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1
Guo, Hailong. "Antigenic epitope composition and protectivity of avian hepatitis E virus (avian HEV) ORF2 protein and vertical transmission of avian HEV". [Ames, Iowa : Iowa State University], 2006.
2
Ankavay, Maliki. "Étude des modifications post-traductionnelles de la protéine de capside ORF2 du virus de l'hépatite E (HEV)". Thesis, Lille 2, 2019. http://www.theses.fr/2019LIL2S013.
Abstract (sommario):
L’infection par le virus de l’hépatite E (HEV) est un problème majeur de santé publiquequi touche plus de 20 millions de personnes et tue environ 70 000 chaque année à travers lemonde. Le HEV est la cause majeure d’hépatite virale aiguë dans le monde. En France, laséroprévalence du HEV est de 22,4% dans la population générale. Ce virus se transmet parvoie féco-orale ou par consommation de viande contaminée mal cuite. Très récemment, nousavons décrit un système de culture cellulaire permettant d’amplifier efficacement le HEV. Cesystème représente un outil unique pour caractériser les différentes étapes du cycle infectieuxdu HEV qui sont très mal connues à ce jour. Dans le cadre de ma thèse, je me suis intéresséplus particulièrement à la protéine de capside ORF2 qui est l’unité structurale des particulesvirales et est donc un acteur central du cycle infectieux du HEV. La protéine ORF2 est uneprotéine de 660 acides aminés (aa) qui possède un peptide signal (PS) et trois sites potentielsde N-glycosylation (N1, N2 et N3). J’ai donc étudié le rôle de la N-glycosylation de cetteprotéine ORF2 dans le cycle infectieux du HEV. Pour atteindre cet objectif, la soucheinfectieuse HEV-p6 génotype 3 a été utilisée pour infecter les cellules de la lignéehépatocytaire PLC3, sous clone de la lignée PLC/PRF/5. Les résultats obtenus ont montré quela protéine ORF2 est N-glycosylée uniquement au niveau des sites N1 et N3 ; le site N2 estdéfavorable à la N-glycosylation. Nos résultats ont montré que la N-glycosylation de laprotéine ORF2 n’est pas importante pour sa stabilité, son oligomérisation, sa reconnaissancepar des anticorps, l’assemblage et l’infectiosité des particules virales. Nous avons aussimontré que la protéine ORF2 est importée dans le noyau des cellules hôtes au cours du cycleinfectieux du HEV de manière indépendante de la N-glycosylation. Nous avons identifié pourla première fois un signal de localisation nucléaire (NLS) fonctionnel en position N-terminalede la protéine ORF2 lui permettant d'interagir probablement avec l’importine alpha1. Demanière surprenante, ce NLS régule non seulement l’import nucléaire de cette protéine virale,mais aussi sa translocation réticulaire. Nous avons également démontré que la protéine ORF2est exportée du noyau des cellules hôtes en interagissant avec l’exportine1 et possède 3signaux d’export nucléaire (NES) en position C-terminale. Ces résultats améliorent laconnaissance du trafic intracellulaire de la protéine ORF2 et pourraient orienter les stratégiesantivirales dirigées contre le HEVHepatitis E virus (HEV) infection is a major public health problem that affects more than20 million people and kills approximately 70,000 worldwide each year. HEV is the leadingcause of acute viral hepatitis in the world. In France, the seroprevalance of HEV is 22.4% inthe general population. This virus is transmitted by fecal-oral route or by consumption ofundercooked contaminated meat. Very recently, we have described an efficient cell culturesystem to amplify HEV. This system represents a unique tool for characterizing the variousstages of the infectious HEV lifecycle that are very poorly understood to date. As part of mythesis, I focused on the ORF2 capsid protein which is the structural unit of viral particles andis therefore a central player in the HEV lifecycle. ORF2 is a 660 amino acids protein thatcontains a signal peptide and three potential N-glycosylation sites (N1, N2 and N3). My workaimed to identify the role of the ORF2 N-glycosylation in the HEV lifecycle. Our resultsrevealed that the ORF2 protein is N-glycosylated on the N1 and N3 sites, whereas the N2 siteis not N-glycosylated. Also, the N-glycosylation has no significant relevance neither for thestability, oligomerization, antibody recognition of the ORF2 protein nor for viral assemblyand viral infectivity. We also revealed that, the ORF2 protein is translocated into the nucleusof infected cells, independently of N-glycosylation. We identified for the first time afunctional nuclear localization signal (NLS) located at the N-terminus of the ORF2 proteinthat interacts probably with the importin alpha1. Surprisingly, this NLS regulates both nuclearimport and endoplasmic reticulum translocation of the ORF2 protein. Finally, wedemonstrated that, the ORF2 protein possesses three nuclear export signals (NES) located atits C-terminus. The ORF2 protein interacts with the exportin1 and is exported from the cellnuclei. This interaction drives the ORF2 protein from the nucleus to the cytoplasm of theinfected cells. Hence, this study led to new insights into the molecular mechanisms of theORF2 protein and could help to the design of novel antiviral strategies against HEV
3
Ferrié, Martin. "Étude de la maturation protéolytique et du trafic intracellulaire de la protéine de capside ORF2 du virus de l'hépatite E (HEV)". Electronic Thesis or Diss., Université de Lille (2022-....), 2023. http://www.theses.fr/2023ULILS067.
Abstract (sommario):
L'infection par le virus de l'Hépatite E (HEV) est un problème majeur de santé publique qui toucherait 100 millions de personnes et tuerait 100 000 personnes chaque année dans le monde. Le HEV est la première cause d'hépatite aigüe dans le monde. En France, la séroprévalence s'élève à 22,4%. Ce virus se transmet par voie féco-orale ou par la consommation de viande contaminée mal cuite. Au cours de ma thèse, je me suis intéressé plus particulièrement à la protéine de capside ORF2, qui est l'unité structurale des particules virales et un acteur central du cycle infectieux du HEV. La protéine ORF2 est une protéine de 660 acides aminés qui possède un peptide signal N-terminal et trois sites potentiels de N-glycosylation. Au cours de son cycle infectieux, le HEV produit au moins trois formes de sa protéine de capside : (i) la forme ORF2g (pour glycosylée) et (ii) la forme ORF2c (pour clivée), abréviées ORF2g/c, qui sont des formes glycosylées et massivement sécrétées dans les surnageants de culture ou le sérum des patients infectés, et (iii) la forme ORF2i (pour infectieuse), qui n'est pas glycosylée et qui est associée aux particules virales.Dans le cadre de mes travaux de thèse, j'ai étudié les mécanismes de maturation protéolytique et de trafic intracellulaire de la protéine ORF2. Plus précisément, j'ai d'abord montré que la protéine ORF2 était importée dans le noyau des cellules infectées par le biais d'un mécanisme dépendant de l'Importine-alpha1, ceci grâce à un motif riche en résidus arginine situé à l'extrémité N-terminale de l'ORF2 et nommé ARM. La protéine ORF2 est ensuite exportée vers le cytoplasme par un mécanisme dépendant de l'exportine CRM1, ceci grâce à trois sites d'export nucléaire (NES9, NES10 et NES12) que nous avons identifiés dans la séquence de l'ORF2. J'ai également montré que le trafic nucléo-cytoplasmique de l'ORF2 régule probablement l'expression de certains gènes de l'immunité antivirale aux temps précoces de l'infection.Dans un second temps, j'ai participé à l'identification et à la caractérisation des usines virales du HEV. En ce sens, nous avons montré que les protéine virales ORF1, ORF2 et ORF3, ainsi que l'ARN viral sont enrichis dans des structures vésiculaires et tubulaires localisées dans les régions périnucléaires des cellules infectées. Nous avons montré que ces structures sont également enrichies en marqueurs du compartiment endosomal de recyclage (ERC), comme Rab11 et CD71, indiquant que les usines virales du HEV dérivent probablement de l'ERC. En réalisant des expériences d'extinction de Rab11 avec des ARN interférents, nous avons confirmé l'importance de l'ERC dans la production des particules virales du HEV.Dans un troisième temps, j'ai démontré que la protéine ORF2i, qui est associée aux membranes de la voie de sécrétion, est adressée aux usines virales par un mécanisme faisant intervenir le complexe adapteur AP-1.Dans un quatrième temps, j'ai caractérisé les mécanismes de maturation protéolytique des formes ORF2g/c et ORF2i. J'ai montré que la furine, une proproprotéine convertase de la voie de sécrétion, est impliquée dans la maturation protéolytique des glycoprotéines ORF2g/c. J'ai également montré que la préséniline, qui est la sous-unité catalytique du macro-complexe Gamma-sécrétase, est impliquée dans la maturation protéolytique de la forme infectieuse ORF2i. De manière intéressante, j'ai montré que l'inhibition pharmacologique de la préséniline réduit drastiquement l'infectiosité virale dans des lignées d'hépatocarcinome humain et dans des hépatocytes primaires humains. Ces données suggèrent que l'inhibition pharmacologique de la préséniline représente une stratégie antivirale prometteuse.En conclusion, les résultats obtenus dans le cadre de ma thèse permettent de mieux comprendre les mécanismes d'adressage subcellulaire et de maturation protéolytique de la protéine de capside ORF2, et ouvrent la voie au développement de nouvelles thérapies pour lutter contre le HEVHepatitis E virus (HEV) infection is a major public health problem, affecting an estimated 100 million people and killing 100,000 every year worldwide. HEV is the leading cause of acute hepatitis worldwide. In France, seroprevalence is 22.4%. This virus is transmitted via the feco-oral route, or by eating contaminated undercooked meat. During my thesis, I focused on the ORF2 capsid protein, which is the structural unit of viral particles and a central player in the HEV lifecycle. ORF2 is a 660 amino acid protein with an N-terminal signal peptide and three potential N-glycosylation sites. During its lifecycle, HEV produces at least three forms of its capsid protein: (i) the ORF2g (for glycosylated) form and (ii) the ORF2c (for cleaved) form, abbreviated ORF2g/c, which are glycosylated forms and massively secreted in culture supernatants or serum from infected patients, and (iii) the ORF2i (for infectious) form, which is not glycosylated and is associated with viral particles.As part of my thesis work, I studied the mechanisms of proteolytic maturation and intracellular trafficking of the ORF2 protein. More specifically, I first showed that ORF2 is imported into the nucleus of infected cells via an Importin-alpha1-dependent mechanism, thanks to an arginine-rich motif located at the N-terminus of ORF2 and named ARM. The ORF2 protein is then exported to the cytoplasm via a CRM1 exportin-dependent mechanism, thanks to three nuclear export sites (NES9, NES10 and NES12) that we have identified in the ORF2 sequence. I have also shown that nucleocytoplasmic trafficking of ORF2 probably regulates the expression of certain antiviral immunity genes in the early stages of infection.Secondly, I participated in the identification and characterization of HEV viral factories. We have shown that the viral proteins ORF1, ORF2 and ORF3, as well as viral RNA, are enriched in vesicular and tubular structures located in the perinuclear regions of infected cells. We have shown that these structures are also enriched in markers of the endosomal recycling compartment (ERC), such as Rab11 and CD71, indicating that HEV viral factories probably derive from the ERC. By performing Rab11 silencing experiments with siRNAs, we confirmed the importance of the ERC in the production of HEV viral particles. Thirdly, I demonstrated that the ORF2i protein, which is associated with the membranes of the secretion pathway, is addressed to viral factories by a mechanism involving the AP-1 adaptor complex.In a fourth step, I characterized the proteolytic maturation mechanisms of the ORF2g/c and ORF2i forms. I showed that furin, a proproprotein convertase of the secretory pathway, is involved in the proteolytic maturation of ORF2g/c glycoproteins. I have also shown that presenilin, which is the catalytic subunit of the macro-complex Gamma-secretase, is involved in the proteolytic maturation of the ORF2i form. Interestingly, I have shown that pharmacological inhibition of presenilin drastically reduces viral infectivity in human hepatocarcinoma lines and in primary human hepatocytes. These data suggest that pharmacological inhibition of presenilin represents a promising antiviral strategy. In conclusion, the results obtained during my thesis provide a better understanding of the mechanisms of subcellular addressing and proteolytic maturation of the ORF2 capsid protein, and pave the way for the development of new therapies to combat HEV
4
Horridge, Jackie J. "RNA-protein interactions of the adenovirus proteins E1B 55K and E4 Orf6". Thesis, University of Warwick, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322435.
