binds to ligands more strongly than Tri a 14. The gastroduodenal digestion of these LTPs revealed that both are stable to gastric digestion and while Pru p 3 is susceptible to duodenal digestion, Tri a 14 digestion is negligible. Ligand binding did not affect the digestibility of Pru p 3 but improved the duodenal digestibility of Tri a 14. The IgE binding studies using sera from peach allergic individuals confirmed that both Pru p 3 and its digestion fragments in the presence and absence of ligand were IgE reactive. Model processing conditions were employed to treat these LTPs. It was found that heat treatment destroys the secondary structure of Pru p 3 at 121°C and slightly affects that of Tri a 14. Heat treatment also increased the susceptibility of Pru p 3 to gastric digestion while Tri a 14 was less affected. The IgE binding studies showed that heat treatment of Pru p 3 appeared to reduce its IgE recognition while its digestion fragments lost all of their IgE reactivity. To investigate the effect of the food matrix on the digestibility of these LTPs, peach peel containing Pru p 3 and wheat flour containing Tri a 14 were digested under simulated conditions. It was found that they were resistant to proteolysis in their native matrices. Effect of heat treatment to the food matrix again confirmed that both of these proteins were more stable to heat in the matrix and were less digestible. In conclusion, this study shows that there are factors in food matrices which enhance structural stability of LTPs to both processing and digestion. Thus factors such as the effect of food matrix and effect of processing should be taken into account in assessing the allergenic risk posed by foods and not simply rely on data from purified proteins.
18
tw, spchen@mail atit org, i Shih-Ping Chen. "Epidemiology, pathogenesis and surveillance of the pig adapted strain of foot and mouth disease in Taiwan". Murdoch University, 2008. http://wwwlib.murdoch.edu.au/adt/browse/view/adt-MU20080813.104029.
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Foot-and-mouth disease (FMD) is one of the most contagious infectious diseases of domestic and wild cloven-hoofed animals, particular in cattle, sheep, pigs, goats and domestic buffalo, as well as wild ruminants such as deer. In Taiwan, there was a severe outbreak of FMD after more than 60 years freedom from the disease. The virus strain, O Taiwan 97 from the March 1997 outbreak of FMD in Taiwan, however, has been shown to have a species-specific adaptation to pigs. Although there are 7 distinct serotypes of FMD found in different regions of the world, this study focuses on the pig-adapted type O strain of FMD.
After the FMD outbreak commenced in Taiwan, the spread of disease was very rapid and the whole of the western parts of Taiwan was affected within a few days after the diagnosis of FMD was confirmed. In some situations airborne transmission of FMD virus was suspected and it was speculated that this was the explanation for such rapid spread in Taiwan. Therefore, studies were conducted to investigate the transmissibility of O Taiwan/97 FMDV to susceptible pigs by direct and indirect spread including airborne spread in an enclosed animal house. This study showed that pigs in direct contact with challenged pigs became infected but none of the close-contact pigs became infected. These experiments clearly demonstrated that the pig adapted strain O Taiwan/97 was only efficiently transmitted by direct contact. This indicates that effective control against future outbreaks of pig adapted FMDV strains could be achieved by restriction of pig movement and stamping out if the outbreak has been detected in the early stages and prior to the movements of pigs from the infected premises.
The measures used to control the Taiwanese FMD outbreak in 1997 were initially the slaughter of whole herds in the infected premises. However, with the rapid spread and large numbers of cases, the decision was taken to use universal compulsory vaccination of pig herds to control the outbreak when sufficient supply of vaccines was organized. Type O FMD vaccines were imported from a number of major FMD vaccine manufactures from around the world. Initially, vaccine efficacy for the imported vaccines was tested by measurement of neutralizing antibody titers in vaccinated pigs. To establish the relationship between serum neutralizing titers and protection from foot and mouth disease in pigs after vaccination, challenge studies were conducted with O/Taiwan/97 FMD in vaccinated pigs. Additionally, antibody responses to structural (neutralizing antibody) and non-structural proteins (NSP) were evaluated in vaccinated pig herds after primary and secondary vaccination in herds infected before and after vaccination.
In order to be able to monitor the circulation of virus in vaccinated pig populations, valid diagnostic kits based on the detection of antibody against NSP were required. These tests needed to be evaluated against pig sera derived from challenge studies and natural FMD outbreaks. Three commercially available ELISAs (Cedi, UBI and Checkit), which were available to differentiate infected from vaccinated pigs, were tested and results showed that the Cedi test had the optimal sensitivity and specificity for pig adapted type O FMD testing. This test was used to retrospectively evaluate the sera collected from infected and non-infected pig herds collected sequentially in the year after the 1997 FMD outbreak in Taiwan. These studies also showed that the early vaccines used, stimulated NSP antibody production in swine herds that were vaccinated but not infected. This resulted in the requirement for purified FMD vaccines to be used when monitoring programs for FMD infection by NSP testing were in place. In these studies, it was also demonstrated that the purified FMD vaccines used later in the control program did not induce NSP antibody after multiple double dosage to pigs.
