African horsesickness virus (AHSV) belongs to the Orbivirus genus in the family Reoviridae. This non-enveloped virus consists of an outer capsid formed by two structural proteins, VP2 and VP5, and an inner core formed by structural proteins VP7 and VP3. Three additional structural proteins associated with viral replication, as well as ten dsRNA molecules responsible for replication, are found inside the core. VP7 is the smallest of the structural proteins and each monomer consist of two domains, a hydrophilic top and hydrophobic bottom domain. Upon expression of VP7, the protein spontaneously assembles into trimers. Recombinant expression of the core protein VP7 results in large hexagonal structures formed by a double layer of these VP7 trimers with the hydrophobic bottom domains on the inside and hydrophilic top domains on the outside. The use of these crystal structures as a general display system for the display of foreign peptides/epitopes is being investigated in our group. In this regard, sites for the insertion of foreign peptides/epitopes were constructed at amino acid positions 177, 144 and 200 of the top domain of the VP7 protein and the resultant proteins named vectors AHSV-9 VP7-177, AHSV-9 VP7-144 and AHSV-9 VP7-200. Various inserts ranging from the HIV-1 ELDKWA epitope and FMDV VP1 epitopes to the eGFP peptide were inserted and subsequently analysed for immunogenicity. Results showed that a significant immune response was only elicited if the soluble trimer component of a chimeric VP7 protein was used for inoculation purposes. The crystal particles initially investigated as a display system did not result in any immune response. These results emphasized the importance of protein solubility for eliciting a significant immune response. The importance of solubility prompted an investigation into the use of the Bluetongue virus (BTV) VP7 protein as a vaccine display system. This protein is inherently more soluble than AHSV VP7 and does not result in crystal hexagonal structures if recombinantly expressed. An insertion site analogous to that of the AHSV-9 VP7-177 vector, located at amino acid 177 within an RGD loop in the top domain of VP7 was constructed. This new BTV VP7 vector, BTV-10 VP7-169, was characterised with regard to solubility and the ability to form trimers. In order to investigate the effect on solubility and trimerisation, FMDV VP1 epitope and eGFP were inserted into the BTV-10 VP7-169 vector. Results showed that following the construction of the insertion site, the vector was largely insoluble compared to the AHSV VP7 vectors and that insertion of the abovementioned peptides/epitopes did not have a significant effect on solubility. Although trimers were present, the yield was low compared to that of the AHSV chimeric VP7 proteins. Methods of improving the solubility of the chimeric VP7 proteins were investigated by treatment with solubilisation agents, sarkosyl and L-arginine. The results indicated that a strong denaturant such as sarkosyl can solubilise the particulate component of all chimeric VP7 proteins whereas Larginine had limited effect. The effect of these agents on the folding of the proteins were evaluated using fluorescence, since the ability to fluoresce is regarded as an indicator of correct folding. A comparison of the different VP7-eGFP proteins treated with these solubilisation agents showed that the sarkosyl solubilised proteins were not necessarily correctly folded. These results combined with the previously performed solubility assays indicated that a large proportion of correctly folded chimeric VP7 proteins associate with the particulate fraction. Investigation showed that expression of a large amount of correctly folded chimeric proteins results in the aggregation of these proteins within the expressing host cell. Once harvested these proteins remain associated with the insoluble fraction but can be solubilised by arginine treatment, or in some cases mere resuspension in a low-salt buffer, and used for vaccination purposes. In conclusion, the comparative analyses of solubility and trimerisation for the display vectors indicated that AHSV-9 VP7-144 vector may be most suitable for the display of foreign epitopes/peptides as it consistently yielded the largest component of correctly folded proteins. Furthermore, considering that large amounts of correctly folded chimeric VP7 proteins occurred in the insoluble component of each the VP7 display proteins, this study emphasize that use of solubility assays alone does not provide adequate information regarding the potential of a display vector for vaccination purposes. / Dissertation (MSc)--University of Pretoria, 2013. / Genetics / unrestricted

No description available.