5
Baghbadorani, G. Ahmadian. "Analysis of the utilisation of second ORFs in pneumovirus mRNA". Thesis, University of Warwick, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.323416.
6
Chand, Puran. "Molecular and immunological characterisation of a major envelope protein of capripoxvirus". Thesis, University of Surrey, 1992. http://epubs.surrey.ac.uk/2774/.
Abstract (sommario):
Analysis of the proteins of capripoxvirus (KS-1) revealed a 32kd protein that is one of the major structural proteins of the virus and is localised in the virus envelope. Monospecific serum prepared against the 32kd envelope protein neutralised the virus indicating that this protein contains neutralising epitopes. Lymphocyte proliferation studies, using the 32kd protein and peripheral blood mononuclear cells from capripoxvirus (KS-i) vaccinated sheep, showed that this protein strongly induced cellmediated immune responses. The 32kd protein is capripoxvirus specific and induced antibodies in early stages of capripoxvirus infections. Immunoblot analysis of antibody responses against this protein has provided a basis for the differential diagnosis of capripoxvirus and orf virus infections. The 32kd protein bound to the surface of cultured lamb testis cells. The binding of the 32kd protein was completely inhibited by prior incubation of cells with purified capripoxvirus (KS-1) but not by bovine serum albumin. Trypsin treatment of capripoxvirus (KS-1) degraded the majority of the 32kd protein with a minimal effect on a few other virus proteins. Trypsin removed an external 10kd fragment from the 32kd protein, leaving a 22kd fragment associated with the virus. In addition, the trypsin treatment reduced the virus infectivity by at least ten fold, suggesting that the cell surface binding domain of the 32kd protein is located within the external 10kd fragment. The monospecific serum to the 32kd protein had no effect of the infectivity titre of the trypsin treated virus further supporting the concept that the external 10kd fragment of the 32kd protein is involved in binding of the virus particle to the cell surface. A degenerate oligonucleotide probe, based on an internal amino acid sequence obtained from V8 protease cleavage products of the 32kd protein, was used to identify the gene encoding the 32kd protein. The gene encoding the 32kd protein was identified within the 2.8kb HindI1l Q1 fragment of the capripoxvirus (KS-1) genome. The nucleotide sequence analysis of the Hindu Q1 fragment revealed five open reading frames (Q11L, Q12R, Q13L, Q14R and Q15L), one of these open reading frames, Q13L, is capable of encoding a 30.6kd protein and contains the complete internal amino acid sequence obtained from the V8 protease cleavage products of the 32kd protein, indicating that the Q13L encodes the 32kd envelope protein of capripoxvirus (KS-1). The deduced amino acid sequence of the Q13L shows a 34.1% identity and 61.3% similarity with that of H3L open reading frame of vaccinia virus.
7
Koyuncu, Emre. "Expression, Purification, And Functional Analysis Of Adenovirus Type 5 E4 Orf3 Protein". Master's thesis, METU, 2004. http://etd.lib.metu.edu.tr/upload/12605415/index.pdf.
Abstract (sommario):
In this study, structural and functional aspects of adenovirus type 5 (Ad5) E4orf3 protein were analyzed by biophysical and biochemical methods. Ad5 is one of the mostly used gene therapy vectors to date. However, some of its proteins possess oncogenic potential and their presence comprises safety risks. E4orf3 is one of the oncoproteins of Ad5. It also takes important roles in viral infection, and is beneficial for therapy vectors. Therefore, understanding the functions of E4orf3 is very important for developing efficient and safe adenovirus vectors.
Most of the present knowledge about the functions of E4orf3 comes from its mutational analysis. It has never been expressed or purified successfully due to its extreme insolubility. Therefore, this study focused on the optimization of expression of E4orf3 protein. As a result, full-length E4orf3 was obtained in soluble form as a Glutathione-S-transferase (GST) fusion protein and purified by GST affinity chromatography for the first time. Subsequently, the interaction of E4orf3 with four different proteins, DNA-PK, Aup1, E1B-55 kDa and E4orf6 was analyzed in detail by GST-pulldown technique. In these experiments, E4orf3 was shown to associate with Aup1, E1B-55 kDa and E4orf6 in vitro, and the C-terminal of E4orf3 was determined to be responsible for these interactions. Finally, basic structural information about E4orf3 protein was also obtained for the first time by the direct analysis of the fusion protein in glutathione beads with Fourier Transform Infrared (FTIR) spectroscopy. Since the purified E4orf3 protein could not be separated from the glutathione beads due to its hydrophobic regions, the secondary structures in this protein were determined after subtracting glutathione and H2O absorption bands, and the GST moiety.
8
Bowers, Laura Yvonne. "Orf protein modulates phage and bacterial pathways of genetic recombination". Thesis, Durham University, 2008. http://etheses.dur.ac.uk/2345/.
Abstract (sommario):
The emergence of novel pathogenic organisms due to the acquisition of virulence determinants from bacteriophages has generated significant interest in the pathways responsible for genomic rearrangements. Phageλ encodes its own recombination system, the Red system, comprising Exo, β and γ proteins. In addition,λ encodes another recombinase, Orf, which participates in the initial stages of genetic exchange and supplies a frmction equivalent to that of the Escherichia coli RecFOR proteins. This thesis focuses on determining the function of Orf in phage and bacterial recombination pathways by analysing its impact on recombinases encoded by λ and E. coli. Experiments revealed that Orf interacts with bacterial and phage recombination proteins in the initial exchange step of recombination, modulating the activities of both Exo and RecA. Orf, along with β, attenuates the 5'-3' exonuclease activity of Exo, a feature that depends largely on the ability of Orf to bind DNA. Orf also facilitates loading of RecA onto ssDNA pre-coat SSB but only if a ssDNA:dsDNA intersection is incorporated in the substrate. A motif similar to that found at the BRCA2-Rad51 interface may be responsible for Orf mimicking a RECA monomer to initiate nucleoprotein filament formation. Significantly, this would direct recombination down the bacterial RecA pathway of break restoration rather than the phage Red pathway with potentially important consequences for the outcome of the exchange reaction.
9
Liedeman, Kerwin. "Transient transgene expression of human Coronavirus nl63 orf3 protein in a baculovirus system". University of the Western Cape, 2020. http://hdl.handle.net/11394/8042.
Abstract (sommario):
>Magister Scientiae - MScInsect-derived baculoviruses have been used extensively as a safe and versatile research model for transgenic protein expression. Preclinical studies have revealed the promising potential of Baculoviruses as a delivery vector for a variety of therapeutic applications, including vaccination, tissue engineering and cancer treatments. Coronaviruses are enveloped viruses containing linear, non-segmented ribonucleic acid. Human coronavirus NL63 was first discovered in the Netherlands in January 2004, where a 7-month-old girl presented with an acute respiratory tract infection that was later established to predominantly infect infants, the elderly and immunocompromised individuals. In addition to the known non-structural and structural proteins of coronaviruses, an accessory protein known as open reading frame 3 which is conserved in the Coronaviridae family has not been extensively researched. Open reading frame 3 encodes a putative membrane-bound protein. This study cloned the open reading frame 3 viral gene of 741 base pairs into the baculovirus expression construct via competent bacterial cell lines. Open reading frame 3-Baculovirus particles were generated in Spodoptera frugiperda insect cells. Recombinant cells containing the viral protein gene were used to infect healthy Spodoptera frugiperda 9 cells at varying ratios of multiplicity of infection over a fixed time-course. The open reading frame 3 viral protein was not detected by quantification methods at a molecular weight of 26 kilo Dalton, due to polyclonal antibody degradation.
10
Yadav, Kush Kumar. "Genotype 1 hepatitis E virus (HEV) ORF4 protein enhances genotype 3 HEV replication". The Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1574781581580768.
11
Ali, Ahmed Said. "Investigation of epoxide hydrolase activity in Saccharomyces cerevisiae ORF YNR064c protein". Thesis, Uppsala universitet, Institutionen för kemi - BMC, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-203473.
12
Dickinson, Victoria Jane. "The cloning and subcellular localisation of maize streak virus ORF V1". Thesis, University of Hull, 1996. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.321050.
13
Mo, Min, e n/a. "Characterization of an Orf virus RING-H2 protein, B5L : a mimic of cellular anaphase promoting complex subunit 11". University of Otago. Department of Microbiology & Immunology, 2009. http://adt.otago.ac.nz./public/adt-NZDU20090220.085825.
Abstract (sommario):
The anaphase promoting complex (APC/C) is an ubiquitin ligase that is an essential regulator of multiple steps in the cell cycle. The complex consists of at least 12 subunits with a catalytic core formed by a scaffold protein, APC2, and a RING-H2 protein, APC11. The Parapoxvirus, Orf virus (OV), encodes a RING-H2 protein, B5L, with clear sequence similarities to APC11. The disruption of APC/C function leads to pre-mature entry into S phase and a delayed M phase exit and, potentially, apoptosis. This investigation explored the functional significance of the similarity between B5L and APC11 and specifically sought to determine if B5L manipulates cell cycle regulation by targeting APC/C function.
Co-immunoprecipitation experiments from lysates of cells expressing a range of constructs revealed an interaction between B5L and APC2 in the same manner as seen with APC11. Furthermore, B5L was found to associate with endogenous APC/C. However, although APC11 promoted the formation of polyubiquitin chains in substrate-independent in vitro assays, B5L was inactive in this assay. Bioinformatics comparisons of APC11 and other known RING ubiquitin ligases with B5L and its poxviral homologues revealed some subtle differences. In particular a domain of APC11 (amino acids 61-74), that is essential for its ubiquitin ligase activity is not conserved in B5L or its homologues. When this APC11 domain was incorporated in place of the corresponding region of B5L (amino acids 59-67), the mutated B5L acquired ubiquitin ligase activity. On the other hand, APC11 protein in which the domain was replaced with that of B5L lost ubiquitin ligase activity.
Stable cell lines expressing B5L showed an increased number of cells in G2/M phase (30�4%) compared with cell lines expressing APC11 (11�2%, n=3, p<0.05, ANOVA, Tukey�s), consistent with impaired APC/C function. APC/C substrates such as cyclin A, cyclin B and the thymidine kinase were stablized in B5L-expressing cells compared with control cells. Furthermore, transient hyper-expression of B5L induced apoptosis in 25�2% (n=3, p<0.05) of the cell population compared with only 6�1% apoptotic cells when APC11 was hyper-expressed. Analysis of the DNA content of OV-infected cells revealed enhanced DNA synthesis compared with cells infected with a B5L knockout OV.
These observations indicate that B5L is a non-functional mimic of APC11. It associates with APC/C, but lacks ubiquitin ligase activity, and hence disrupts APC/C function. These abilities may enable OV to induce a cellular environment that enhances viral replication.