Although clinical FMD appeared to be successfully controlled with vaccination program in Taiwan it was essential for the eradication plan to maintain active surveillance for NSP reactors in the pig population. The UBI and Cedi NSP kits were applied as screening and confirmatory tests, respectively, to pig sera collected in auction markets distributed around Taiwan to monitor for evidence of the circulation of FMD virus. Herds with positive reactors were followed-up by clinical inspection and 15 sera from suspected herds were further sampled. Negative results were obtained from all these investigation. With the absence of clinical outbreaks and the lack of evidence of FMDV circulation in the field from the NSP reactor surveillance, the Taiwanese government has progressed the eradication plan to a progressive cessation of vaccination, commencing with banning of vaccination on one isolated island in December 2006. The absence of outbreaks on that island, paved the way for further cessation of FMD vaccination in Taiwan from July 2008.
19
Gamlen, Toby Philip Edward. "Non-structural proteins derived from non-cytopathic or cytopathic biotypes of bovine viral diarrhoea virus have distinct effects on cell survival and antiviral signalling pathways". Thesis, University of Leeds, 2006. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.435778.
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Gosal, Walraj Singh. "Morphological, structural and mechanical properties of self-assembled fibrillar aggregates, networks and gels derived from non-disease associated globular proteins". Thesis, King's College London (University of London), 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.400636.
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To, Thuan. "PDZ Binding Motif of NS1 Proteins of Influenza A Viruses: : A Virulent Factor in the Expression of Interferon-β?" Thesis, Uppsala universitet, Institutionen för medicinsk biokemi och mikrobiologi, 2012. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-177337.
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Background: The PDZ domain is a peptide sequence of 80-90 amino acids and can be found in e.g. bacteria, animals and plants. These domains are commonly part of the cytoplasmic and membrane adapter proteins and its function are important in protein-protein interactions. The NS1 proteins of influenza A viruses play an important role in inhibiting the IFN-β production in many ways. In the C-terminus of the NS1 protein, a peptide sequence of four amino acids had been demonstrated to bind to the PDZ domain termed as PDZ binding motif (PBM). Objective: The aim of this study is to determine whether the PBM sequence of the NS1 protein of influenza A virus plays a key roll in the expression of interferon-β. Methods: The open reading frame of the NS1 protein was amplified and cloned into expressing vector and transfected into A549 cells along with a reporter plasmid containing ISRE promoter, driving expression of firefly luciferase. Dual luciferase reporter assay was performed to measure luciferase activity which represented expression of IFN-β. The assay was performed only once and unfortunately the result can not be trusted since the negative control showed positive value. Therefore, to understand the interaction between the PBM sequence of NS1 proteins and the production of IFN-β, further experiments are needed.
22
Capelli, R. "COMPUTATIONAL MODELING OF PROTEINS: FROM STATISTICAL MECHANICS TO IMMUNOLOGY". Doctoral thesis, Università degli Studi di Milano, 2017. http://hdl.handle.net/2434/527950.
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One of the biggest revolutions occurred during the second half of the 20th century in physics was the introduction of computers in research. In particular, the use of fast computing machines opened the possibility to study complex systems by simulating their dynamics, without the need to pursue analytical solutions, otherwise impossible to tackle. The consequences of this breakthrough were huge both in the study of equilibrium and non-equilibrium many-body problems, with the strong limitation given by the number of atoms involved in the calculation.
The first technique used in biology-related problems was the Monte Carlo Method, and some years later Molecular Dynamics (MD) was formalized. In MD, for each atom of the system one can solve its Newton equations of motion, obtaining a trajectory in the phase space for the entire system, and study its behavior in equilibrium and non-equilibrium conditions. The constant rise in computational power gave the possibility to scientists to study larger and larger systems, while the advances in experimental techniques enhanced the possibility for direct comparisons between wet and in silico data at similar levels of resolution. Despite the validity of Moore’s Law (i.e., the exponential growth of the computing power due to transistors miniaturization) until now, the timescale of the events that can be simulated has an upper limit of the millisecond with tailor-made computers, which is not enough to study all the biologically-relevant phenomena. Since the birth of computational chemistry, a huge number of different statistical mechanics-based methods has been implemented to permit, given the computing power limit, an effective reliable use of MD simulations in biochemistry.
One of the most relevant problems tackled by MD is the calculation of free energy differences, both in conformational changes and in sequence mutations of a protein. The main reason of this difficulty is represented by the frustrated nature of interactions in proteins and the size of these systems: this leads to a complex energy landscape which in principle needs very long sampling times to overcome all possible energy barriers. In the present thesis, we studied and improved a path-independent and system-independent free energy calculation technique, called Simplified Confinement Method. We describe this work in Chapter 1.
Although MD has been successful in most of its applications, there are still many open problems: as mentioned before, the available parametrizations of interaction potentials (called force fields) are not completely reliable. In particular, the choice of force field parameters is performed comparing experimental data on a fixed set of (usually small) molecules with computed data on the same molecules. This raises a significant problem: large molecules can have a more complex behavior, and using these potentials can lead to a systematic error; furthermore, the timescale in which the force field is tested needs to be limited. Another strong limitation of MD depends on the equilibrium experiments used for parametrization: the kinetic properties of a system are not considered. Given the impossibility to reparametrize a general force field with non-equilibrium experimental data, we implemented a technique that uses equilibrium-based force fields, adding a potential term based on time series resulting from kinetic experiments. This approach, based on the principle of Maximum Caliber, restrains the system with an experimental-based bias, returning a more realistic behavior of the simulation in condition where the usual force fields show their limitations. We describe this work in Chapter 2.
