Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid (N) protein may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. In the infected cell, N was the only viral protein that was phosphorylated as shown by 32P-orthophosphate labeling and Western blot analysis and with IBV specific polyclonal chicken antibody. Using pulse-labeling with 32Porthophosphate, the IBV N protein was found to be phosphorylated in the virion, as well as at all times during infection of Vero cells. One-hour pulse-chase analysis followed by immunoprecipitation of IBV N using rabbit anti-IBV N polyclonal antibody showed that the phosphate on the protein did not fall below 70% of the maximum and remained stable. The small but reproducible drop in phosphorylation could modulate the various functions of the N protein in the infected cell. Simultaneous labeling with 32Porthophosphate and 3H-leucine of infected CEK cells indicated a 3.5-fold increase in the ratio of the 32P:3H counts per minute (cpm) on the virion N protein as compared to the 32P:3H cpm ratio of the N protein from lysates at 7 h p.i. The 32P:3H cpm ratio of the N protein from virion from infected-Vero cell lysates was 10.5X more than the 32P:3H cpm ratio of the N protein obtained at 7 h p.i. It has been shown that the N proteins from the measles and rabies viruses form helical nucleocapsid-like structures when expressed in bacteria (Schoehn et al., 2001; Warnes et al., 1995). The ability of E. coli expressed IBV N protein to form helical-nucleocapsid-like structures was investigated using transmission electron microscopy. Full-length, purified histidine-tagged IBV N protein formed nucleocapsid-like structures when expressed in bacteria. Because E. coli -expressed histidine-tagged fragments of the IBV N protein did not form helical nucleocapsid-like structures, the full-length protein is probably required for assembly of these structures. The highly conserved IBV N protein was also used as a diagnostic tool in an ELISA for detecting anti-IBV antibody in chicken serum using a specialized microwave called the BIOWAVE. The BIOWAVE improves the processing time for an ELISA.

Because phosphorylation of the infectious bronchitis virus (IBV) nucleocapsid (N) protein may regulate its multiple roles in viral replication, the dynamics of N phosphorylation were examined. In the infected cell, N was the only viral protein that was phosphorylated as shown by 32P-orthophosphate labeling and Western blot analysis and with IBV specific polyclonal chicken antibody. Using pulse-labeling with 32Porthophosphate, the IBV N protein was found to be phosphorylated in the virion, as well as at all times during infection of Vero cells. One-hour pulse-chase analysis followed by immunoprecipitation of IBV N using rabbit anti-IBV N polyclonal antibody showed that the phosphate on the protein did not fall below 70% of the maximum and remained stable. The small but reproducible drop in phosphorylation could modulate the various functions of the N protein in the infected cell. Simultaneous labeling with 32Porthophosphate and 3H-leucine of infected CEK cells indicated a 3.5-fold increase in the ratio of the 32P:3H counts per minute (cpm) on the virion N protein as compared to the 32P:3H cpm ratio of the N protein from lysates at 7 h p.i. The 32P:3H cpm ratio of the N protein from virion from infected-Vero cell lysates was 10.5X more than the 32P:3H cpm ratio of the N protein obtained at 7 h p.i. It has been shown that the N proteins from the measles and rabies viruses form helical nucleocapsid-like structures when expressed in bacteria (Schoehn et al., 2001; Warnes et al., 1995). The ability of E. coli expressed IBV N protein to form helical-nucleocapsid-like structures was investigated using transmission electron microscopy. Full-length, purified histidine-tagged IBV N protein formed nucleocapsid-like structures when expressed in bacteria. Because E. coli -expressed histidine-tagged fragments of the IBV N protein did not form helical nucleocapsid-like structures, the full-length protein is probably required for assembly of these structures. The highly conserved IBV N protein was also used as a diagnostic tool in an ELISA for detecting anti-IBV antibody in chicken serum using a specialized microwave called the BIOWAVE. The BIOWAVE improves the processing time for an ELISA.

