SUMMARY (To be printed on this form) Replication of the -30-kb positive-sense RNA genomes of the Coronaviridae and transcription of a set of subgenomic mRNAs is mediated by a large protein complex of nonstructural proteins (nsps) involving two RNA-dependent RNA polymerase activities that, in coronaviruses, are associated with nonstructural proteins (nsp) 8 and 12. To further characterise the RNA polymerase (primase) activity suggested to be associated with coronavirus nsp8, homologs from Human coronavirus 229E (HCoV- 229E) and the bafinivirus White bream virus (WBV), along with a set of mutant proteins containing alanine substitutions of conserved residues, were expressed in E. coli and the polymerase activities of the purified proteins were characterised in vitro. The data revealed robust metal ion-dependent polymerase activities for both HCoV-229E nsp8 and the WBV homologue. The mutant proteins retained varying degrees of activity, with single or double substitutions of three conserved Asn and Lys residues (ppla/ppJab N3672A, N3672A/K37 I lA, K3687A/K371IA) displaying the most detrimental effects on HCo V -229E nsp8 activity. The polymerase activities were further characterised using a range of homopolymeric and virus-specific template RNAs, revealing insight into substrate preferences of the coronavirus primase. HCo V -229E nsp8 substitutions were subsequently studied in the context of HCoV-229E replication in cell culture using a reverse genetics system. The data show that nsp8 activity is essential for coronavirus RNA synthesis, with substitutions of functionally important residues causing lethal phenotypes or defects in viral RNA synthesis and production of infectious virus progeny. The data provides interesting insight into the enzymatic and functional properties of two as yet poorly characterized polymerase/primase activities from distantly related viruses from the Alphacoronavirus and Bafinivirus genera. Furthermore, the study identified a number of WBV main protease cleavage sites, providing information on the substrate specificity of the WBV main protease and the proteolytic processing events used to release the putative WBV primase from larger polyprotein precursors.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:557407 |
| Date | January 2012 |
| Creators | Ferguson, L. J. |
| Publisher | Queen's University Belfast |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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