The application of computational methods in the study of proteins confirms its efficacy in other fields of life sciences: an actual and emerging topic is represented by vaccinology. With techniques developed by Louis Pasteur at the end of the 19 th century (isolation of the pathogen, its inactivation and subsequent inoculation in the host), various scientists developed vaccines for deadly diseases like poliomyelitis, diphterite and measles. None of the mentioned was developed with molecular biology-based approaches. Almost 50 years after the birth of molecular biology, the Human Genome Project decoded human DNA and, at the same time, the genome of the most dangerous pathogen was screened. This has laid the foundation of Reverse Vaccinology (RV), where the proteins responsible for immune reaction can be identified from the pathogen DNA and tested directly on animal models, obtaining a new vaccine candidate with little or no risk for the host, having removed the pathogen itself. At the beginning of the 21st century the first vaccine against Meningococcus B, responsible for the 50% of the meningococcal meningitis, was developed using this protocol. Since then, crystallographic data was inserted in RV workflow to exploit conformational data, creating the so-called Structural Vaccinology (SV). To enhance its efficacy, SV exploits all the aspects of molecular modeling like computer-aided drug/protein design and MD to integrate information that come from experimental sources. One of the most promising technique in this field is the grafting of an immunogenic sequence (i.e., a portion of a protein recognized by the immune system) on a foreign protein; this approach could lead to a new vaccine component which have no risk for the patient. To date, the grafting technique has been carried out by human-driven workflows. Motivated by this reason, we studied immunogenic peptides from a family of pathogens involved in respiratory diseases, exploiting Structural Vaccinology principles with both computational and experimental approach. Furthermore, we developed and implemented an unsupervisionated automated tool to design grafted protein sequences. We describe this work in Chapter 3.
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Schult, Philipp [Verfasser], i Volker [Akademischer Betreuer] Lohmann. "Functional Dissection of the Hepatitis C Virus Non-Structural Proteins and miR-122 in Viral Replication and Translation / Philipp Schult ; Betreuer: Volker Lohmann". Heidelberg : Universitätsbibliothek Heidelberg, 2017. http://d-nb.info/1180735722/34.
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Flügel, Veronika M. Verfasser], Sabine [Akademischer Betreuer] Schneider i Aymelt [Akademischer Betreuer] [Itzen. "Expressed Cyclopeptide Libraries as Selective Inhibitors for RNA-Protein Interactions. Structural Basis for the Site-Specific Incorporation of Non-Natural Amino Acids into Peptides and Proteins / Veronika M. Flügel. Gutachter: Aymelt Itzen ; Sabine Schneider. Betreuer: Sabine Schneider". München : Universitätsbibliothek der TU München, 2014. http://d-nb.info/1064075541/34.
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Manning, Benjamin J. "ATP-Dependent Heterochromatin Remodeling: A Dissertation". eScholarship@UMMS, 2015. https://escholarship.umassmed.edu/gsbs_diss/795.
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Eukaryotic DNA is incorporated into the nucleoprotein structure of chromatin. This structure is essential for the proper storage, maintenance, regulation, and function of the genomes’ constituent genes and genomic sequences. Importantly, cells generate discrete types of chromatin that impart distinct properties on genomic loci; euchromatin is an open and active compartment of the genome, and heterochromatin is a restricted and inactive compartment. Heterochromatin serves many purposes in vivo, from heritably silencing key gene loci during embryonic development, to preventing aberrant DNA repeat recombination. Despite this generally repressive role, the DNA contained within heterochromatin must still be repaired and replicated, creating a need for regulated dynamic access into silent heterochromatin. In this work, we discover and characterize activities that the ATP-dependent chromatin remodeling enzyme SWI/SNF uses to disrupt repressive heterochromatin structure.
First, we find two specific physical interactions between the SWI/SNF core subunit Swi2p and the heterochromatin structural protein Sir3p. We find that disrupting these physical interactions results in a SWI/SNF complex that can hydrolyze ATP and slide nucleosomes like normal, but is defective in its ability to evict Sir3p off of heterochromatin. In vivo, we find that this Sir3p eviction activity is required for proper DNA replication, and for establishment of silent chromatin, but not for SWI/SNF’s traditional roles in transcription. These data establish new roles for ATP-dependent chromatin remodeling in regulating heterochromatin.
Second, we discover that SWI/SNF can disrupt heterochromatin structures that contain all three Sir proteins: Sir2p, Sir3p and Sir4p. This new disruption activity requires nucleosomal contacts that are essential for silent chromatin formation in vivo. We find that SWI/SNF evicts all three heterochromatin proteins off of chromatin. Surprisingly, we also find that the presence of Sir2p and Sir4p on chromatin stimulates SWI/SNF to evict histone proteins H2A and H2B from nucleosomes. Apart from discovering a new potential mechanism of heterochromatin dynamics, these data also establish a new paradigm of chromatin remodeling enzyme regulation by nonhistone proteins present on the substrate.
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Manning, Benjamin J. "ATP-Dependent Heterochromatin Remodeling: A Dissertation". eScholarship@UMMS, 2009. http://escholarship.umassmed.edu/gsbs_diss/795.