Le virus de la Rougeole reste le plus meurtrier des virus contre lesquels il existe un vaccin, avec environ 350000 décès par an dans le monde. Ce virus appartient à la famille des Paramyxoviridae, qui sont des virus enveloppés de forme sphérique dont le génome est composé d’un seul brin d’ARN de polarité négative. L’élément central de la réplication et de la transcription du génome viral est le complexe, de forme hélicoïdale, entre l’ARN du virus et la nucléoprotéine. Cette association intime appelée nucléocapside a des propriétés étonnantes non encore élucidées. En effet, l’ARN des virus à ARN négatif a la particularité de n’être jamais nu, même lors des étapes de réplication/transcription nécessitant pourtant le passage de la polymérase virale. On suppose que l’interaction avec la phosphoprotéine, cofacteur de la polymérase, provoque un changement de la conformation de la nucléoprotéine pour rendre l’ARN viral accessible à la polymérase. Lorsque la nucléoprotéine est exprimée dans des cellules d’insectes, elle se fixe aux ARNs cellulaires et forme des nucléocapsides recombinantes. Les études précédentes sur d’autres virus à ARN négatif (Rage, Marbourg, Sendaï) ont montré que les nucléocapsides recombinantes sont semblables aux nucléocapsides virales. Au sein de la nucléocapside, le domaine C-terminal de la nucléoprotéine joue un rôle crucial en interagissant avec de nombreux partenaires viraux et cellulaires, notamment avec la phosphoprotéine dans les étapes de réplication/transcription du génome viral. Cependant, des observations en microscopie électronique à transmission avaient montré que les nucléocapsides recombinantes contenant la nucléoprotéine entière était trop flexibles pour envisager leur reconstruction tridimensionnelle par analyse d’image, ce qui avait conduit à les rigidifier par un traitement protéasique dont l’effet latéral est justement l’élimination du domaine C-terminal de la nucléoprotéine. Nous avons mis au point des conditions de préparation en coloration négative permettant de rigidifier la nucléocapside intacte, afin d’en calculer une reconstruction tridimensionnelle à basse résolution et de la comparer avec celle de la nucléocapside protéolysée. Nous avons ainsi montré que les nucléocapsides de la Rougeole changeaient radicalement de structure tridimensionnelle en réponse au traitement protéolytique, non seulement en terme de pas de l’hélice ou de nombre de sous-unités par tour, mais aussi au niveau de la conformation de la nucléoprotéine et de ses contacts avec les sous-unités adjacentes, ce qui n’avait encore jamais été observé aussi clairement. / Flexible helical protein polymers exemplified by actin filaments, microtubules and bacterial flagella areubiquitous in biology. Due to their size and intrinsic irregularities, the structure of these polymers cannot be solved by Xraycrystallography. Since half a century, three-dimensional (3D) reconstruction from two-dimensional (2D) ElectronMicroscopy (EM) images appears as a method of choice to solve the structure of large helical polymers. However,depending on the degree of flexibility of the analyzed helices, the 3D reconstruction process can still be a daunting task.For the most regular helices, the classical reciprocal space-based Fourier-Bessel approach can allow both to determinethe helical symmetry and to calculate 3D structures. For more flexible structures, recent “single-particle” approachesconsist in segmentation of long irregular helices into short (i.e. locally more regular) segments and their processing asasymmetrical objects with defined symmetry-imposed constraints (Egelman, 2000; Sachse et al., 2007). However, twomajor difficulties remain: the heterogeneous data must be sorted into homogeneous populations and the helical symmetryfor each population has to be determined. In the presented work, we explored various single-particle approaches,developed new analysis methods, and implemented most of them into a user-friendly processing pipeline. The targetbiological objects were helical nucleocapsids of two negative strand RNA viruses, Measles (MeV) and VesicularStomatitis Virus (VSV ; the prototype for Rabies), the latter being particularly flexible in terms of helical parameters(diameter, number of subunits per turn). Nucleocapsids are formed by the viral genomic RNA coated by thenucleoprotein and serve as a template for viral replication and transcription. To overcome the heterogeneity problem, weused 2D classification, described general processing protocols and applications for helical segments, and introduced anew classification method based on the power spectra of the images. The determination of helical symmetry(ies) wasaddressed by a novel approach relying on ab initio exhaustive search of helical parameters whereby we start from asingle 2D image, reconstruct as many 3D structures as parameters to test by cropping the image and assigning views tothe obtained segments, and calculate the