Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.390524
Title: Orbivirus non-structural protein NS2 : its role in virus replication
Author: Horscroft, Nigel John
ISNI:       0000 0001 3581 8636
Awarding Body: University of Oxford
Current Institution: University of Oxford
Date of Award: 1997
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Abstract:
Bluetongue virus (BTV) is an economically important arthropod-borne virus that causes Bluetongue disease in sheep and other ruminants. BTV is the prototypical orbivirus and is a member of the Reoviridae family. Other arthropod-borne orbiviruses include African horse-sickness virus (AHSV) and epizootic haemorraghic disease virus (EHDV). The virus contains 10 dsRNA segments that encode for both structural and non-structural proteins. The structural proteins of orbiviruses and other members of the Reoviridae, such as reoviruses and rotaviruses, are arranged in a very similar layered arrangement. The roles of the non-structural proteins are largely unknown. This thesis describes the functional characterisation of one of these nonstructural proteins, NS2. The NS2 protein of BTV-10 was purified to a high degree of homogeneity from insect cells. This purified protein was used in crystal screening experiments and in circular dichroism (CD) experiments. Although a wide range of conditions were tried for crystallography, no crystals were obtained. This may be due to sequence or conformational heterogeneity. Size-exclusion chromatography was used to show that purified NS2 exists in large multimers, supporting the conclusion that conformational heterogeneity was responsible for the lack of crystal formation. CD revealed that NS2 has a high α-helical content. NS2 is a ssRNA-binding protein. To identify the nucleotides within the viral RNA segments that are important for recognition by NS2, gel-shift and uvcrosslinking experiments with various labelled synthetic RNA probes and in vitro transcripts were performed. Initial gel shift analysis with short synthetic RNAs indicated that NS2 bound a sequence corresponding to the conserved 5' terminus of rotavirus genomic RNA. Similar sequences, called the NS2 target sequence or NTS, were identified in all 10 BTV segments. A number of truncations and deletions of BTV segments M4 and S10 were created to investigate the role of this aptamer in NS2 binding. The results indicate that the NTS is required for the binding of NS2 to each BTV viral RNA segment. These results were confirmed by using short synthetic RNA probes corresponding to the aptamer identified in segments M4 and S10 and by showing that the aptamer could confer NS2 binding to non-BTV RNA. Further biochemical analysis of NS2 revealed previously undescribed features of the protein. Chemical crosslinking and co-immunoprecipitation studies showed that NS2 can interact with VP1 the viral RNA polymerase. The inclusion of NS2 in in vitro transcription-translation and translation reactions demonstrated a specific shutdown of BTV protein synthesis, suggesting a role for the protein in a switch from viral RNA translation to RNA packaging. This translation inhibition is dependant on the concentration of NS2 present and may be related to the formation of viral inclusion bodies. NS2 was also found to bind nucleotide triphosphates and to have phosphohydrolase activity. Kinase inhibitors were used to identify casein kinase n as the cellular kinase that phosphorylates NS2 in vivo.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.390524  DOI: Not available
Keywords: Proteins ; Bluetongue virus ; Genetic aspects
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