Pełny tekst źródłaStreszczenie:
Eukaryotic DNA is incorporated into the nucleoprotein structure of chromatin. This structure is essential for the proper storage, maintenance, regulation, and function of the genomes’ constituent genes and genomic sequences. Importantly, cells generate discrete types of chromatin that impart distinct properties on genomic loci; euchromatin is an open and active compartment of the genome, and heterochromatin is a restricted and inactive compartment. Heterochromatin serves many purposes in vivo, from heritably silencing key gene loci during embryonic development, to preventing aberrant DNA repeat recombination. Despite this generally repressive role, the DNA contained within heterochromatin must still be repaired and replicated, creating a need for regulated dynamic access into silent heterochromatin. In this work, we discover and characterize activities that the ATP-dependent chromatin remodeling enzyme SWI/SNF uses to disrupt repressive heterochromatin structure.
First, we find two specific physical interactions between the SWI/SNF core subunit Swi2p and the heterochromatin structural protein Sir3p. We find that disrupting these physical interactions results in a SWI/SNF complex that can hydrolyze ATP and slide nucleosomes like normal, but is defective in its ability to evict Sir3p off of heterochromatin. In vivo, we find that this Sir3p eviction activity is required for proper DNA replication, and for establishment of silent chromatin, but not for SWI/SNF’s traditional roles in transcription. These data establish new roles for ATP-dependent chromatin remodeling in regulating heterochromatin.
Second, we discover that SWI/SNF can disrupt heterochromatin structures that contain all three Sir proteins: Sir2p, Sir3p and Sir4p. This new disruption activity requires nucleosomal contacts that are essential for silent chromatin formation in vivo. We find that SWI/SNF evicts all three heterochromatin proteins off of chromatin. Surprisingly, we also find that the presence of Sir2p and Sir4p on chromatin stimulates SWI/SNF to evict histone proteins H2A and H2B from nucleosomes. Apart from discovering a new potential mechanism of heterochromatin dynamics, these data also establish a new paradigm of chromatin remodeling enzyme regulation by nonhistone proteins present on the substrate.
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Murray, Lindsay. "Localisation of Theiler's Murine Encephalomyelitis virus non-structural proteins 2B, 2C, 2BC and 3A in BHK-21 cells, and the effect of amino acid substitutions in 2C on localisation and virus replication". Thesis, Rhodes University, 2007. http://hdl.handle.net/10962/d1007722.
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The picornavirus family includes significant human and animal viruses such as poliovirus (PV), human rhinovirus (HRV) and foot-and-mouth-disease virus (FMDV). Current disease treatment and control strategies are limited by an incomplete understanding of the interactions between the non-structural, replicative picornavirus proteins and host cell components. To investigate these interactions, Theiler's murine encephalomyelitis virus (TMEV) 2B, 2C, 2BC and 3A proteins were transiently expressed in BHK-21 cells and detected by indirect immunostaining and laser-scanning or epifluorescence microscopy. The signal of the 2B protein overlapped with that of the ER marker protein, ERp60, as well as that of the peripheral Golgi marker protein, β-COP. The 2C protein overlapped with ERp60 in a faint reticular stain, and localised to large punctate structures that partially overlapped with β-COP at higher levels of expression. The 2BC protein located to large perinuclear structures that overlapped exclusively with β-COP. The TMEV 3A protein signal overlapped with both ERp60 and β-COP stains, in addition in cells expressing the 3A protein the ER appeared swollen and bulbous while the Golgi was dispersed in some cells. 2C and 2BC proteins with C-terminal deletions localised in the same manner as the wild type proteins indicating that the localisation signals that determine subcellular localisation of the proteins are within the N-terminal 60 amino acids of the 2C protein. The significance of the high degree of conservation of the N-terminal domain of the 2C protein throughout the Picornaviridae was investigated through the introduction of amino acid substitution mutations at highly conserved residues in the N-terminal domain of 2C into the viral cDNA. Upon transfection of the viral RNA into BHK-21 cells, it was observed that substitution of amino acid residues 8, 18 and 29 abolished the ability of TMEV to induce cytopathic effect (CPE), while substitution of residues 4, 14 and 23 only attenuated the ability of TMEV to induce CPE. To determine whether amino acid substitution mutations would affect the localisation of the 2C protein, 2C proteins with substitution mutations at amino acids 4, 8, 14, 18, 23 and 29 were transiently expressed in BHK-21 cells and detected by indirect imrnunostaining and examination by laser-scanning confocal and epifluorescence microscopy. The 2C mutant 4, 8 and 29 proteins showed slightly altered localisation patterns compared to the wild type protein with a significant portion of the proteins localising in a perinuclear stain suggesting possible localisation to the nuclear envelop. The 2C mutant 14 and 18 proteins localised to a diffuse pattern in BHK-21 cells while the 2C mutant 23 protein located to small punctate structures that partially overlapped with the ERp60 stain but were completely separate from the β-COP stain. Finally, a hydrophilic, antigenic region of the 2C protein was expressed in frame with an N-terminal GST tag and was successfully purified on a pilot-scale and detected by Western analysis. This 2C178 peptide will be used to generate antibodies against the 2C and 2BC proteins for use in future studies. This study has furthered our knowledge of the localisation of the picornavirus 2B, 2C, 2BC and 3A proteins in host cells and identified a possible link between this localisation and an ability of TMEV to replicate in BHK-21 cells.