cross-correlation (CC) of the reprojection of the 3D model with the initialimage. Applied to artificial data sets, the method was effectively able to detect a maximum of CC for the true symmetryparameters, but also showed intrinsic ambiguities of helical symmetry determination on which we extensively comment.Altogether, the result of this method-oriented work allowed us to address several biological questions. First, the 3Dreconstruction by negative stain EM of two forms of nucleocapsids of MeV coupled to a docking of a homologouscrystal structure enabled us to determine the orientation of the nucleoprotein and of the RNA in the nucleocapsids.Secondly, we assessed the structure of in vitro formed nucleocapsids of VSV and showed that assemblies close to thenative viral nucleocapsids can be formed in the absence of any other viral proteins, thus providing new insights into theassembly of this virus. As a perspective of this work, our pipeline of flexible helical analysis is being extended andsuccessfully used for other projects.

The methodologies of systems design, rooted in engineering and in cognitivist conceptions of human action, have been stretched to the limit by the complexity of uses to which information and communication technologies are being turned. Within segments of the broader design community there has been a `turn to the social' - a perception that there is a need now for richer stories about the everyday practices systems designers build tools to support. This thesis is presented as a contribution to the corpus of `richer stories' about the what, how, why, when and where of information gathering. The thesis presents findings from an ethnographic study of newsroom information gathering at a UK daily newspaper. Adopting an analytical perspective based upon cultural-historical activity theory (CHAT), it describes and analyses journalistic information gathering on two mutually constitutive levels; that of activity and that of artefact mediation. Its starting point is that neither information gathering, nor the artefacts of information gathering, can be understood without consideration of the social, cultural and historical contexts within which they are situated. Ethnographic data is drawn upon to argue that journalistic information gathering can only be understood within the particular context of the `story lifecycle'. Stories are the principal object of journalistic enterprise, and the thesis examines in detail how everyday working practices are oriented towards this lifecycle. Based on an analysis of the artefacts of newsroom information gathering, and of the discourses of information systems designers, it is also argued that the discourses of systems designers over-emphasise the importance of the category `information'. In particular it is argued that sources are how journalists orient themselves in the vast, heterogeneous information spaces they simultaneously inhabit and populate. The background to these discussions is the often controversial relationship between ethnography, theory and systems design. This relationship is examined and it is argued that the CHAT perspective provides design ethnographers with an opportunity to move from ethnographic intuition to design insight. It is also argued that at a more pragmatic level, CHAT helps the fieldworker navigate the apparently never-ending mass of `potentially interesting material' any field experience throws up.

Avian influenza H5N1 causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. NP is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymophcyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physiochemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.

Dissertation (Ph.D.).--University of Texas Graduate School of Biomedical Sciences at San Antonio, 2007. / Vita. Includes bibliographical references.

Avian influenza H5N1 causes occasional but serious infections in humans and efforts to produce vaccines against this strain continue. Current influenza vaccines are prophylactic and utilize the two major antigens, hemagglutinin and neuraminidase. NP is an attractive alternative antigen because it is highly conserved across all influenza strains, has been shown to increase the rate of viral clearance, and potential therapeutic vaccines would elicit cytotoxic T lymophcyte responses in an infected person. The NP antigen from H5N1 was characterized using a variety of physiochemical methods to gain insights into both the biological and physical properties of the antigen which are important from a regulatory viewpoint when considering therapeutic vaccines. Results obtained to date show that NP is relatively unstable and indicate that the conformation of the H5N1 NP antigen is highly dependent upon purification procedure, buffer conditions, pH and the presence or absence of RNA. These factors will need to be clearly defined and taken into consideration when manufacturing and regulating NP vaccine preparations.

