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Title: Cytoplasmic localisation of classical swine fever virus N-terminal autoprotease (Npro) is essential for modulation of the host cell innate immune response
Author: Jackson , Ben
ISNI:       0000 0004 5918 8247
Awarding Body: St George's, University of London
Current Institution: St George's, University of London
Date of Award: 2015
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The N-terminal autoprotease (NPro) of classical swine fever virus (CSFV) is a potent inhibitor of type I IFN and dsRNA-mediated apoptosis. Npro has been shown to localise to the cytoplasm, nucleus and nucleolus in vitro, however the mechanism and functional significance of the observed localisation have not been characterised. Proteins can be actively directed to the nucleus or cytoplasm by encoded nuclear localisation signals (NLS) or nuclear export signals (NES), respectively. Computational analysis, predicted that NPro encodes a bi-partite classical NLS (cNLS) in its C-terminal domain. GFP-NPro was shown to interact with the nuclear import receptor, importin-a, in reciprocal coimmunopricipitation experiments, suggesting that NPro does indeed encode a cNLS. The putative cNLS contained two conserved KIR motifs which are often critical for nuclear localisation of bipartite NLS. Not surprisingly, the interaction with importin-a was abolished when both of these KIR motifs were mutated. Interestingly, the nucleolar localisation of GFP-NPro was also abolished by these mutations. NPro was also found to encode a predicted NES that aligned with a "classical" CRM I-dependent NES consensus. A segment of NPro encoding the NES was found to direct GFP to the cytoplasm, suggesting that NPro encodes localisation signals that target the protein to both the cytoplasm and nucleus. GFP-NPro fusion proteins were localised to the nucleus or cytoplasm using NLS or NES, to investigate the importance of nuclear localisation for the characterised functions of NPro. NPro could no longer block dsRNA-mediated apoptosis when it was expressed in the nucleus, although interestingly, it did show a partial block on induction of type I IFN. In addition, cells expressing cytoplasmic NPro showed a significant reduction in the induction of type I IFN, but this function was incomplete suggesting that nuclear NPro may also be required to subvert the type I IFN response of the host cell.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available