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Schwardt, Malte. "BDV X protein is a non-structural protein". [S.l. : s.n.], 2006. http://nbn-resolving.de/urn:nbn:de:bsz:25-opus-60677.
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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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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.
31
Nethisinghe, Suran. "Further characterisation of bluetongue virus non-structural protein 2". Thesis, London School of Hygiene and Tropical Medicine (University of London), 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.497276.
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Ward, Rebecca. "Bluetongue virus non-structural protein 1 : virus-host interactions". Thesis, London School of Hygiene and Tropical Medicine (University of London), 2006. http://researchonline.lshtm.ac.uk/4646527/.
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Bluetongue virus (BTY) is an orbivirus of the Reoviridae family that infects sheep and other ruminants. BTY has three non-structural proteins, NS I, NS2 and NS3/3A. NS I forms tubular structures and its function is currently unknown. To investigate the role of NS I in BTY infection, the interactions of NS I with mammalian and insect cellular proteins, and BTY viral proteins, were examined. BTY NS I was identi tied as interacting with aldolase A, NUBP 1, Pyruvate kinase M2, cathespin B, SUM 0-1 and peptide TY7 using the yeast two-hybrid system, ELISA and immunofluorescence analysis. TY7 and NS I caused extensive cell death within 24h of co-expression; this cell death was not apoptosis and reduced BTY yield by 37%. The interaction of NS I with SUMO-I and its importance in BTY infection was confinned using siRNA to knockdown SUMO-I during BTY-IO infection. Knockdown of SUMO-I elicited a dramatic reduction in virus yield by 73%. NS I interactions with proteins of the insect vector Culicoides were also examined. A putative interaction between NS 1 and the ubiquitin activating enzyme El (UBA EI) ofCulicoides was identified during screening of a phage library, this has not been confirmed by other means. NS 1 interactions with other BTY proteins were analysed using immunoprecipitation and a strong interaction between NS 1 and YP7 was identified; this was confim1ed using the yeast two-hybrid system and immunoflourescence. Two main roles have been hypothesised for NS I from this data; firstly it is likely that NS I interaction with SUMO-I and UBA E I allows the targeting of specific proteins for sumoylation and ubiquitination allowing NS 1 to modify the host response to BTY infection. Secondly it is possible that NS I serves as an anchor for YP7 and virus cores allowing the build up of cores at the cytoskeleton in close proximity to YP2 for subsequent assembly and release. RNAi against NS J eliminated tubule formation but did not affect virus yield or YP7 and SUMO-J distribution and expression. It is therefore likely that the function of NS I does not rely on tubule fom1ation and that tubules are a form of storage for the active monomer of NSI.
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Bhatt, Veer Sandeep. "Non-lectin type Protein-carbohydrate Interactions: A Structural Perspective". The Ohio State University, 2011. http://rave.ohiolink.edu/etdc/view?acc_num=osu1306858684.
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Butan, Carmen Crina. "Structural characterization of the RNA binding domain of BTV non-structural protein 2". Thesis, Birkbeck (University of London), 2004. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.414198.
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Bennett, Gwendolyn M. "Chromatin Regulators and DNA Repair: A Dissertation". eScholarship@UMMS, 2014. https://escholarship.umassmed.edu/gsbs_diss/742.
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DNA double-strand break (DSB) repair is essential for maintenance of genome stability. However, the compaction of the eukaryotic genome into chromatin creates an inherent barrier to any DNA-mediated event, such as during DNA repair. This demands that there be mechanisms to modify the chromatin structure and thus access DNA. Recent work has implicated a host of chromatin regulators in the DNA damage response and several functional roles have been defined. Yet the mechanisms that control their recruitment to DNA lesions, and their relationship with concurrent histone modifications, remain unclear. We find that efficient DSB recruitment of many yeast chromatin regulators is cell-cycle dependent. Furthering this, we find recruitment of the INO80, SWR-C, NuA4, SWI/SNF, and RSC enzymes is inhibited by the non-homologous end joining machinery, and that their recruitment is controlled by early steps of homologous recombination. Strikingly, we find no significant role for H2A.X phosphorylation (γH2AX) in the recruitment of chromatin regulators, but rather that their recruitment coincides with reduced levels of γH2AX. We go on to determine the chromatin remodeling enzyme Fun30 functions in histone dynamics surround a DSB, but does not significantly affect γH2AX dynamics. Additionally, we describe a conserved functional interaction among the chromatin remodeling enzyme, SWI/SNF, the NuA4 and Gcn5 histone acetyltransferases, and phosphorylation of histone H2A.X. Specifically, we find that the NuA4 and Gcn5 enzymes are both required for the robust recruitment of SWI/SNF to a DSB, which in turn promotes the phosphorylation of H2A.X.
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Bennett, Gwendolyn M. "Chromatin Regulators and DNA Repair: A Dissertation". eScholarship@UMMS, 2012. http://escholarship.umassmed.edu/gsbs_diss/742.