Philosophiae Doctor - PhD / Human Coronavirus NL63 (HCoV-NL63) is one of seven coronaviruses (CoVs) that cause respiratory disease in the global population. The Membrane (M) and Nucleocapsid (N) proteins are part of the core CoV-structural proteins, crucial in viral replication and virion assembly. Here the expression of HCoV-NL63 M and N was characterized across multiple in vitro systems including bacterial, insect and mammalian. To detect untagged proteins in viral structural studies, anti-peptide antibodies were generated in a mouse model. Polyclonal antisera and hybridoma-secreted antibodies exhibited specific binding to their respective full length protein antigens. Anti-peptide monoclonal antibodies were successfully generated against the HCoV-NL63 M and N proteins. During CoV infection, the interaction of CoV M and N is necessary for the production of infectious virions. For the first time, co-expressed, full length HCoV-NL63 M and N were assayed for protein-protein interaction in a mammalian cell system, allowing for native protein folding and modification. M protein formed higher order homomultimers in the presence and absence of co-expressed N.

No description available.

Poon Wing Ming Jodie. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (leaves 207-233). / Abstracts in English and Chinese. / Acknowledgements --- p.i / Abstract --- p.ii / 論文摘要 --- p.iv / Abbreviations --- p.v / List of Figures --- p.x / List of Tables --- p.xiii / Contents --- p.xiv / Chapter CHAPTER ONE --- INTRODUCTION --- p.1 / Chapter 1.1. --- Severe Acute Respiratory Syndrome (SARS) --- p.1 / Chapter 1.1.1. --- Background of SARS --- p.1 / Chapter 1.1.2. --- Etiology and pathology of SARS --- p.3 / Chapter 1.1.3. --- Genome organization and expression of SARS-CoV --- p.5 / Chapter 1.1.4. --- Current molecular advances of SARS-CoV --- p.13 / Chapter 1.1.5. --- Current research advances on SARS-CoV nucleocapsid --- p.18 / Chapter 1.1.6. --- Current diagnostic assays of SARS-CoV infection --- p.23 / Chapter 1.1.7. --- Current treatment --- p.25 / Chapter 1.1.8. --- Vaccine development --- p.27 / Chapter 1.2. --- Aims of study --- p.30 / Chapter CHAPTER TWO --- MATERIALS AND METHODS --- p.33 / Chapter 2.1. --- Subcellular localization study of the SARS-CoV nucleocapsid protein --- p.33 / Chapter 2.1.1. --- "Cloning of SARS-CoV nucleocapsid cDNA into the green fluorescence protein (GFP) mammalian expression vector, pEGFP-C1" --- p.33 / Chapter 2.1.1.1. --- Amplification of SARS-CoV nucleocapsid gene by polymerase chain reaction (PCR) --- p.33 / Chapter 2.1.1.2. --- Purification of PCR products --- p.35 / Chapter 2.1.1.3. --- Restriction digestion of purified PCR products and the circular pEGFP-C 1 vector --- p.36 / Chapter 2.1.1.4. --- Ligation --- p.36 / Chapter 2.1.1.5. --- Preparation of chemically competent bacterial cell E.coli strain DH5a for transformation --- p.37 / Chapter 2.1.1.6. --- Transformation of ligation product into chemically competent bacterial cells --- p.38 / Chapter 2.1.1.7. --- Small-scale preparation of bacterial