Viral protein seven (VP7) is a major core protein and a group-reactive antigen that can be used for the diagnosis of bluetongue virus. VP7 gene of bluetongue virus serotype 4 was expressed in E. coli. Using phage display technology, anti-VP7st4 scFvs were selected from a chicken scFv library (Nkuku®) following different panning strategies. Polyclonal phage ELISA showed that VP7st4-specific scFvs were enriched after three rounds of panning. Six different scFvs (A1, H2, TA8, TC9, TD12 and SA12) were identified by sequence analysis. Stability of these scFvs was determined by incubation at different temperatures and after several freeze/thaw cycles. The scFvs were also tested in an inhibition ELISA. Inhibition with an anti-bluetongue virus guinea pig serum resulted in a 30% decrease in ELISA signal of A1. No inhibition was obtained with the rest of the scFvs when guinea pig and sheep serum were used. An anti-bluetongue virus chicken IgY inhibited the scFvs by 50% to 86%. A fragmented-gene library displaying peptides of VP7st4 was constructed. The library was subjected to three rounds of affinity selection against the anti-VP7st4 scFvs. Enrichment of clones specific to each scFv was observed. The clones were identified by sequence analyses. The regions on VP7st4 to which the scFvs bind could not be identified since no duplicate clones were selected. / Dissertation (MSc)--University of Pretoria, 2008. / Veterinary Tropical Diseases / unrestricted

Les Rotavirus appartiennent à la famille des Reoviridae, famille du groupe III des virus à ARN double brin. Identifiés en 1973 par Ruth Bishop, ces virus non enveloppés sont la première cause de diarrhée aiguë sévère du jeune enfant dans le monde. La capside virale icosaédrique est constituée de 3 couches protéiques de structure : la couche externe formée par la glycoprotéine VP7 d’où émergent les spicules de protéine VP4, la couche intermédiaire constituée par la protéine VP6 représentant près de 50 % du poids du virus et enfin, la couche interne appelée core, résultant de l’assemblage des protéines VP2, d’où émergent vers l’intérieur les protéines VP1 et VP3. Cette capside renferme un génome divisé en 11 segments d’ARN bicaténaires. A ces 6 protéines structurales s’ajoutent les protéines non structurales qui interviennent lors de la réplication du virus. Les deux protéines structurales, VP4 et VP7 sont essentielles pour la fixation de la particule triple couche (TLP) aux membranes des cellules hôtes, par interaction aux récepteurs intégrines, elle sont également impliqués dans la déstabilisation des membranes endosomales, indispensable à la libération de la particule double couche (DLP) infectieuse dans le cytoplasme. Actuellement, contrairement au mécanisme d’action de la protéine VP5*, celui de la glycoprotéine VP7 est inconnu. L’objectif de cette thèse, a été de comprendre le mécanisme moléculaire de déstabilisation des membranes par les peptides dérivés de VP7. Dans un premier temps nous avons montré, par des études in silico, l’existence d’un domaine prédit en hélice membranaire bordé de résidus arginine et lysine hautement conservés, situé à l’extrémité C-terminale de la glycoprotéine VP7. Ces résultats ont conduit à la synthèse de quatre peptides avec lesquels des tests de perméabilisation de membranes modèles de larges vésicules unilamellaires (LUVs) ont été menés. Ceux-ci ont permis d’identifier le domaine minimum le plus actif, VP723, parmi les peptides sélectionnés. Dans un second temps nous avons déterminé la structure de ces peptides par RMN, dans des conditions mimant l’environnement hydrophobe de la membrane. Le peptide minimal VP723 s’organise en hélice α-amphipathique, structure souvent impliquée dans la déstabilisation des membranes cellulaires. La comparaison de sa structure obtenue par RMN à celle du domaine correspondant dans la structure cristallographique de la protéine native montre le réarrangement conformationnel de ce segment après maturation par la trypsine. Ces résultats ont été confirmés par deux mutants de synthèse, dont l’un est inactif pour la perméabilisation des membranes modèles. Ces travaux ont été complétés par des expériences de Résonance Plasmonique aux Ondes guidée (PWR). Des études par RMN du solide sont en cours afin de déterminer l’orientation du peptide dans les membranes modèles. En conclusion, nos résultats mettent en évidence l’importance du domaine C-terminal VP723 de la protéine VP7 dans la déstabilisation des membranes, permettant d’assurer la translocation de la particule virale infectieuse (DLP) de l’endosome vers le cytoplasme. Un modèle du mécanisme d’entrée du virus, médié par les peptides dérivés de la maturation par la trypsine de la glycoprotéine VP7 est proposé. / Rotaviruses belong to the Reoviridae family, belonging to the group III of dsRNA viruses. Identified in 1973 by Ruth Bishop, these non-enveloped viruses are the leading cause of severe diarrhea in young children worldwide. The icosahedral capsid is composed of three structural protein layers: the outer one, formed by the glycoprotein VP7, emerges spicules protein VP4, the intermediate one consists of VP6 protein representing nearly 50% of the weight of the virus and finally, the inner one called core, results from the assembly of proteins VP2, emerges towards the inside of proteins VP1 and VP3. The capsid contains a genome divided into 11 segments of dsRNA. To these six structural proteins are added nonstructural proteins involved in virus replication. The two structural proteins, VP4 and VP7, are involved in the interaction of the triple layer particle (TLP) to integrin receptors, necessary for the release of the infectious double layer particle (DLP) into the cytoplasm following the permeabilization of the membrane of the endosome compartments. Currently, unlike the mechanism of action of the protein VP5*, the glycoprotein VP7 remains unknown. The objective of this work was to understand the molecular mechanism involved in the destabilization of membranes by peptides derived from VP7. In a first step, we have shown, by in silico studies, the existence of a helical trans-membrane domain predicted containing a highly conserved arginine and lysine residues, located at the C-terminus of the VP7 glycoprotein. These results led to the synthesis of four peptides with which permeabilizing tests of model membranes were conducted. We have identified the minimum of the most active domain, named VP723, among the selected peptides. In a second step, we determined the structure of these peptides by NMR under conditions mimicking the hydrophobic environment of the membrane. The VP723 peptide is organized like an α-helical amphipathic structure often involved in the destabilization of cell membranes. The comparison of the structure obtained by NMR to that of the corresponding domain in the crystallographic structure of the native protein shows a conformational rearrangement of the segment after trypsin maturation. These results were confirmed by two synthetic mutants, one of which is inactive for the permeabilization of model membranes. These studies were complemented by experiments Plasmon Resonance guided the Waves (PWR). Studies by solid state NMR are in progress to determine the orientation of the peptide in model of membranes. In conclusion, our results highlight the importance of the C-terminal domain of the VP7 protein, named VP723, in the destabilization of membranes, to ensure the translocation of the infectious viral particle (DLP) from the endosome into the cytoplasm compartments. A mechanism of virus entry mediated by peptides derived from trypsin maturation of the VP7 glycoprotein is proposed in this study.