14
Butarbutar, Nunut. "Analysis of yeast codon usage patterns using the movable ORF collection /". Online version of thesis, 2007. http://hdl.handle.net/1850/5700.
15
Laitinen, Saara. "Family of human oxysterol binding protein homologues : ORP2 is a new regulator of cellular lipid metabolism". Helsinki : University of Helsinki, 2002. http://ethesis.helsinki.fi/julkaisut/laa/kansa/vk/laitinen/.
16
Öhrmalm, Christina. "Functional Characterization of the Cellular Protein p32 : A Protein Regulating Adenovirus Transcription and Splicing Through Targeting of Phosphorylation". Doctoral thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2006. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-6794.
Abstract (sommario):
Cellular processes involved in the conversion of the genetic information from DNA into a protein are often regulated by reversible phosphorylation reactions. By modulating the phosphorylated status of key proteins their activity can either be enhanced or repressed. In this thesis I have studied the significance of phosphorylation in the regulation of transcription and splicing using human adenovirus as a model system.
The results show that the activity of the cellular SR family of splicing enhancer or repressor proteins are reduced in adenovirus infected nuclear extracts by a virus-induced hypophosphorylation. The viral E4-ORF4 was shown to induce SR protein dephosphorylation by recruiting the cellular protein phosphatase PP2A. The E4-ORF4/PP2A complex was shown to relieve the SR protein-mediated repression of late virus-specific splicing and further activate alternative splicing in transiently transfected cells. Collectively, these results showed that alternative splicing, like many other biological processes, is regulated by reversible protein phosphorylation.
Similarly, the cellular p32 protein was shown to cause hypophosphorylation of the SR protein ASF/SF2 resulting in a reduced RNA binding capacity of ASF/SF2. This change in ASF/SF2 RNA binding also had a drastic effect on the function of ASF/SF2 as a regulatory protein affecting splice site choice. The cellular p32 protein and the viral E4-ORF4 protein both target the same cellular splicing factor, ASF/SF2. However, they regulate splicing by different mechanisms. E4-ORF4 recruits a phosphatase to dephosphorylate ASF/SF2, while p32 sequester ASF/SF2 in an inactive complex.
Further, we demonstrated that overexpression of p32 during a lytic infection suppressed transcription from the adenovirus major late transcription unit. p32 induced a selective repression of CAAT-box containing promoters indicating the involvement of the transcription factor CBF/NF-Y in this regulation. A further analysis showed that p32 caused a hyperphosphorylation of the CTD of RNA Pol II, which resulted in a significant reduction in the processivity of Pol II during the elongation phase of transcription.
In summary, we have shown that E4-ORF4 regulates the activity of splicing regulatory SR proteins, and that p32 regulates the activity of the SR protein ASF/SF2 in splicing and Pol II processivity during transcription elongation. Mechanistically, both E4-ORF4 and p32 appears to function by regulating the phosphorylated status of key cellular proteins involved in these processes.
17
D'Angelillo, Anna. "Studio dell'attività biologica della proteina ORF-A del virus dell'immunodeficienza felina". Doctoral thesis, Università degli studi di Padova, 2013. http://hdl.handle.net/11577/3423592.
Abstract (sommario):
Feline Immunodeficiency Virus (FIV), a non-primate lentivirus, causes an immunodeficiency syndrome in domestic cats that is strikingly similar to AIDS in humans. Although diverse in term of evolution, FIV is similar to Human Immunodeficiency Virus Type-1 (HIV-1) in many respects including genome organization, target cells infected in vivo, course of infection and disease state. Thus, for these reasons, FIV represents an attractive model for AIDS research. Similarly to HIV-1 and other lentivirus, the FIV genome encodes three large open reading frame gag, pol, env that encode for the structural and enzymatic proteins. HIV-1 encodes six additional small open reading frames that express regulatory and accessory proteins: vif, rev, tat, vpr, vpu and nef. However, the FIV genome contains only three additional open reading frames, two of which include rev, that encodes a viral post transcriptional regulatory protein and vif that encodes an accessory protein necessary for viral infectivity. The primary function of the third gene designated orf-A remains unclear but it is believed to represent a multifunctional accessory protein similar to HIV-1 Vpr, Tat and Nef accessory proteins.
This study is part of a research project aimed to clarify FIV biology, focusing on the functions performed by ORF-A. Firstly, we evaluated Orf-A expression and its involvement in the cell cycle progression using different systems of over-expression. Secondly, in order to explore Orf-A involvement in cell cycle progression in the context of more physiological conditions, we obtained Petaluma proviral constructs containing a tagged version of Orf-A. Moreover, we employed the same constructs in order to evaluate i) the intracellular localization of Orf-A and Cyclin B1 proteins with immunofluorescence assays; ii) the replicative capacity of Orf-A tagged plus and minus viruses; iii) the incorporation of the Orf-A protein in viral particles.
Taken together, the results suggest that Orf-A expression leads to a cell cycle arrest in G2/M phase, indicating a role of Orf-A similar to the one described for HIV-1 Vpr. Furthermore, for the first time we report the Orf-A incorporation into viral particles.
This study contributes to dissect FIV Orf-A functions and to clarify the mechanisms underlying Feline Immunodeficiency Virus and, generally, lentiviruses biologyIl Virus dell'Immunodeficienza Felina (FIV) è un Lentivirus dei non primati che causa nel gatto domestico una sindrome cronica progressiva molto simile alla Sindrome da Immunodeficienza Acquisita (AIDS) generata dal Virus dell'Immunodeficienza Umana di tipo 1 (HIV-1). Le analogie tra FIV ed HIV-1 coinvolgono numerosi aspetti, tra cui l'organizzazione genomica, il ciclo replicativo, i bersagli cellulari ed i meccanismi patogenetici. Per questi motivi FIV ed il suo ospite naturale rappresentano un interessante modello per la ricerca sull'AIDS. Similmente ad HIV-1, il genoma di FIV contiene i geni gag, pol, env che codificano le proteine strutturali ed enzimatiche del virione e i geni accessori e regolatori che giocano un ruolo chiave nella regolazione e nell'infettività virale. A differenza del genoma di HIV-1 caratterizzato dalla presenza di quattro geni accessori, il genoma di FIV ne presenta due: vif ed orf-A. Di questi, il primo codifica il fattore di infettività virale, molto simile a quello di HIV-1, mentre il secondo gene codifica la proteina Orf-A. Il ruolo biologico di Orf-A non è stato ancora chiarito, ma si ritiene rappresenti una proteina accessoria multifunzionale con caratteristiche simili alle proteine accessorie di HIV-1 quali Vpr, Tat e Nef. Il presente lavoro di dottorato si inserisce in un progetto di ricerca più ampio, volto a chiarire la biologia di base di FIV, focalizzandosi sulle funzioni svolte dalla proteina Orf-A. Inizialmente è stata valutata l'espressione della proteina ed il suo coinvolgimento nel ciclo cellulare mediante diversi sistemi di over-espressione. Successivamente sono stati condotti gli stessi esperimenti in un contesto più simile a quello fisiologico. Pertanto, sono stati ottenuti plasmidi basati sul genoma provirale, Orf-A plus e minus, dove l'estremità Carbossi-terminale della proteina Orf-A è stata fusa al tag HA. I vettori sono stati testati sia in cellule umane che feline per valutarne l'espressione ed il coinvolgimento nella progressione del ciclo cellulare. Gli stessi plasmidi sono stati impiegati per valutare i) la localizzazione della proteina Orf-A e della Ciclina B1 target del blocco del ciclo cellulare in saggi di immunofluorescenza; ii) la capacità replicativa del virus; iii) l'incorporazione della proteina Orf-A nelle particelle virali. Nel complesso, i risultati suggeriscono che, laddove la proteina Orf-A è espressa, si verifica un arresto del ciclo cellulare in fase G2/M indicando un ruolo di Orf-A simile a quello descritto per la proteina Vpr di HIV-1. Inoltre, per la prima volta in letteratura, è stata dimostrata l'incorporazione della proteina Orf-A nelle particelle virali. Questo studio contribuisce a dissezionare le funzioni della proteina Orf-A di FIV ed a chiarire i meccanismi alla base della biologia del Virus dell'Immunodeficienza Felina e, più in generale, di quella dei lentivirus
18
Tan, Joanne Li-Ching, e n/a. "Development of Orf virus as a vaccine vector : manipulation of structural proteins for surface display of immunogenic peptides". University of Otago. Department of Microbiology & Immunology, 2009. http://adt.otago.ac.nz./public/adt-NZDU20090427.144304.
Abstract (sommario):
Orf virus (ORFV) has the potential to be developed as a vaccine vector. Its ability to stimulate non-specific as well as specific immune responses in permissive and non-permissive hosts stands it in good stead to be utilised as such a tool. The fusion of immunogenic peptides to vaccinia virus (VACV) structural proteins have been shown to improve their immunogenicity due to presentation of the foreign antigens in a particulate form that can stimulate both B and T cells. The aims of this study were to fuse foreign antigens to ORFV structural proteins to demonstrate proof-of-concept that such surface display could also render the foreign antigens more immunogenic.
Little is known about ORFV structure and morphogenesis. When this study commenced, the ORFV genome had recently been sequenced and this revealed a large number of homologues in common with VACV. It was thus assumed that both viruses may share structural similarities and that ORFV also assumes the different morphological forms such as the mature virion (MV) and extracellular virion (EV) that are present in VACV. The MV and EV forms are both infectious, with the EV containing an additional membrane acquired from the trans-Golgi network during viral morphogenesis. Furthermore, specific viral proteins are associated with both the MV and EV membranes.
Six ORFV structural proteins ORFV 089, 10 kDa, F1, that are homologues of structural membrane proteins A13, A27 and H3 of VACV MVs, together with ORFV 109, ORFV 110 and B2, that are homologues of structural membrane proteins A33, A34 and F13 of VACV EVs were selected as possible candidates for manipulation. At present, there is some information available only for 10 kDa, F1 and B2. The 10 kDa is required for virus assembly, F1 for mediating cell attachment while B2 has been shown to induce significant antibody responses in sheep. Indeed proteomic analyses predicted similarities in the topologies of all of these proteins with their VACV counterparts.
Using this information, preliminary studies were conducted to generate recombinant ORFVs (rORFVs) which had FLAG fused to the terminus of the protein that was exposed on the surface of the virus particle. Three rORFVs 10 kDa, F1L and 110 were successfully generated. Immunogold labelling of FLAG proteins on virus particles isolated from lysed cells showed that FLAG-10 kDa and FLAG-F1 were displayed on the surface of MV particles whereas FLAG-ORFV 110 could not be detected. Western blot analyses of solubilised recombinant ORFV 110-FLAG particles revealed that FLAG-ORFV 110 was abundant and undergoes post-translational modification indicative of endoplasmic reticulum trafficking whereas FLAG-10 kDa and FLAG-F1 did not appear to be subjected to post-translational modifications. Fluorescent microscopy confirmed the prediction that ORFV 110-FLAG localised to the Golgi in virus-infected cells and immunogold labelling of EVs showed that ORFV110-FLAG became exposed on the surface of EV-like particles as a result of egress from the cell, suggesting that the membranes had been acquired from the Golgi. These modifications also appeared to have minimal effect on the infectivity of these rORFVs.