Pełny tekst źródłaStreszczenie:
DNA double-strand break (DSB) repair is essential for maintenance of genome stability. However, the compaction of the eukaryotic genome into chromatin creates an inherent barrier to any DNA-mediated event, such as during DNA repair. This demands that there be mechanisms to modify the chromatin structure and thus access DNA. Recent work has implicated a host of chromatin regulators in the DNA damage response and several functional roles have been defined. Yet the mechanisms that control their recruitment to DNA lesions, and their relationship with concurrent histone modifications, remain unclear. We find that efficient DSB recruitment of many yeast chromatin regulators is cell-cycle dependent. Furthering this, we find recruitment of the INO80, SWR-C, NuA4, SWI/SNF, and RSC enzymes is inhibited by the non-homologous end joining machinery, and that their recruitment is controlled by early steps of homologous recombination. Strikingly, we find no significant role for H2A.X phosphorylation (γH2AX) in the recruitment of chromatin regulators, but rather that their recruitment coincides with reduced levels of γH2AX. We go on to determine the chromatin remodeling enzyme Fun30 functions in histone dynamics surround a DSB, but does not significantly affect γH2AX dynamics. Additionally, we describe a conserved functional interaction among the chromatin remodeling enzyme, SWI/SNF, the NuA4 and Gcn5 histone acetyltransferases, and phosphorylation of histone H2A.X. Specifically, we find that the NuA4 and Gcn5 enzymes are both required for the robust recruitment of SWI/SNF to a DSB, which in turn promotes the phosphorylation of H2A.X.
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Lymperopoulos, Konstantinos. "Functional characterisation of the bluetongue virus non-structural protein NS2-protein and RNA-protein interactions". Thesis, University of Oxford, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.424733.
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Jacobs, Michael Graham. "Membrane association of dengue 2 virus non-structural protein 1". Thesis, Imperial College London, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.325917.
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Chauché, Caroline Marie. "Molecular evolution of equine influenza virus non-structural protein 1". Thesis, University of Glasgow, 2018. http://theses.gla.ac.uk/8877/.
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Influenza A viruses (IAVs) are common infections of certain avian reservoir species, and they periodically transfer to mammalian hosts. These cross-species jumps are usually associated with sporadic outbreaks, and on rare occasions lead to the establishment of a lineage in the new host species. The immune pressure exerted by the new host on the emergent virus forces it to evolve and adopt strategies to evade immunity in order to survive in nature. Understanding the biological mechanisms that allow successful inter-species transmission and adaptation to mammals is crucial to develop the theoretical tools required to predict and/or control emergence of new viruses in humans and animals. H3N8 equine influenza virus (EIV) represents an interesting model to study the dynamic of within-host variation of an avian-origin IAV. Indeed, this virus has emerged from birds in 1963 and has circulated in horse populations for more than fifty years despite the availability of vaccines. Evidence of evolution of EIV virulence factor non-structural protein 1 (NS1) also exists. NS1 is the main viral antagonist of the host interferon (IFN) response, and it relies on different strategies for overcoming these responses, which varies depending on the viral strain. While some NS1 proteins effectively block the induction of IFN and IFN stimulated genes (ISGs), others block general gene expression at a post-transcriptional level, and therefore reduce the synthesis of IFN and ISGs indirectly. Importantly, little is known about the contribution of these NS1 functions to EIV infection phenotype and adaptation to horses. In this work, we characterised NS1 proteins spanning the entire EIV lineage and showed that NS1s from different time periods after EIV emergence counteract the IFN response using different and mutually exclusive mechanisms. While EIVs circulating in the early 1960s blocked general gene expression by a NS1-mediated blockade of the cleavage and polyadenylation specificity factor 30 (CPSF30), NS1s from contemporary EIVs specifically inhibit the induction of ISGs by interfering with the JAK/STAT pathway. These contrasting anti-IFN strategies are associated with two mutations that appeared sequentially during EIV evolution, E186K substitution and C-terminal truncation. These changes in NS1 allowed contemporary EIVs to replicate in the presence of high levels of IFN. The results shown here with EIV indicate that the interplay between virus evolution and immune evasion plays a key role in IAV mammalian adaptation.
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Poonsiri, T. "Structural study of the C-terminal domain of non-structural protein 1 and capsid protein from Japanese encephalitis virus". Thesis, University of Liverpool, 2018. http://livrepository.liverpool.ac.uk/3021941/.
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Japanese encephalitis virus (JEV) is a mosquito-transmitted Flavivirus that is closely related to other emerging viral pathogens including dengue (DENV), West Nile (WNV) and Zika viruses (ZIKV). JEV infection can result in meningitis and encephalitis, which in severe cases cause permanent brain damage and death. JEV occurs predominantly in rural areas throughout South East Asia, the Pacific islands, and the Far East, causing around 68,000 cases worldwide each year. There is no specific treatment for JEV. This study aims to determine the molecular structure of new potential drug targets for JEV. In this study, the JEV non-structural protein 1 C-terminal β-ladder domain (C-NS1) is presented at 2.1 Å resolution. The crystal structure of C-JEVNS1 shares a conserved fold with flavivirus C-NS1 domains. The surface charge distribution of C-JEVNS1 is similar to WNV and ZIKV but is significantly different from DENV. Analysis of the C-JEVNS1 structure, in silico molecular dynamics simulations and experimental solution small angle X-ray scattering, indicate extensive loop flexibility on the exterior of the protein. It is proposed that this together with charge distribution on the exterior of the protein influence NS1-host protein interaction specificity which may impact on pathogenicity. These factors may also affect the interaction with the monoclonal antibody, 22NS1, which is protective against WNV infection. Liposome and heparin binding assays indicate that only the N-terminal region of NS1 participates in the interaction with lipidic membranes and that sulphate binding sites are not the glycosaminoglycans binding interfaces. For the first time, the crystal structure of the JEV capsid protein at 1.98 Å is also reported and compared to the existing flavivirus capsid protein. JEV capsid shows helical secondary structure (α helixes 1-4) and protein folding similar to DENV and WNV capsid proteins. It forms a homodimer by antiparallel pairing with another subunit (‘), α helix 1-1’, 2-2’, and 4-4’. The capsid dimer is believed to be the building block of the nucleocapsid. The flexibility of the N-terminal α helix 1 of the capsid could be important for its function. This dimer model agrees with a previous suggestion that the capsid protein interacts with RNA via the basic rich C-terminal, α4-α4’, and associates with lipid bilayers at the opposite hydrophobic, α2-α2’.