No description available.

Bluetongue virus belongs to the Orbivirus genus from the Reoviridae family. It infects predominantly domestic and wild ruminants and is economically significant worldwide. Bluetongue virus VP7 forms the intercepting layer between the outer capsid (VP2 and VP5) and VP3 which surrounds the genomic material. BL21(DE3), NiCo21(DE3), C43(DE3) pLysS and KRX Escherichia coli cells were transformed with a pET28a plasmid with the cDNA sequence encoding Bluetongue virus VP7. Expression of Bluetongue virus VP7 was tested at post induction temperatures between 16˚C and 37 ˚C, at inducer concentrations between 0.1 mM and 1.0 mM isopropyl-β-D-thiogalactopyranoside in BL21(DE3), NiCo21(DE3) and C43(DE3) pLysS cells and 0.05 % and 0.15 % rhamnose for KRX cells, in two types of growth media (LB and 2xYT) and post-induction growth times between two and 16 hours. Under all conditions tested; Bluetongue virus VP7 expression was found to be predominantly in the insoluble fraction (pellet). BL21(DE3) and NiCo21(DE3) cells were chosen and grown for five hours post induction, induced with 0.1 mM isopropyl-β-D-thiogalactopyranoside and grown at a post-induction temperature of 37 ˚C. Bluetongue virus VP7 in bacterial cell inclusion bodies was solubilised using urea and a freeze-thaw step. Solubilisation was tested with urea concentrations between 2 M and 8 M, with solubilisation efficiency not increasing past 5 M urea. Solubilized Bluetongue virus VP7 was purified using nickel-affinity chromatography. Purified Bluetongue virus VP7 was then probed with far-UV circular dichroism and intrinsic fluorescence in several buffer conditions including different urea and guanidinium chloride concentrations as well as in the presence of glycerol and sodium chloride. Guanidinium chloride was able to cause Bluetongue virus VP7 unfolding, and the unfolding transition had 94 % and 89 % reversibility at 218 nm and 222 nm respectively. Bluetongue virus VP7 was shown to contain a native-like structure in 20 % glycerol and in up to 8 M urea and was found to be stable till at least 55 ˚C, even in the presence of 5 M urea. Glycerol and sodium chloride influenced the conformation of the protein resulting in different unfolding transitions. Thermal unfolding of Bluetongue virus VP7 was found to be irreversible. / Life and Consumer Sciences / M. Sc. (Life Sciences)