The study was extended by replacing the small FLAG peptide with an immunogenic protein (EG95), derived from the oncosphere of the zoonotic parasite Echinococcus granulosus. This protein is known to confer protection in immunised animals. Three rORFVs were generated in which a truncated version of the protein, EG95[Delta]TM, was fused to 10 kDa in the absence (rORFV 699) or presence (rORFV 700) of a linker, and also to F1 (rORFV 701). Western blot analyses of these solubilised particles demonstrated that the fusion proteins appeared to be post-translationally modified while immunogold labelling using anti-EG95 monoclonal antibodies successfully demonstrated the surface labelling on these rORFVs.
In order to test the immunogenicity of these rORFVs, prime-boost experiments in sheep were conducted using rORFVs 699, 700 and 701 and a glutathione-S-transferase (GST-EG95) based vaccine. The results showed the production of EG95-specific antibodies. In particular, antibody production by group rORFV 701 compared favourably with a control group that was primed and boosted by GST-EG95 vaccine. This was despite the slightly slower growth rates of rORFVs 700 and 701 and the decreased infectivity of all three rORFVs discovered in in vitro experiments.
In conclusion, these studies indicated the feasibility of this strategy to manipulate ORFV structural proteins for use as an agent for vaccine delivery.
19
Diekmann, Ulrike Verfasser], e Lothar [Akademischer Betreuer] [Jänsch. "Characterisation of the KSHV protein ORF20 and its role in innate immunity / Ulrike Diekmann ; Betreuer: Lothar Jänsch". Braunschweig : Technische Universität Braunschweig, 2019. http://d-nb.info/1179909984/34.
20
Diekmann, Ulrike [Verfasser], e Lothar [Akademischer Betreuer] Jänsch. "Characterisation of the KSHV protein ORF20 and its role in innate immunity / Ulrike Diekmann ; Betreuer: Lothar Jänsch". Braunschweig : Technische Universität Braunschweig, 2019. http://d-nb.info/1179909984/34.
21
Estmer, Nilsson Camilla. "Viral Control of SR Protein Activity". Doctoral thesis, Uppsala : Acta Universatis Upsaliensis : Univ.-bibl. [distributör], 2001. http://publications.uu.se/theses/91-554-5124-1/.
22
White, Ian Alexander. "Hematopoietic stem cell expansion : under serum free and cytokine-limited conditions using primary endothelial cells transfected with the adenoviral E4-ORF1 gene /". Access full-text from WCMC, 2009. http://proquest.umi.com/pqdweb?did=1692100331&sid=5&Fmt=2&clientId=8424&RQT=309&VName=PQD.
23
Mardegan, Catarina. "Caracterização funcional da ORF XAC0239 de Xanthomonas citri subsp. citri /". Jaboticabal, 2018. http://hdl.handle.net/11449/155873.
Abstract (sommario):
Orientador: Maria Inês Tiraboschi FerroCoorientador: Helen Alves Penha
Banca: José Belasque Júnior
Banca: Daniel Guariz Pinheiro
A Xanthomonas citri subsp. citri (Xac) é a bactéria causadora da doença cancro cítrico. A doença atinge todas as variedades comerciais de citros e, o entendimento da relação fitopatogênica permitirá conhecer formas mais eficientes de controle da bactéria. Com o sequenciamento e análise do genoma e transcriptoma da Xac, foram identificadas ORFs que, provavelmente, estão envolvidas em processos de ataque e colonização da bactéria. Para iniciar a caracterização de uma destas ORFs foram utilizadas, neste estudo, as estratégias de mutação sítio-dirigida de PCR por sobreposição e extensão e modelagem molecular. A ORF XAC0239, é predita como proteína de membrana plasmática, e por homologia, possivelmente, pode fazer parte de uma proteína transportadora de zinco, além disso, há indícios de que é regulada por um fator sigma. A mutação teve efeito negativo sobre a ação de celulases e motilidade swimming e efeito positivo sobre a formação de biofilme e a multiplicação bacteriana in vivo. Aqui é sugerido que na falta desta proteína, houve pouca absorção de zinco, necessário para a formação de biofilme e consequentemente, para a patogenicidade. Além disso, sugestiona-se que, como subterfúgio, a bactéria superexpressou genes para produção e/ou ação de celulases, tentando atacar o hospedeiro de forma alternativa.
Xanthomonas citri subsp. citri (Xac) causes the citrus canker disease. The disease reaches all commercial varieties of citrus and, the understanding of the phytopathogenic relationship allow knowing the ways of control of bacteria. With sequencing of the Xac genome and the realization of a transcriptome, ORFs have been identified that are probably involved in bacterial attack and colonization processes. To initiate the characterization of one of these ORFs, in this study, the strategies of site-directed mutagenesis by overlap extension PCR and molecular modeling were used. The ORF XAC0239 is predicted as plasma membrane protein, and by homology may possibly be part of a zinc transporter protein, moreover, there are indications that it is regulated by a sigma factor. The mutation had a negative effect at the cellulases action and swimming motility and a positive effect on biofilm formation and in vivo multiplication. Here it is suggested that in the absence of this protein, there was little absorption of zinc, necessary for biofilm formation and consequently for pathogenicity. In addition, it is suggested that, as a subterfuge, the bacteria overexpressed genes for production and / or cellulases action, trying to attack the host on alternative way.
Mestre
24
Deane, David Leslie. "The characterisation of a GM-CSF and IL-2 inhibitory protein encoded by Orf virus". Thesis, University of Edinburgh, 2001. http://hdl.handle.net/1842/23323.
Abstract (sommario):
In this study, the gene encoding the GM-CSF inhibitory factor (GIF) was isolated and mapped to the right terminal quarter of the orf virus genome. The orf virus GIF cDNA was expressed as a secreted protein in Chinese hamster ovarian cells as detected by GM-CSF inhibition ELISA. Recombinant GIF was purified by ovine GM-CSF affinity chromatography and gel filtration. Sequence analysis of the 20 N-terminal amino acids was performed on the purified GIF. The GIF gene encodes a 28 kDa protein that exhibits 32% amino acid sequence similarity to the predicted sequence of the A41L gene product encoded by vaccinia virus. Although the vaccinia virus A41L protein has sequence similarity to the T1 secreted chemokine-binding proteins of leporipoxviruses, its function is not known. GIF did not share any homology with any cytokine receptor molecule identified to date. In contrast to other parapoxvirus immunomodulatory proteins that are products of early viral genes, GIF was found to be the product of an intermediate/late viral gene of orf virus infected cells. GIF formed homodimers and homotetramers in solution and bound ovine GM-GSF with a Kd of 369 pM. In addition GIF bound ovine IL-2 with a Kd of 1.04 nM. Although orf virus infects humans, GIF did not bind human GM-CSF or IL-2. GIF was shown to inhibit the binding of ovine GM-CSF labelled with 125I to its receptor on isolated sheep neutrophils and it inhibits the haematopoietic activity of ovine GM-CSF in a soft agar bone marrow colony assay. GIF also inhibited the binding of ovine IL-2 labelled with 125I to CD4+ T cells and inhibited the stimulatory activity of ovine IL-2 in a T cell proliferation assay. This inhibitory activity was neutralised by a rabbit antiserum raised against purified GIF. GIF was produced in vivo during orf virus reinfection. GIF was detected in skin, localised to the area of orf virus infected cells and in afferent lymph draining the skin site of infection. The presence of GIF, 3-7 days after virus infection was associated with reduced levels of GM-CSF in the lymph plasma and the period of maximum viral replication in the skin.
25
Melling, Michael [Verfasser], e Thomas [Akademischer Betreuer] Dobner. "The influence of SUMOylation on the adenoviral early region 4 protein Orf6/7 / Michael Melling ; Betreuer: Thomas Dobner". Hamburg : Staats- und Universitätsbibliothek Hamburg, 2018. http://d-nb.info/1163013692/34.
26
Craft, Jennifer Leigh. "Generating an expression construct and soluble protein for characterization studies of a putative RNA m5C methyltransferase, yeast ORF YNLO22c". Virtual Press, 2005. http://liblink.bsu.edu/uhtbin/catkey/1319220.
Abstract (sommario):
RNA m5C methyltransferases are a group of enzymes that catalyze the transfer of a methyl group to a cytosine nucleotide of RNA. Only two of these enzymes have been well characterized: Fmu from E. coli and Trm4p from S. cerevisiae. YNLO22c is one of three ORFs identified in S. cerevisiae that have homology with both known and putative RNA m5C methyltransferases, but its encoded protein, YNLO22p, has not been confirmed to have enzyme activity. Verifying that YNLO22c encodes an RNA m5C methyltransferase will require adequate amounts of soluble YNLO22p for enzyme assays. A bacterial expression plasmid for YNLO22c was developed, but the result was insoluble protein. Therefore, several methods known to improve protein solubility were tested to develop a system in which a sufficient amount of soluble YNLO22p could be produced. Results of this study found that coexpression of YNLO22c with chaperone proteins can provide sufficient quantities of soluble YNLO22p.Department of Biology
27
Scholz, Kai. "Immunmodulation durch Parapocken-Viren: Identifikation und Analyse funktionaler Viruskomponenten". Doctoral thesis, Saechsische Landesbibliothek- Staats- und Universitaetsbibliothek Dresden, 2003. http://nbn-resolving.de/urn:nbn:de:swb:14-1061969873968-07930.
Abstract (sommario):
Fusionspeptid-, Redox-, Viruscore- und sonstige Proteine. Alle analysierten Single ORF (SO)-VVOV Rekombinanten vermittelten einen signifikanten Schutz vor einer tödlichen Belastung mit Aujeszky-Virus. Zwei der Rekombinanten (SO 93-, SO 94-VVOV) enthalten ORFs, die für ATI/Fusionspeptid-Proteine kodieren. In SO 19- und SO 70-VVOV sind dagegen für Redoxproteine kodierende ORFs integriert. Weiterführende Untersuchungen zeigten, dass SO 94- und SO 19-VVOV in zwei weiteren Modellsystemen immunstimulatorisch aktiv sind. Im Baculo-Virussystem exprimierte Proteine waren nur in Kombination mit Vaccinia Lister-Virus (VV) wirksam. Dabei zeigten jeweils Virus-Protein-Gemische mit dem geringsten Proteinanteil den stärksten immunstimulatorischen Effekt. Proben in denen VV durch bovines Herpes-Virus-1 ersetzt wurde, sind dagegen nicht wirksam. Dies lässt auf eine Beteiligung VV-spezifischer Faktoren schließen. Übereinstimmend mit diesen Ergebnissen führte eine Frameshift-Mutation in ORF 94r von SO 94mut-VVOV nur zur Abschwächung und nicht zum vollständigen Verlust der immunstimulatorischen Wirkung. Beide in Schizosaccharomyces pombe exprimierten Proteine, sp-ORF19 und sp-ORF94r, induzierten keinen signifikanten Schutz im Aujeszky Maus Modell. Mit der Identifikation einzelner immunstimulatorisch aktiver PPVO-Komponenten ist es erstmals gelungen, den paramunisierenden Effekt von Parapox-Viren einzelnen viralen Genen zu zuordnen. Insbesondere stellen SO 94- und SO 19-VVOV viel versprechende Kandidaten für die prophylaktische bzw. therapeutische Anwendung in verschiedenen Indikationen als auch für weitere Untersuchungen des Wirkmechanismus dar.
28
Key, Kijona Farthing. "Molecular characterization of the major envelope protein of porcine reproductive and respiratory syndrome virus (PRRSV) and evaluation of its use for a diagnostic assay, vaccine development, and the examination of quasispecies evolution". Diss., Virginia Tech, 2007. http://hdl.handle.net/10919/27282.