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Yang, Weiming. "Functional studies of the group A rotavirus non-structural protein NSP4". Thesis, University of Warwick, 2010. http://wrap.warwick.ac.uk/35683/.
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NSP4, encoded by rotavirus genome segment 10 has been shown to be a transmembrane, endoplasmic reticulum (ER) specific N-linked glycoprotein. Consistent with its localization to the ER membrane, NSP4 was first shown to have a role in the morphogenesis of the infectious virion. The protein has also been reported to have cytotoxic activity when applied extracellularly to cells. Consequently it has been earmarked as an enterotoxin being secreted from virus-infected cells to cause early cellular pathology in the gut. The effect of expressing the NSP4 protein of group A rotaviruses in cells has been studied. It led to the rapid appearance of long cytoplasmic extrusions. Site-directed mutagenesis was used to block N-linked glycosylation at both of the known glycosylation sites near the amino terminus of NSP4. This revealed that the NSP4 induced formation of the cytoplasmic extrusions was dependent on the protein’s ability to become fully glycosylated. The cytoplasmic extrusions seen in cells expressing glycosylated NSP4 were also evident in virus-infected cells. Using real-time confocal microscopy a dynamic elongation of the cytoplasmic extrusions with a growth speed of 2 μm/min was observed in virus-infected cells. The cytoplasmic extrusions were found to contain β-tubulin and F-actin. Inhibiting their polymerization prevented the formation of the extrusions from virus-infected cells. Functional studies using Cell Tracker dyes showed that the cytoplasmic extrusions could disseminate vesicles from virus-infected cells onto the plasma membrane surface of uninfected cells. The vesicles were then found in the interior of the uninfected cells. Mono-specific antibody to NSP4 revealed the presence of the protein in the vesicles suggesting that the cytoplasmic extrusions facilitated the direct cell-cell spread of NSP4. The effect of NSP4 expression on the microtubular network of cells was analysed. It was found that NSP4 de-polymerized the microtubular network from the centre of cells and promoted the assembly of microtubules at the periphery of the cells in a glycosylation independent manner. Similar de-polymerization and re-assembly of the microtubules was observed in the virus-infected cells. Interestingly in the presence of nocodazole, tubular structures containing tubulin and viral proteins excluding NSP4 were found in virus-infected cells. A YFP-PCA assay was established to screen for cellular partners of NSP4. The functionality and the sensitivity of the assay were examined, but only two false positive colonies were isolated in the first screening. In conclusion, the function of glycosylated and unglycosylated NSP4 was examined with the former possessing the ability to promote the formation of the cytoplasmic extrusions from cells and both being capable of disrupting the microtubular network indicating that two forms of NSP4 play different roles in NSP4 function. The cytoplasmic extrusions seen in our studies may be relevant to rotavirus infection and pathogenesis.
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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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Sung, Po-yu. "Functional analysis of the non-enzymatic properties of the dengue virus non-structural protein 5". Thesis, University of Bristol, 2011. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.556732.
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The four serotypes of dengue virus (DENV 1-4) cause the most important arthropod-borne viral disease of humans. The DENV RNA genome is translated into a polyprotein that is cleaved into three structural and seven nonstructural proteins. Nonstructural protein 5 (NSS) has enzymatic activities required for capping and replication of the viral genome. NSS is phosphorylated and with some serotypes can accumulate in the nucleus of infected cells. However the role of NSS phosphorylation or nuclear trafficking in the virus lifecycle, is not well understood. Using a combination of proteomic and bioinformatic approaches, potential NSS phosphorylation sites were identified and analysed for their role in the virus lifecycle using a DENV reverse genetic system. Mutation of the NSS amino acid Thr-39S was found to decrease NSS nuclear localisation and viral replication in a cell specific manner. Mutations preventing and mimicking phosphorylation at Thr-39S had similar effects on NSS localisation and viral growth, showing that the effects were not caused by phosphorylation. Mutation of Thr-39S did not influence NSS mediated suppression of interferon signalling. For the first time this thesis revealed that there are serotype specific differences in NSS localisation. DENV-1 and DENV-4 NSS are predominantly localised to the cytoplasm, whereas DENV-2 and DENV-3 NSS accumulate in the nucleus. The role of the NSS nuclear localisation signal (NLS) in mediating these differences was investigated. NSS of all DENV serotypes bound to importin-a, the protein responsible for active nuclear import, but exchanging the NLSs of DENV-2 and 4 NSS didn't fully confer the properties of the NLS to the background protein, suggesting that protein context was also important for NSS nuclear localisation. Chimeric DENV-2 containing sequences encoding the DENV-3 NSS or polymerase subdomain were produced, whilst DENV-2 containing the DENV-4 NSS sequence failed to replicate. The two DENV-2/3 chimeric viruses showed decreased replication especially in the insect cell line C6/36; continued passaging resulted in the accumulation of mutations in NS1 and NS2B that increased viral replication.
