Coronaviruses are the causative agents of various mammalian diseases which have crucial economic and health-related problems and are mainly respiratory and gastrointestinal pathogens. They are positive strand RNA viruses which may require nuclear functions for replication. The nucleocapsid (N) protein of s~veral members has previously been shown to localise to the nucleus/nucleolus and the cytoplasm during infection and after transient transfection. The coronavirus N protein is a viral RNA binding protein with several functions during the virus life cycle, especially with regard to RNA replication and transcription and controlling cell signalling pathways. In order to localise to the cytoplasm and nucleus/nucleolus N protein must contain appropriate trafficking motif(s). This thesis focused on characterising the newly emerged severe acute respiratory syndrome coronavirus (SARS-CoV) N protein. The sub-cellular localisation of the SARS-CoV N protein was determined· in virus infected and transfected cells using antibody labelling and C-terminally tagged fluorescent fusion proteins, respectively. Comparison with the avian coronavirus N protein indicated that in contrast to other coronavirus N proteins, SARS-CoV N protein localised mainly in the cytoplasm with low frequency localisation in the nucleolus. Live cell, confocal microscopy and fluorescence loss in photo-bleaching (FLIP) was used to investigate the presence of any potential trafficking signals. Based on amino acid sequence conservation with the other coronavirus N proteins the SARS-CoV N protein was divided into three regions and mutation and bioinformatic analysis was used to potential nuclear import and export motifs. This approach delineated a cryptic nucleolar localisation signal in the central portion of the protein and a novel nuclear export signal in the C-terminal part, which may be the predominant trafficking signal. These motifs may be exposed by differential phosphorylation. The N protein was expressed in vitro and experiments formally demonstrated that it was a phosphoprotein which could bind viral RNA and an RNA binding region spanned the N-terminal and central·part ofthe protein. Together, the data in this study has provided insights into the expression and sub-cellular localisation ofthe SARS Cov N protein.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:489883 |
| Date | January 2008 |
| Creators | You, Jae Hwan |
| Publisher | University of Leeds |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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