Abstract (sommario):
Porcine reproductive and respiratory syndrome (PRRS) is a viral disease that has devastated the global swine industry since the mid 1980s. Although modified live vaccines (MLVs) are typically used for the prevention of clinical disease, they are not always fully effective. Additionally, acute PRRS outbreaks, characterized by more severe clinical signs, have appeared in herds that were previously vaccinated. In this dissertation, we further analyzed the pathogenesis of PRRSV through genetic characterization, assay development, and quasispecies evaluation using the PRRSV ORF5 gene while also attempting to develop an improved PRRS vaccine.
To explore the possible mechanism for the emergence of acute PRRS, the open reading frame 5 (ORF5) gene encoding the major envelope protein (GP5) of acute PRRSV isolates was characterized. Sequence and phylogenetic analyses revealed that seven of the acute PRRS virus (PRRSV) isolates were related to other N. American PRRSV isolates while one isolate, 98-37120-2, was very closely related to and may have been derived from the MLV, RespPRRS. We also developed a heteroduplex mobility assay (HMA) for quickly identifying PRRSV field isolates with significant nucleotide sequence identities (â d98%) with the MLVs based on the amplification, denaturation, and reannealing of the ORF5 gene of the field isolates with those of MLV reference strains. All of the field isolates that were highly related to RespPRRS (â T2% nucleotide sequence divergence) were identified by the HMA to form homoduplexes with the reference RespPRRS MLV.
We also developed a unique strategy for infecting pigs with PRRSV, known as in vivo transfection, by bypassing the traditional in vitro cell culture step required for in vivo studies. We demonstrated that inoculation of RNA transcripts of a PRRSV infectious cDNA clone directly into the lymph nodes and tonsils of pigs produces active PRRSV infection. Using this method, we also examined the quasispecies populations of PRRSV. Finally, we evaluated the ability of Salmonella choleraesuis to express the PRRSV GP5, and tested its immunogenicity in mice. Based on our data, there was no indication of Salmonella replication in the mice or any evidence of antibody production against S. choleraesuis or PRRSV GP5.Ph. D.
29
Chi, Xiuling. "BIOSYNTHETIC PATHWAY OF THE AMINORIBOSYL COMPONENT OF LIPOPEPTIDYL NUCLEOSIDE ANTIBIOTICS". UKnowledge, 2013. http://uknowledge.uky.edu/pharmacy_etds/23.
Abstract (sommario):
Several lipopeptidyl nucleoside antibiotics that inhibit bacterial translocase I (MraY) involved in peptidoglycan cell wall biosynthesis contain an aminoribosyl moiety, an unusual sugar appendage in natural products. A-90289 and muraminomicin are the two representative antibiotics that belong to this family. Bioinformatic analysis of the biosynthetic A-90289 gene clusters revealed that five enzymes are likely involved in the assembly and attachment of the aminoribosyl unit. These enzymes of A-90289 are functionally assigned by in vitro characterization. The results reveal a unique ribosylation pathway that highlighted by uridine-5′-monophosphate as the source of the sugar, a phosphorylase strategy to generate a sugar-1-phosphate, and a primary amine-requiring nucleotidylyltransferase that generates the NDP-sugar donor. Muraminomicin, which has a structure similar to A-90289, holds the distinction in that both ribose units are 2-deoxy sugars. The biosynthetic gene cluster of muraminomicin has been identified, cloned and sequenced, and bioinformatic analysis revealed a minimum of 24 open reading frames putatively involved in the biosynthesis, resistance, and regulation of muraminomicin. Similar to the A-90289 pathway, fives enzymes are still likely involved in the assembly of the 2,5-dideoxy-5-aminoribose saccharide unit, and two are now functionally assigned and characterized: Mra20, a 5′-amino-2′,5′-dideoxyuridine phosphorylase and Mra23, a UTP:5-amino-2,5-dideoxy-α-D-ribose-1-phosphate uridylyltransferase. The cumulative results are consistent with the incorporation of the ribosyl appendage of muraminomicin via the archetypical sugar biosynthetic pathway that parallels A-90289 biosynthesis
30
Huang, Yu-Liang, e 黃有良. "Immuno-protection of porcine circovirus type 2 ORF1 and ORF2 recombinant protein". Thesis, 2004. http://ndltd.ncl.edu.tw/handle/12062489232254298987.
Abstract (sommario):
碩士國立屏東科技大學
獸醫學系
92
Porcine circovirus type 2 (PCV2) has been reported to be associated with the development of porcine multisystemic wasting syndrome (PMWS), which often results in the death of pigs due to secondary infection. The genome of PCV2 might contain eleven open reading frames (ORFs). ORF 1 and ORF 2 encode the replication-associated proteins and capsid protein, respectively. In this study, the immunogenicity of E. coli-expressed PCV2 ORF 1 and PCV2 ORF 2 and baculovirus-expressed PCV2 ORF 2 were compared in mice and pigs. In addition, ORF 2 gene with Pseudomonas aeruginosa exotoxin A (expressed as PE407 and PE532) gene added at 5’ terminal, and/or the KDEL amino acids gene (expressed as K) added at 3’ terminal were constructed and expressed in E. coli. A total of seven recombinant proteins including PCV2 ORF1 (21 KDa), PCV2 ORF 2 (54 KDa), PCV2 ORF2K (56 KDa), PE407-ORF2 (81 KDa), PE407-ORF2K (100 KDa), PE532-ORF2 (95 KDa) and PE532-ORF2K (97 KDa) were successfully expressed in E. coli. A recombinant protein of 34 KDa (Bac-PCV2ORF2) was also expressed in baculovirus. Antibodies against PCV2 could be detected when pigs or mice were immunized with PCV2ORF1, PCV2ORF2, PE407-ORF2K and Bac-PCV2ORF2. Among the recombinant proteins tested, Bac-PCV2ORF2 induces the best antibody titer in animals. The aforementioned recombinant proteins can be used as the components for the development of PCV2 subunit vaccine in the future.
31
Chen, Yu-San, e 陳裕森. "Cloning, Expression, and Antigenesity Analysis of the PCV2 Coat Protein Gene (ORF2)". Thesis, 2006. http://ndltd.ncl.edu.tw/handle/99558729710906776501.
Abstract (sommario):
碩士國立宜蘭大學
生物技術研究所碩士班
94
Porcine circovirus type 2 belongs to the Circoviridae family. PCV2 is a small, nonenveloped virus with a single-stranded circular DNA genome of about 1.76 kb. The open reading frame ORF2 of the PCV2 genome encodes the immunogenic structural capsid protein. The expression for this immunogenic protein using full length ORF2 DNA was difficult to obtain in large quantity in prokaryotic expression system. Therefore, an alternative approach was to divide ORF2 DNA into three segments for expression, F1, F2, and F3. It was discovered that expression in E.coli was obtained in segments other than F1, which contained the N-terminal sequences. When F1+F2 (named F6) and F2+F3 (named F5) fragments were cloned and expressed, F6, similar to F1, was not expressed due to the N-terminal sequences. On the other hand, F5 was still expressed in large quantity. When dividing the full length ORF2 into half, F4 with C-terminal sequences was again expressed in large quantity as predicted. Analysis on PCV2 ORF2 full length sequence showed that within F1, there were 21 arginine codons, far more than the 9 arginine codons in F2 and F3 combined. Therefore, five additional constructs with the first 9, 21, 36, 48, 111 nucleotides missing of ORF2 (named F10, F22, F37, F49, and F112 respectively) were cloned and subsequently expressed. It was discovered that PCV2 ORF2 recombinant protein expression increased as arginine codons decreased. When immunizing rat with purified F5 recombinant protein, high antibody titer against PCV2 was induced, as confirmed by the IPMA method in PCV2 infected PK-15 cells. Currently, since PCV2 vaccine is not commercially available, this research project was also aimed for the development of a PCV2 subunit vaccine. Rats were used as an experimental animal model to evaluate the various recombinant proteins, F2, F3, F5, and F49, for their abilities to induce antibodies against PCV2. In addition, F5 and F49 recombinant proteins, in conjunction with ORF1 and/or ORF2 recombinant eukaryotic expression plasmids, were injected in rats to evaluate their abilities to induce antibodies against PCV2. Other than using IPMA for the detection of neutralizing antibodies, I used ELISA to measure the levels of antibodies induced in the collected rat serum of different experimental groups. The result of ELISA indicated that F2 recombinant protein induced the highest level of IgG antibodies in rat. As for neutralizing antibodies, neither the recombinant proteins nor when used in conjunction with recombinant eukaryotic expression plasmids were induced when using rat as an animal model.
32
"Analysis Of The Line-1 Orf2 Protein Using An Evolutionarily-informed Genetic Approach". Tulane University Digital Library, 2016.
Abstract (sommario):
Long Interspersed Element 1(LINE1 or L1), along with the parasitic Short Interspersed Element (SINE), are the only two currently active retrotransposons in the human genome. These genetic elements are capable of causing DNA damage through their mobilization and the enzymatic activities of the L1-encoded Open Reading Frame 2 (ORF2) protein (ORF2p). The L1 ORF2p contains four annotated domains important to retrotransposition. These include the endonuclease (EN) and reverse transcriptase (RT) domains, as well as the Z domain and the cysteine rich domain (Cys). While much is known about the enzymatic activities of the EN and RT domains, and individual amino acids important to retrotransposition have been identified in the Z and Cys domains, more than 50% of the 150kDa ORF2p amino acid sequence serves no known function. I hypothesized that the unannotated areas of the ORF2p, specifically the sequence C-terminal to the EN domain and N-terminal to the Z domain as well as the sequence C-terminal to the Cys domain, contained amino acids important to the retrotransposition process. Specifically, I hypothesized that they contained amino acids involved in the activity of the EN domain, RT domain, or interaction with the L1 ORF1p. To test this hypothesis, I developed a technique termed Bipartile Alu Retrotransposition (BAR) that utilized EN and RT-containing ORF2 fragments combined with the Alu retrotransposition reporter construct. This system allowed me to define a new ORF2p region, which I termed Cryptic. This region contains an essential WD pair important for cDNA syntheses by the ORF2p. This WD pair is also involved in the regulation of the EN domain activity. I also discovered a putative PCNA binding domain that is essential for retrotransposition. Additionally, I identified the region in Cryptic that is involved in the previously reported differences in subcellular localization and cytotoxic potential of EN-containing ORF2p fragments. Using truncated ORF2p fragments generated for use in BAR, I also made several discoveries concerning the extreme C-terminus of the ORF2p. Notably, I discovered that the extreme C-terminal end of the ORF2p is dispensable for retrotransposition. I also identified a human-specific Y residue that is important for Alu retrotransposition driven by the ORF2p.1
Claiborne Magnant Christian
33
Chowdhury, Soumya Roy. "Molecular Characterization Of Movement Protein Encoded By ORF-1 Of Sesbania Mosaic Virus (SeMV)". Thesis, 2010. https://etd.iisc.ac.in/handle/2005/1465.
34
Chowdhury, Soumya Roy. "Molecular Characterization Of Movement Protein Encoded By ORF-1 Of Sesbania Mosaic Virus (SeMV)". Thesis, 2010. http://hdl.handle.net/2005/1465.
35
Tseng, Yeu-Yang, e 曾宇揚. "Functional analysis of orf virus OV20.0 protein isoforms". Thesis, 2015. http://ndltd.ncl.edu.tw/handle/19898262310985605669.