plasmid DNA --- p.39 / Chapter 2.1.1.8. --- Screening for recombinant clones --- p.40 / Chapter 2.1.1.9. --- DNA sequencing of cloned plasmid DNA --- p.41 / Chapter 2.1.1.10. --- Midi-scale preparation of recombinant plasmid DNA --- p.42 / Chapter 2.1.2. --- Cell culture --- p.44 / Chapter 2.1.2.1. --- Sub-culture of VeroE6 and HepG2 cell lines --- p.44 / Chapter 2.1.2.2. --- Transient transfection of GFP fusion construct --- p.45 / Chapter 2.1.3. --- Epi-fluorescent microscopy --- p.46 / Chapter 2.2. --- Study on differential gene expression patterns upon SARS-CoV nucleocpasid induction by cDNA microarray analysis --- p.48 / Chapter 2.2.1. --- Cloning of SARS-CoV N gene into mammalian expression vector pCMV-Tagl --- p.48 / Chapter 2.2.2. --- Cell culture --- p.50 / Chapter 2.2.2.1. --- Sub-culture of VeroE6 cell line --- p.50 / Chapter 2.2.2.2. --- Transient transfection of pCMV-Tag1 -SAR-CoV N construct --- p.50 / Chapter 2.2.3. --- Total RNA isolation --- p.51 / Chapter 2.2.3.1. --- Total RNA isolation by RNeasy Mini Kit --- p.51 / Chapter 2.2.3.2. --- Checking of RNA integrity --- p.53 / Chapter 2.2.3.3. --- Checking of RNA purity --- p.54 / Chapter 2.2.3.4. --- Determinations of total RNA concentrations and precipitation --- p.54 / Chapter 2.2.4. --- cDNA microarray (done by Affymetrix Inc. as a customer service) --- p.55 / Chapter 2.2.4.1. --- Precipitation of RNA --- p.55 / Chapter 2.2.4.2. --- Quantification of RNA --- p.56 / Chapter 2.2.4.3. --- Synthesis of double-stranded cDNA from total RNA --- p.56 / Chapter (i) --- First stand cDNA synthesis --- p.56 / Chapter (ii) --- Second cDNA synthesis --- p.57 / Chapter 2.2.4.4. --- Clean-up of double stranded cDNA --- p.58 / Chapter (i) --- Phase lock gel-phenol/ chloroform extraction --- p.58 / Chapter (ii) --- Ethanol precipitation --- p.58 / Chapter 2.2.4.5. --- Synthesis of biotin-labeled cRNA --- p.59 / Chapter 2.2.4.6. --- Clean-up and quantification of in vitro transcription (IVP) products --- p.59 / Chapter (i) --- In vitro transcription clean-up --- p.59 / Chapter (ii) --- Ethanol precipitation --- p.60 / Chapter (iii) --- Quantitation of cRNA --- p.60 / Chapter (iv) --- Sample checking --- p.60 / Chapter 2.2.4.7. --- cRNA fragmentation for target preparation --- p.60 / Chapter 2.2.4.8. --- Eukaryotic target hybridization --- p.61 / Chapter 2.2.4.9. --- "Probe array washing, staining and scanning" --- p.62 / Chapter 2.2.5. --- Confirmation of results by RT-PCR --- p.62 / Chapter 2.2.5.1. --- First-strand cDNA synthesis --- p.62 / Chapter 2.2.5.2. --- RT-PCR of candidate gene --- p.63 / Chapter 2.3. --- In vitro RNA interference of SARS-CoV nucleocapsid --- p.66 / Chapter 2.3.1. --- siRNA target site selection --- p.66 / Chapter 2.3.2. --- Cloning of target siRNA sequences into pSilencer 3.1-H1 vector --- p.71 / Chapter 2.3.3. --- Cell culture --- p.72 / Chapter 2.2.3.1. --- Sub-culture ofVeroE6 cells --- p.72 / Chapter 2.3.3.2. --- Transient co-transfection --- p.72 / Chapter 2.3.4. --- Detection of SARS-CoV nucleocapsid mRNA expression level by RT-PCR --- p.73 / Chapter 2.3.4.1. --- Total RNA isolation by TRIzol reagent --- p.73 / Chapter 2.3.4.2. --- First-strand cDNA synthesis --- p.74 / Chapter 2.3.4.3. --- RT-PCR assays --- p.74 / Chapter 2.3.5. --- Detection of SARS-CoV nucleocapsid protein expression level by Western blotting --- p.75 / Chapter 2.3.5.1. --- Total protein extraction --- p.75 / Chapter 2.3.5.2. --- Protein quantification --- p.75 / Chapter 2.3.5.3. --- Protein separation by SDS-PAGE and Western blot --- p.76 / Chapter 2.3.5.4. --- Western blot analysis --- p.78 / Chapter 2.4. --- Human fgl2 prothrombinase promoter analyses --- p.80 / Chapter 2.4.1. --- Cloning of the full-length human fgl2 prothrombinase promoter construct into a promoterless mammalian expression vector-pGL3-Basic --- p.80 / Chapter 2.4.2. --- Cloning of SARS-CoV Membrane gene into the mammalian expression vector pCMV-Tagl --- p.82 / Chapter 2.4.3. --- Cell culture --- p.84 / Chapter 2.4.3.1. --- Sub-culture of HepG2 and VeroE6 cell lines --- p.84 / Chapter 2.4.3.2. --- "Transient co-transfection of the full-length human fgl2 prothrombinase promoter construct with the pCMV-Tagl empty vector, pCMV-Tagl-SARS-CoV M expression vector, or pCMV-Tag1 -SARS-CoV N expression vector" --- p.84 / Chapter 2.4.4. --- Dual-luciferase reporter assay --- p.85 / Chapter 2.4.5. --- Detection of fgl2 mRNA expression level under the induction of SARS-CoV nucleocapsid protein by RT-PCR --- p.86 / Chapter 2.4.5.1. --- Total RNA isolation by TRIzol reagent --- p.86 / Chapter 2.4.5.2. --- First strand cDNA synthesis --- p.86 / Chapter 2.4.5.3. --- RT-PCR of fgl2 gene --- p.87 / Chapter CHAPTER THREE --- RESULTS --- p.88 / Chapter 3.1. --- Computer analysis of SARS-CoV Nucleocapsid --- p.88 / Chapter 3.2. --- Subcellular localization of SARS-CoV nucleopcasid protein in VeroE6 cells and HepG2 cells --- p.102 / Chapter 3.3. --- cDNA microarray analysis on differential gene expression pattern upon the over-expression of SARS-CoV Nucleocapsid gene --- p.114 / Chapter 3.4. --- In vitro RNA Interference of SARS nucleocapsid --- p.129 / Chapter 3.5. --- Transactivation of fgl2 prothrombinase gene promoter by SARS-CoV nucleocapsid protein in HepG2 and VE6 cells --- p.138 / Chapter CHAPTER FOUR --- DISCUSSION --- p.155 / Chapter 4.1. --- "The EGFP-tagged SARS-CoV N protein was localized in the cytoplasm only in VE6 cells, but translocated into both cytoplasm and nucleus in HepG2 cellsin the epi-fluorescence microscopy study" --- p.155 / Chapter 4.2. --- cDNA microarray demonstrated alternations of mRNA transcript level on a number of genes belonging to various functional classes upon over expression of SARS-CoV nucleocapsid gene --- p.162 / Chapter 4.3. --- RNA interference demonstrated effective gene silencing of SARS-CoV nucleocapsid gene --- p.171 / Chapter 4.4. --- SASR-CoV nucleocapsid protein induced the promoter activity of the prothrombinase fibrinogen-like protein2/ fibroleukin (fgl2) gene --- p.191 / Chapter 4.5. --- Conclusion --- p.196 / Chapter 4.6. --- Future work --- p.198 / Appendices --- p.199 / References --- p.207