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Wardell, Andrew D. "Expression and characterisation of the hepatitis C virus non-structural protein 3". Thesis, Imperial College London, 1999. http://hdl.handle.net/10044/1/11239.
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Bürckstümmer, Tilmann. "Identification of cellular targets of hepatitis C virus non-structural protein 5A". [S.l. : s.n.], 2005. http://www.diss.fu-berlin.de/2005/150/index.html.
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Marongiu, Michela. "Studies on the hyperphosphorylation of hepatitis C virus non structural protein NSSA". Thesis, University of Southampton, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.548251.
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Savage, Jason Eric. "Investigating a putative non structural protein of the birnavirus Drosophila-X virus". College Park, Md. : University of Maryland, 2004. http://hdl.handle.net/1903/1841.
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Thesis (M.S.) -- University of Maryland, College Park, 2004.Thesis research directed by: Dept. of Cell Biology and Molecular Genetics. Title from t.p. of PDF. Includes bibliographical references. Published by UMI Dissertation Services, Ann Arbor, Mich. Also available in paper.
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Kognole, Abhishek A. "UNDERSTANDING CARBOHYDRATE RECOGNITION MECHANISMS IN NON-CATALYTIC PROTEINS THROUGH MOLECULAR SIMULATIONS". UKnowledge, 2018. https://uknowledge.uky.edu/cme_etds/80.
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Non-catalytic protein-carbohydrate interactions are an essential element of various biological events. This dissertation presents the work on understanding carbohydrate recognition mechanisms and their physical significance in two groups of non-catalytic proteins, also called lectins, which play key roles in major applications such as cellulosic biofuel production and drug delivery pathways. A computational approach using molecular modeling, molecular dynamic simulations and free energy calculations was used to study molecular-level protein-carbohydrate and protein-protein interactions. Various microorganisms like bacteria and fungi secret multi-modular enzymes to deconstruct cellulosic biomass into fermentable sugars. The carbohydrate binding modules (CBM) are non-catalytic domains of such enzymes that assist the catalytic domains to recognize the target substrate and keep it in proximity. Understanding the protein-carbohydrate recognition mechanisms by which CBMs selectively bind substrate is critical to development of enhanced biomass conversion technology. We focus on CBMs that target both oligomeric and non-crystalline cellulose while exhibiting various similarities and differences in binding specificity and structural properties; such CBMs are classified as Type B CBMs. We show that all six cellulose-specific Type B CBMs studied in this dissertation can recognize the cello-oligomeric ligands in bi-directional fashion, meaning there was no preference towards reducing or non-reducing end of ligand for the cleft/groove like binding sites. Out of the two sandwich and twisted forms of binding site architectures, twisted platform turned out to facilitate tighter binding also exhibiting longer binding sites. The exterior loops of such binding sites were specifically identified by modeling the CBMs with non-crystalline cellulose showing that high- and low-affinity binding site may arise based on orientation of CBM while interacting with non-crystalline substrate. These findings provide various insights that can be used for further understanding of tandem CBMs and for various CBM based biotechnological applications.
The later part of this dissertation reports the identification of a physiological ligand for a mammalian glycoprotein YKL-40 that has been only known as a biomarker in various inflammatory diseases and cancers. It has been shown to bind to oligomers of chitin, but there is no known function of YKL-40, as chitin production in the human body has never been reported. Possible alternative ligands include proteoglycans, polysaccharides, and fibers such as collagen, all of which make up the mesh comprising the extracellular matrix. It is likely that YKL-40 is interacting with these alternative polysaccharides or proteins within the body, extending its function to cell biological roles such as mediating cellular receptors and cell adhesion and migration. We considered the feasibility of polysaccharides, including cello-oligosaccharides, hyaluronan, heparan sulfate, heparin, and chondroitin sulfate, and collagen-like peptides as physiological ligands for YKL-40. Our simulation results suggest that chitohexaose and hyaluronan preferentially bind to YKL-40 over collagen, and hyaluronan is likely the preferred physiological ligand, as the negatively charged hyaluronan shows enhanced affinity for YKL-40 over neutral chitohexaose. Collagen binds in two locations at the YKL-40 surface, potentially related to a role in fibrillar formation. Finally, heparin non- specifically binds at the YKL-40 surface, as predicted from structural studies. Overall, YKL-40 likely binds many natural ligands in vivo, but its concurrence with physical maladies may be related to the associated increases in hyaluronan.
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NESREEN, HAMAD ABDELGAWWAD HAMAD. "Structural analysis of the interaction between FUS/TLS protein and non-coding RNA". Kyoto University, 2020. http://hdl.handle.net/2433/259065.
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Thomas, Claire Philippa. "The expression of bluetongue virus non-structural protein NS2 and its structure-function relationship". Thesis, University of Oxford, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.292328.
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