Abstract (sommario):
碩士國立中興大學
微生物暨公共衛生學研究所
103
Orf virus (ORFV) infection causes pustule and ulceration around muzzle of small ruminants. Although it often occurs with low mobility and mortality, orf may be fatal in juvenile hosts. ORFV, belonging to genus parapoxvirus of the family Poxviridae, is an enveloped virus with double-stranded DNA genome. OV20.0 protein is produced from OV20.0L gene, an E3L ortholog, which is conserved in the genome of most members in Poxviridae. Vaccinia E3 (VV E3) has been studied extensively in these days; however sequences of VV E3 shares low identity with those of OV20.0. According to previous publication, OV20.0L could be translated into to two isoforms, full-length OV20.0 and N-terminal truncated one (sh20). Due to limited information of OV20.0 protein, our research focused on the translation mechanism of the isoform, comparative analysis of their cellular distribution and realizing their functions as well as their contribution to pathogenicity in mice model. First, we proved sh20 was translated from the third start codon of OV20.0L gene. In in vitro and in virus infection condition, both ORFV 20.0 and sh20 can hold the ability to bind double-stranded RNA (dsRNA), sequester the substrate of dsRNA-dependent protein kinase (PKR) that in turns inhibits the activity of downstream factor, eukaryotic initiation factor 2 (eIF2α), and influences cytokine releasing in different levels. Moreover, constructing recombinant virus and animal experiments clarify the role ORFV OV20.0 played in vivo. Although full-length OV20.0 and sh20 shared most functions in vitro, the ORFV recombinant virus which only expressed sh20, was attenuated in vivo. This data implied N-terminus of OV20.0 was required to induce intact pathogenicity in live animals.
36
Cilloniz, Cristian. "Role of the varicella zoster virus ORF9 protein in viral replication". 2007. http://proquest.umi.com/pqdweb?did=1331418241&sid=10&Fmt=2&clientId=39334&RQT=309&VName=PQD.
Abstract (sommario):
Thesis (Ph.D.)--State University of New York at Buffalo, 2007.Title from PDF title page (viewed on Nov. 27, 2007) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Ruyechan, William T. Includes bibliographical references.
37
Huang, Yu-Yuan, e 黃俞淵. "Characterization of SARS-CoV encoded accessory proteins ORF8a and ORF8". Thesis, 2009. http://ndltd.ncl.edu.tw/handle/74372663873642985655.
Abstract (sommario):
碩士國立陽明大學
生物藥學研究所
97
SARS-CoV, a coronavirus, was identified as the etiologic agent for severe acute respiratory syndrome. It is established that human SARS-CoV originated from animals, such as palm civets and raccoon dogs. SARS-CoV genome shares about 99.8% homology with its animal counterpart. The major difference of the two viruses is the 29 nucleotide deletion found in the corresponding open reading frame (ORF) 8 of human SARS-CoV, given rise to ORF8a and ORF8b. Our previous data had shown that ORF8a localizes to mitochondria. Using yeast two-hybrid screening, ORF8a, but not ORF8 and ORF8b, was shown to interact with calcium-modulating cyclophilin ligand (CAML), a protein had been known to regulate the intracellular Ca2+ concentration. Using Hela cell Tet/on system, ORF8a can promote the apoptosis initiated by staurosporine. The main goal of this dissertation is to determine the biological functions of ORF8 and ORF8a that might give better understandings to the pathogenesis of both viruses. With immunofluorescence assay and subcellular fractionation, ORF8a and ORF8 are shown to be located in mitochondria and ER, separately, in the presence of endogenous CAML. However, when we ectopically expressed CAML, both ORF 8a and ORF8 co-localized with exogenous CAML and displayed a similar pattern of distribution, which differed from that in the presence of endogenous CAML. ORF8a was shown to have higher binding capacity for CAML than that of ORF8 in an immunoprecipitation assay, which likely contributed to the redistribution of ORF8a. In light of reports indicating that in SARS patient lung cells showed readily apoptosis than that of enterocytes, although both tissues are permissive for viral infection and growth. We proceeded to evaluate the apoptotic effect of either ORF8a or ORR8 on an alveolar epithelium cell, A549 and an enterocyte, Caco-2. A549 was showed to have DNA fragementation in the presence of ORF8a or ORF8 by propidium iodide staining, while both proteins also facilitated staurosporine-induced apoptosis using TMRM staining assay that targeting at changes in mitochondrial membrane potential. A549 was also showed low grade of apoptosis in the DMSO control group in the presence of ORF8a or ORF8, which coincided with the observed induction of DNA fragmentation by both proteins. Interestingly, Caco-2 with ectopically expressed ORF8a or ORF8 failed to exhibit apoptotic phenotype enhancement regardless of the presence or absence of staurosporine. Currently, the contribution of interaction between CAML and ORF8a or even ORF8 in apoptosis remains unclear. In the future, we will re-examine the above stated observations in both A549 and Caco-2 cells with CAML knockdown for the evaluation of the contribution of the interactions between CAML and ORF8a or CAML and ORF8.
38
Li, ChiWei, e 李誌偉. "A novel mechanism to regulate IS1550 putative transposase production by ORF1 protein". Thesis, 2002. http://ndltd.ncl.edu.tw/handle/22403527173198471760.
Abstract (sommario):
碩士國立陽明大學
醫學生物技術研究所
90
We have previously identified a transposable element, IS1550 from Mycoplasma fermentans. This element encodes two open reading frames, ORF1 and ORF2. Two proteins were detected with the molecular weight of 16 kDa and 50 kDa, respectively, when expression of the IS1550 genes was examined in E. coli T7 expression system. The 16-kDa protein was determined to be encoded by ORF1, whereas the 50-kDa protein was produced by forming a fusion of ORF1 and ORF2, ORF12’, through an efficient —1 translational frameshift mechanism. Our previous studies have confirmed a framseshift signal in IS1550. It contains an essential shifty site AAAAAAG (A6G) which is located near the 3’ end of ORF1, and an enhancing element, the internal inverted repeat (IIR) to form a RNA stem-loop structure, which is located 5 bp downstream of the essential shifty site. In this study, we examined the effect of ORF1 protein on the -1 translational frameshift product by using our previously established dual-reporter gene (lacZ and luc) system. The results showed that ORF1 protein could repress the -1 frameshift product both in vivo and in vitro with an inhibitory effect of about 60 % and 80 %, respectively. We also showed the ORF1 protein could bind to the frameshift signal DNA region. Furthermore, we proved that a truncated mRNA was formed when the ORF1 protein was present. Thus, we proposed that the repression of ORF1 protein on the -1 frameshift product was possibly caused by ORF1 protein binding to the frameshift signal DNA region, blocking the RNA polymerase proceeding, and resulting in the decreasing of the fusion protein (a putative transposase) production.
39
Wu, Ting-Yun, e 吳亭昀. "Monoclonal Antibody Preparation and Cellular Distribution Analysis of Koi Herpesvirus ORF72 Protein". Thesis, 2018. http://ndltd.ncl.edu.tw/handle/8a7b5r.
40
Ou, Chi-Ming, e 歐啟明. "Expression and Purification of the Major Envelope Protein of Orf Virus". Thesis, 2007. http://ndltd.ncl.edu.tw/handle/50383988977441905067.
Abstract (sommario):
碩士中興大學
獸醫學系暨研究所
95
Abstract Orf is also known as contagious ecthyma, contagiou pustular dermatitis, Scabby mouth and sore mouth. Orf occurs worldwide in sheep and goats, especially in young animal. The disease is characterized by proliferative lesion on the lips, around the nostrils and in the oral mucosa. Orf virus is a member of the Parapoxvirus genus of Poxviridae. The virus particles are ovoid and the viral genome is a linear double-stranded DNA. The viral B2L gene encodes for the highly immunogenic major envelope protein , which is the homologue of vaccinia virus envelope protein antigen p37 . We amplified the B2L gene by polymerase chain reaction (PCR) and cloned the gene into the expression vector pET24a. The plasmid was transformed into the E. coli. strain BL21(DE3) and the expression of recombinant B2L protein was induced by IPTG. The recombinant B2L protein was identified with western blotting and with mass spectrometry analysis. Then, the B2L protein was purified by affinity chromatography. Moreover, we made four truncated constructs to express shorter B2L proteins; although two of these deletion mutants were also expressed, they showed lower level of production. The vaccinia virus envelope protein p37 has phospholipase activity; therefore it has been proposed that the B2L protein has phospholipase function. However, we were not able to detect the phospholipase activity of our purified recombinant protein.
41
LEE, Tzu-Ying, e 李姿穎. "Molecular Mechanism of the type I interferon antagonist function by SARS Coronavirus ORF6 protein". Thesis, 2008. http://ndltd.ncl.edu.tw/handle/75653793395898183769.
Abstract (sommario):
碩士中國醫藥大學
醫學檢驗生物技術學系
96
Abstract Severe acute respiratory syndrome (SARS) is a newly emerged infectious disease. The causative agent of SARS has been identified to be a new type of coronavirus, namely SARS coronavirus (SARS-CoV).SARS-CoV proteins including the ORF6 protein have been reported to inhibit interferon signaling response. The goal of this study is to identify human SARS-CoV ORF6-interacting proteins using the phage displayed human lung cDNA libraries and to investigate their interaction on the type I interferon antagonist function. At first, the recombinant ORF6 protein was generated in BL21(DE3) and purified by using IMAC (immobilized mental-affinity chromatography). After five rounds of biopanning, PI-3 kinase-related kinase SMG-1 protein was identified as SARS-CoV ORF6 interacting protein from phage displayed lung cDNA library. Confocal imaging revealed co-localization of SARS-CoV ORF6 protein with SMG-1 protein in HL-CZ cells. In vivo signaling pathway assay and real time RT-PCR showed that single gene expression of SARS-CoV ORF6-expressing cells blocked the INFα/β-induced responses, such as ISRE-responsive firefly luciferase activity and the expression of PKR. However, both gene expression of SARS-CoV ORF6 and SMG-1 had a significantly higher relative activities of INFα/β-induced ISRE-responsive firefly luciferase than single gene expression of SARS-CoV ORF6 in HL-CZ cells. In addition, confocal imaging and Western blotting assays revealed that the translocation of STAT-1 into nucleus and the STAT-1 phophorylation were found in the transfected cells expressing both genes of ORF6 and SMG-1, but not in the ORF 6-expressing cells in response to INFα/β. Therefore, cell expression of PI-3 kinase-related kinase SMG-1 could restore the INFα /β responses in SARS-CoV ORF6 expressing cells. The interaction of SARS CoV ORF6 and SMG-1 may be responsible for the inhibition of type I IFN response by SARS-CoV.
42
Chao, I.-Chi, e 趙譯棋. "Establishment of Recombinant Orf Virus Expressing Goatpox virus P32 Proteins". Thesis, 2015. http://ndltd.ncl.edu.tw/handle/30630163851365322611.
Abstract (sommario):
碩士國立中興大學
微生物暨公共衛生學研究所
103
Orf virus (ORFV), belonging to parapoxvirus, causes skin lesions in infected sheep and goats. Due to the restricted host range, the skin tropism, the absence of systemic virus spread, and the short-lived ORFV vector-specific immunity allowing repeated immunizations, it has been successfully used as a novel viral vector system for expressing foreign antigens to prevent infectious diseases of swine, rabbit, and avian. Hence, study herein aimed to establish ORFV as a bivalent vaccine platform for goat. In 2008, goatpox (GP) outbreaks caused substantial loss in the production and productivity of goats in Taiwan. Considering that control of goatpox virus (GPV) infection relies on foreign vaccine, the goal of current study is to construct a recombinant ORFV that would express goatpox virus P32, the major immunogenic protein. To do so, coding regions of GPV P32 and eGFP that serves as a selection marker were inserted between the flanking sequences, i.e. open reading frame 127 and 128 of the ORFV genome. By homologous recombination, recombinant ORFV with GPV p32 gene (GPV P32-ORFV) was generated. At the present, the presence and expression of GPV P32 gene were confirmed by polymerase chain reaction and western blot analysis, respectively. The immune characteristics of GPV P32-ORFV will be further tested in animal models.
