The avian coronavirus infectious bronchitis virus (IBV) expresses four nonstructural, non-gene 1 proteins (3a, 3b, 5a and 5b) which have been shown to be dispensable for virus replication in cell culture. These IBV accessory proteins have no sequence homology to any of the accessory proteins of the group I and II coronaviruses but are highly conserved among the group III coronaviruses. Characterisation of naturally occurring strains of IBV which do not express two or more of the accessory proteins and of genetically modified recombinant IBVs has demonstrated that these accessory proteins contribute at most a minor role to the pathogenicity of the virus. To understand the relevance of these proteins for IBV the subcellular location of the 3a, 5a and 5b proteins have been characterised along with the identification of potential protein-protein interactions for the 3a protein. The subcellular location and protein-protein interactions of the 3b protein were attempted but specific problems were encountered. Indirect immunofluorescence with confocal microscopy of IBV-infected chick kidney cells was used to study the subcellular location of the accessory proteins. The 3a protein displayed a punctate, cytoplasmic distribution pattern which colocalised with virally-induced double-stranded RNA. A diffuse, cytoplasmic distribution was observed for the 5b protein which produced limited colocalisation with an IBV structural protein. Expression of a FLAG-tagged 5a protein in transfected Vero cells resulted in a punctate, cytoplasmic pattern. The protein interactions of the 3a protein were identified using FLAG-tag pull-down experiments with tandem mass spectrometry. Six cellular proteins were identified as interacting with the FLAG/3a protein within transfected Vero cells, three of which, GCN1, PP2A and Exportin-1, may interact with native 3a protein in IBV-infected cells. The 3a protein could sequester the viral dsRNA to hide it from the innate immune system and the potential interactions with three cellular proteins indicate that the IBV 3a protein may contribute to attenuation of host cell translation, induce cell cycle arrest and/or attenuate the nuclear export of a specific subset of mRNAs.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:521308 |
| Date | January 2009 |
| Creators | Davies, Marc Tudor |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/3145/ |
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