43
Chorghade, Sandip Gulab. "Studies on Interactions between ARE Binding Proteins and Splicing Factors and their Role in Altered Splicing of PDGF-B ORF". Thesis, 2012. http://etd.iisc.ac.in/handle/2005/3227.
Abstract (sommario):
Pre-mRNA splicing is an important level in posttranscriptional gene regulation that is essential for accurate protein synthesis and generating protein diversity. The abundance of cryptic splice sites and long intronic DNA sequences makes their splicing a complex one. The identification of correct exons and introns needs additional information in the form of splicing regulatory elements (SREs) along with canonical splice signals. The interplay among these SREs and the trans factors (which bind to SREs) gives the identity to introns and exons which in turn leads to precise pre-mRNA splicing.
Previous studies from our laboratory showed, that when expressed in mammalian cells from an expression vector, PDGF-B ORF was re-spliced at 4/5 exon junction with the downstream SV40 splice acceptor site in the vector. However, deletion of the 66-nt PDGF-B 3’ UTR region resulted in about 25% reduction in re-splicing. Sequence analysis of this region revealed presence of binding sites for splicing factors ASF/SF2 and SRp55, and an AU-rich element (ARE), mutation each of which affected re-splicing partially. In mammals, AREs are commonly found in the 3’UTR of mRNAs encoding proteins involved in diverse functions and are involved in selective mRNA degradation. Several ARE binding proteins are crucial for ARE’s function. Since mutation of the single ARE in the 3’UTR region altered the re-splicing efficiency, the role of AU-rich elements and ARE-binding proteins (AU-BPs) in modulation of splicing was investigated using siRNAs against AU-BPs, BRF1, hnRNPD, HuR, GAPDH and TTP. Down regulation of expression of these factors indeed affected the level of re-spliced product.
We have studied the interactions between the full-length splicing factors (U1-70K and U2AF35) and the AU-BPs (BRF1, hnRNPD and HuR) as well as among the AU-BPs using three different assay methods: Yeast-two hybrid, co-immunoprecipitation and pull down assays. Our study has revealed that the BRF1 interacts with U1-70K and U2AF35 as well as the other AU-BPs hnRNPD and HuR but with different affinities. We have also analyzed the ability of AU-BPs to interact with SR proteins SRp20 and 9G8. We did find strong interaction of BRF1 with SRp20 and 9G8.
Generation of a large number of nested deletion mutants of all the proteins allowed us to identify the interaction regions on the surface of BRF1, U1-70K, hnRNPD, U2AF35 and HuR. The results of Y2H analyses were further confirmed by pull down assay using purified interacting regions.
It was found that a single region from aa 181-254 in BRF1 interacts with multiple partners i.e., splicing factors and the AU-BP hnRNPD. However, the RNA-binding zinc-finger domain from residue 120-181 independently interacts with HuR. Further, the multiple protein interacting region (MPIR) (aa 181-254) in BRF1 exhibits different affinities towards its interacting partners with that for U1-70K and hnRNPD being stronger than that for U2AF35 and HuR. This observation suggests that BRF1 activity can be modulated by interaction with different partners at different sites.
U1-70K interacted only with BRF1 among the proteins tested in this study and this interaction appears to be RNA independent .This could have implications in splice site selection and RNA stability since BRF1 has been shown to promote RNA degradation. While the Arg/Glu-rich C-terminal region in U1-70K is sufficient for its interaction with BRF1, U2AF35 requires both the zinc-finger 2 and the arg/Gly/Ser-rich C-terminal regions for its association with BRF1.
hnRNPD also interacts with multiple partners that include BRF1, HuR and U2AF35 using the N-terminal region that harbors a Ala-rich domain. The interaction of hnRNPD with HuR is RNA dependent while with BRF1 and U2AF35, it is RNA independentt. Further, its interaction with all the partners is equally strong. This suggests that hnRNPD could exert differential influence depending on the context of its interaction and abundance of the interacting partner.
HuR, primarily known as an mRNA stabilizing factor, interacts with both BRF1 and hnRNPD with equal affinity involving the hinge region, the interaction with the former being RNA-independent and the later being RNA-dependent. This differential RNA-dependent and independent interactions with the two AU-BPs using a single interacting domain suggests a balancing act of HuR on the activities of BRF1 and hnRNPD. These interactions can further be differentially modulated by posttranslational modifications on one or all of the interacting partners depending on the physiological status of the cell.
We have also analyzed the multiple protein complexes formed in absence of cellular RNA. Though we are unable to see direct protein-protein interaction between HuR and U1-70K in Yeast two hybrid analysis, we could detect the presence of U1-70K in HuR immunoprecipitate. It appears that U1-70K associates with HuR via BRF. We also detected the presence of HuR in U1-70K complexes which could be due to its association with BRF1. We are unable to find hnRNPD and U2AF35 in these complexes indicating that they may have been excluded. In anti-U2AF35 immunoprecipitates, we detected the presence of U1-70K as well as hnRNPD but no HuR. This may be due to RNase treatment as hnRNPD and HuR interactions are RNA dependent.
Our findings that AU-rich elements in conjunction with AU-BPs function as intronic splicing modulators or enhancers, reveal hitherto unidentified new players in the poorly understood complex mechanisms that mediate alternative splicing. The possibility of dynamic nature of the interactions among splicing factors and AU-BPs mediated by post-translational modifications provide a basis for rapid cellular responses to changing environmental cues through generation of differentially spliced mRNAs and corresponding protein products that differ in their stability and hence their relative abundance. Our results also unfold enormous possibilities for future investigations on interactions among the many splicing factors and AU-BPs, and in understanding these complex interactions in modulation of pre-mRNA splicing, mRNA translation and degradation. The finding of coupling of AU-BPs to splicing machinery could further lead to better understanding of the mechanism of AU-BP-mediated targeting of mRNAs to processing bodies and ultimate degradation of the mRNAs.
44
Chorghade, Sandip Gulab. "Studies on Interactions between ARE Binding Proteins and Splicing Factors and their Role in Altered Splicing of PDGF-B ORF". Thesis, 2012. http://hdl.handle.net/2005/3227.
Abstract (sommario):
Pre-mRNA splicing is an important level in posttranscriptional gene regulation that is essential for accurate protein synthesis and generating protein diversity. The abundance of cryptic splice sites and long intronic DNA sequences makes their splicing a complex one. The identification of correct exons and introns needs additional information in the form of splicing regulatory elements (SREs) along with canonical splice signals. The interplay among these SREs and the trans factors (which bind to SREs) gives the identity to introns and exons which in turn leads to precise pre-mRNA splicing.
Previous studies from our laboratory showed, that when expressed in mammalian cells from an expression vector, PDGF-B ORF was re-spliced at 4/5 exon junction with the downstream SV40 splice acceptor site in the vector. However, deletion of the 66-nt PDGF-B 3’ UTR region resulted in about 25% reduction in re-splicing. Sequence analysis of this region revealed presence of binding sites for splicing factors ASF/SF2 and SRp55, and an AU-rich element (ARE), mutation each of which affected re-splicing partially. In mammals, AREs are commonly found in the 3’UTR of mRNAs encoding proteins involved in diverse functions and are involved in selective mRNA degradation. Several ARE binding proteins are crucial for ARE’s function. Since mutation of the single ARE in the 3’UTR region altered the re-splicing efficiency, the role of AU-rich elements and ARE-binding proteins (AU-BPs) in modulation of splicing was investigated using siRNAs against AU-BPs, BRF1, hnRNPD, HuR, GAPDH and TTP. Down regulation of expression of these factors indeed affected the level of re-spliced product.
We have studied the interactions between the full-length splicing factors (U1-70K and U2AF35) and the AU-BPs (BRF1, hnRNPD and HuR) as well as among the AU-BPs using three different assay methods: Yeast-two hybrid, co-immunoprecipitation and pull down assays. Our study has revealed that the BRF1 interacts with U1-70K and U2AF35 as well as the other AU-BPs hnRNPD and HuR but with different affinities. We have also analyzed the ability of AU-BPs to interact with SR proteins SRp20 and 9G8. We did find strong interaction of BRF1 with SRp20 and 9G8.
Generation of a large number of nested deletion mutants of all the proteins allowed us to identify the interaction regions on the surface of BRF1, U1-70K, hnRNPD, U2AF35 and HuR. The results of Y2H analyses were further confirmed by pull down assay using purified interacting regions.
It was found that a single region from aa 181-254 in BRF1 interacts with multiple partners i.e., splicing factors and the AU-BP hnRNPD. However, the RNA-binding zinc-finger domain from residue 120-181 independently interacts with HuR. Further, the multiple protein interacting region (MPIR) (aa 181-254) in BRF1 exhibits different affinities towards its interacting partners with that for U1-70K and hnRNPD being stronger than that for U2AF35 and HuR. This observation suggests that BRF1 activity can be modulated by interaction with different partners at different sites.
U1-70K interacted only with BRF1 among the proteins tested in this study and this interaction appears to be RNA independent .This could have implications in splice site selection and RNA stability since BRF1 has been shown to promote RNA degradation. While the Arg/Glu-rich C-terminal region in U1-70K is sufficient for its interaction with BRF1, U2AF35 requires both the zinc-finger 2 and the arg/Gly/Ser-rich C-terminal regions for its association with BRF1.
hnRNPD also interacts with multiple partners that include BRF1, HuR and U2AF35 using the N-terminal region that harbors a Ala-rich domain. The interaction of hnRNPD with HuR is RNA dependent while with BRF1 and U2AF35, it is RNA independentt. Further, its interaction with all the partners is equally strong. This suggests that hnRNPD could exert differential influence depending on the context of its interaction and abundance of the interacting partner.
HuR, primarily known as an mRNA stabilizing factor, interacts with both BRF1 and hnRNPD with equal affinity involving the hinge region, the interaction with the former being RNA-independent and the later being RNA-dependent. This differential RNA-dependent and independent interactions with the two AU-BPs using a single interacting domain suggests a balancing act of HuR on the activities of BRF1 and hnRNPD. These interactions can further be differentially modulated by posttranslational modifications on one or all of the interacting partners depending on the physiological status of the cell.
We have also analyzed the multiple protein complexes formed in absence of cellular RNA. Though we are unable to see direct protein-protein interaction between HuR and U1-70K in Yeast two hybrid analysis, we could detect the presence of U1-70K in HuR immunoprecipitate. It appears that U1-70K associates with HuR via BRF. We also detected the presence of HuR in U1-70K complexes which could be due to its association with BRF1. We are unable to find hnRNPD and U2AF35 in these complexes indicating that they may have been excluded. In anti-U2AF35 immunoprecipitates, we detected the presence of U1-70K as well as hnRNPD but no HuR. This may be due to RNase treatment as hnRNPD and HuR interactions are RNA dependent.
Our findings that AU-rich elements in conjunction with AU-BPs function as intronic splicing modulators or enhancers, reveal hitherto unidentified new players in the poorly understood complex mechanisms that mediate alternative splicing. The possibility of dynamic nature of the interactions among splicing factors and AU-BPs mediated by post-translational modifications provide a basis for rapid cellular responses to changing environmental cues through generation of differentially spliced mRNAs and corresponding protein products that differ in their stability and hence their relative abundance. Our results also unfold enormous possibilities for future investigations on interactions among the many splicing factors and AU-BPs, and in understanding these complex interactions in modulation of pre-mRNA splicing, mRNA translation and degradation. The finding of coupling of AU-BPs to splicing machinery could further lead to better understanding of the mechanism of AU-BP-mediated targeting of mRNAs to processing bodies and ultimate degradation of the mRNAs.
45
Lin, Wei Li, e 林維莉. "Expression and functional analysis of the porcine circovirus type 2 Rep and ORF3 proteins". Thesis, 2011. http://ndltd.ncl.edu.tw/handle/04865829915580224077.
Abstract (sommario):
博士國立中興大學
微生物暨公共衛生學研究所
100
Porcine circovirus type 2 (PCV2) is a member belongs to the genus Circovirus of the Circoviridae family, and closely associated with a disease syndrome in pigs described as “postweaning multisystemic wasting syndrome” (PMWS) now known as porcine circovirus diseases (PCVD). The genome of PCV2 is a single-stranded circular DNA, and contains three major open reading frames (ORFs). They are ORF1, the rep gene, which encodes the Rep proteins responsible for virus replication; and ORF2, the cap gene, which encodes the immunogenic capsid (Cap) protein; and ORF3 encodes the protein for induce cell apoptosis. Synthesis of Rep protein as the first stage after PCV2 infection, is essential to viral DNA replication. The non-pathogenic type 1 PCV (PCV1) Rep protein has been shown to process the nuclear localization signal (NLS) for protein transport into nucleus and has DNA binding activity for viral DNA replication. To characterize the DNA binding potential and the significant region that confers the nuclear localization of the PCV2 Rep protein, the defined coding regions of rep gene were cloned, expressed and analyzed the DNA binding activity. The results demonstrated that the full-length Rep (N314) and the deletion mutants coding the N-terminal 110 amino acid residues had the ability to bind to the 22-mer dsDNA fragment of the PCV2 origin sequence minimal binding site (ori MBS) sequence. Furthermore, to determine the region responsible for nuclear localization, several eukaryotic expression plasmids containing various coding regions of Rep protein identical to those expressed in E. coli were constructed. At 48 h post-transfection of plasmids into PK15 cells, cell were fixed, analyzed by indirect immunofluorescence assay (IFA) using mouse immune serum against PCV2 Rep, and observed by florescent microscopy. The full-length Rep protein (N314) and recombinant deletion mutants with 110 amino acid at N-terminal of Rep protein were dominantly localized in the nucleus. Examination of the 110 amino acid (aa) residues of the N-terminal region of PCV2 Rep revealed three clusters of basic aa with homology to other NLS. These results suggest that the utmost 20 residues in the N-terminal region could sufficiently mediate nuclear import of fusion protein, confirming its role as a functional NLS. Recent report showed that PCV2 induced apoptosis in the cultured porcine kidney cell (PK-15) via a viral protein encoded by ORF3. Monocyte/macrophage lineage cells are the major target cells for PCV2 infection. The purpose of this study was to expressed PCV2 ORF3 protein and further analyzing the role of ORF3 in inducing apoptosis in porcine peripheral blood mononuclear cells (PBMC). The full length of PCV2 ORF3 gene DNA fragment was amplification by PCR and suclone onto pET24 plasmid for preparation of the mouse antiserum against recombinant ORF3 protein. To analyze whether ORF3 is able to induce apoptosis, transient expression of the full length and the N- or C-terminal half of ORF3 protein in porcine PBMC was performed. After transfection, the transfected cells were detected by IFA and apoptosis induced by transient expression of viral protein was confirmed by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-nick end labeling (TUNEL-labeling) which detected the DNA breakage and caspase activity. Quantification of TUNEL-positive cells showed that the percentage of apoptotic cells transfected with pcDNA/ORF3 was significantly higher the C-terminal half of ORF3 retained its ability to induce apoptosis similar to the full length of ORF3. Caspase activity assays demonstrated significant activation of caspase-3, -8, and -9 by the full length and the C-terminal half of ORF3 protein. The results suggest that nuclear localization may be required for apoptosis induction and the C-terminal half region of ORF3 contains either N53-68 or N85-104 regions responsible for nuclear localization. In summary, the functional regions of PCV2 Rep protein responsible for DNA binding activity and nuclear localization appear to be the N-terminal 110 residue portion of the protein. Another nonstructural protein of PCV2, ORF3, is capable of triggering apoptotic response in porcine PBMC maybe associated with the NLS. The apoptotic activity is correlated with the nuclear localization of ORF3, and nuclear localization may play an important role of PCV2 viral protein function and virus life cycle.
46
Starck, Holger [Verfasser]. "Das RNA-bindende Protein Orb2 und seine regulatorische Funktion in der Spermatogenese von Drosophila melanogaster / vorgelegt von Holger Starck". 2008. http://d-nb.info/988337436/34.
47
Tanner, Tricia Lynn. "Analysis of the function of two varicella-zoster virus proteins involved in gene regulation IE63 and ORF29 /". 2007. http://proquest.umi.com/pqdweb?did=1240709031&sid=6&Fmt=2&clientId=39334&RQT=309&VName=PQD.
Abstract (sommario):
Thesis (Ph.D.)--State University of New York at Buffalo, 2007.Title from PDF title page (viewed on July 18, 2007) Available through UMI ProQuest Digital Dissertations. Thesis adviser: Ruyechan, William T. Includes bibliographical references.
48
Dan, Tran Quang, e 陳光瑩. "Studies on the Expression of Recombinant ORF3 Fusion Proteins of Porcine Circovirus Type 2 in Transgenic Tobacco". Thesis, 2014. http://ndltd.ncl.edu.tw/handle/xwceag.
Abstract (sommario):
碩士中國文化大學
生物科技研究所
102
Porcine circovirus type 2 (PCV2) is the causative agent of postweaning multisystemic wasting syndrome in pigs and has impact on swine-producing industry worldwide. The open reading frame 3 (ORF3) of PCV2 which encodes an 11.9 kDa ORF3 protein has been found to be involved in the pathogenesis of PCV2 infection by its apoptotic activity. The ORF3–based antigens thus represent a traditional approach to develop efficacious oral vaccines for immunization against PCV2. In this study, various ORF3-containing expression vectors, namely pGKF/U-ORF3, pGKF/U-L-ORF3, pGKF/U-ORF2-3b, and pGKF/U-L-ORF2-3b, were transformed to tobacco plants in order to produce the respective subunit recombinant vaccines. Following the Agrobacterium tumefaciens-mediated transformation of 200 leaf samples, 20 ORF3, 15 L-ORF3, 14 ORF2-3b, and 14 L-ORF2-3b putative transgenic tobacco plants were regenerated on the selective medium supplied with 200 mg/L kanamycin and showed GUS expression. The expected sequences of respective ORF3-containing trangenes, 359 bp, 369 bp, 1056 bp, and 1439 bp were amplified from genomic DNA of 19 ORF3 (9.5%), 14 L-ORF3 (7.0%), 12 ORF2-3b (6.0%), and 12 L-ORF2-3b (6.0%) GUS-positive transgenic plants, using specific pair of primers in PCR analysis. On the other hand, integration of 1-2 copies of independent ORF3-containing transgenes into tobacco genome was determined by means of Southern hybridization. The mRNA transcripts of particular transgenes were also found in the PCR-positive transgenic plants. Particularly, expression of respective ORF3 fusion proteins was qualitatively examined via immunobloting (Western blot) assay. However, the results have not clearly shown presence of target proteins, due to either low level of expression or interfering background.
49
Lee, Chia-Chun, e 李佳俊. "Expression of modified ORF 5 protein of PRRSV in the baculovirus system and cell surface display system". Thesis, 2010. http://ndltd.ncl.edu.tw/handle/pefp2t.
50
Fabrizio, Jacqueline Alba. "Characterising Novel Substrates of the Asparaginyl Hydroxylase FIH". Thesis, 2017. https://hdl.handle.net/2440/132758.
Abstract (sommario):
Factor Inhibiting the Hypoxia Inducible Factor (FIH) is an oxygen-dependent asparaginyl hydroxylase that plays a role in the cellular response to changes in oxygen concentrations. It was first found to catalyse the post translational hydroxylation of a family of transcription factors known as Hypoxia Inducible Factors (HIFs), and thus acts as a cellular sensor of hypoxia. Subsequently, other protein substrates were discovered containing ankyrin (ANK) repeat domains, although their activities were not affected by hydroxylation. FIH null mice display a metabolic phenotype which is not obviously linked to HIF regulation, supporting the existence of other important substrates of FIH. Hence the search for novel substrates of FIH was pursued, with particular interest in target proteins that upon modification by FIH would have a functional cellular outcome. Methods such as yeast two hybrid and pull down assays were previously employed to discover new substrates of FIH, but this limited searches to proteins with a relatively strong affinity for FIH. In research by a collaborator, a non-biased bioinformatic search was used to identify novel potential FIH substrates. Interestingly, the search identified a family of 5 ANK proteins present in the Orf virus (ORFV) as likely substrates of FIH. Preliminary experiments in our laboratory showed that they could interact with human FIH (hFIH), and some showed activity in CO₂ capture assays, consistent with hydroxylation. The first aim of this thesis was to determine whether the 5 ORFV ANK proteins, 008, 123, 126, 128 and 129 were substrates of FIH. Given that ORFV is an ovine virus, ovine FIH (oFIH) was cloned and purified, with recombinant protein displaying similar activity to recombinant hFIH. The ORFV ANK domains were cloned, expressed in bacteria and purified for analysis with recombinant oFIH. The hydroxylation state of these proteins was first analysed indirectly using in vitro hydroxylation assay and 008, 126 and 129 displayed high activity, consistent with being effectively hydroxylated by FIH, whereas 123 and 128 displayed low activity. Mass spectrometry (MS) of the recombinant proteins confirmed FIH-mediated hydroxylation of N40 in 008, N285 in 126, and N44 in 129, with poor ionisation preventing definitive conclusions regarding hydroxylation of 123 and 128. The ANK proteins of another closely related poxvirus, vaccinia virus (VACV), were also predicted to be substrates of FIH, but analysis of recombinant VACV ANK proteins showed no evidence of hydroxylation. These data indicate that these hydroxylation events are not conserved across the poxvirus family. The second aim was to characterise the functional role for this interaction between ORFV ANK proteins and FIH. Given the poorly characterised roles of the ORFV ANK proteins, and the relatively stable interaction between these proteins and FIH, these studies focused on the hypothesis that the ORFV ANK proteins could sequester FIH and subsequently upregulate HIF activity upon viral infection. Cell-based reporter gene assays confirmed that expression of the ORFV ANKs could sequester FIH and upregulate the HIFα transactivation domain, leading to increased HIF activity. Further supporting this hypothesis, ORFV infected cells that overexpressed or were deficient in FIH showed the induction of well characterised HIF target genes in a FIH-dependent manner. These data identified a novel mechanism of viral induced modulation of HIF activity, via the sequestration of FIH. The final aim of this thesis was to analyse OTUB1, a non-ANK and non-HIF protein discovered by our collaborators as a putative novel substrate of FIH. The hydroxylation of OTUB1 on N22 by FIH in vitro was confirmed using synthetic OTUB1 peptides in CO₂ capture assays.Thesis (Ph.D.) -- University of Adelaide, School of Biological Sciences, 2018