Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.582299
Title: The role of NSP1 in rotavirus pathogenesis
Author: Zhang, Fan
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2012
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Abstract:
NSP1, a non-structural protein encoded by rotavirus gene segment 5, has been suggested as a virulence determinant for rotavirus and to function as an antagonist of the interferon signalling pathway. Although non-essential for rotavirus replication in cell culture, and is the least conserved in all rotavirus proteins, NSP1 from different rotavirus strains of different species has been demonstrated to interact with several cellular proteins involved in the IFNβ induction pathway. NSP1 from a bovine rotavirus strain (UKtc) has been shown to interact with and to degrade IRF3 in a proteasome dependent manner whereas NSP1 from a porcine rotavirus strain (OSU) fails to target IRF3 but is able to interfere with IFNβ production via similar targeting of β-TrCP. The research presented in this thesis sought to gain a better understanding of the molecular determinants of NSP1 specificity for targeting the IFNβ pathway by mapping the regions in NSP1 sequences responsible for targeting specific cellular proteins. NSP1 hybrid constructs with sequences from both UKtcNSP1 and OSUNSP1 were generated and their interactions with both IRF3 and β-TrCP were tested in a series of assays. The initial attempts to map interaction sites using the mammalian two-hybrid assay were not successful. No reporter plasmid signal was generated indicating the expected interaction. The failure of this assay might be due to the insufficient expression of the NSP1 proteins as subsequent modification of the expression vector was shown to improve the expression level of NSP1 proteins in subsequent reporter assay analysis. Using IFNβ promoter reporter assays to demonstrate the functional consequence of NSP1 action in IRF3, it was found that the constructs containing the entire Cterminal part of UKtcNSP1 were able to reduce IRF3-induced IFNβ promoter activity. Such constructs also caused IRF3 degradation in a proteasome dependent manner in agreement with previous studies. However, the sequence containing the last 135 amino acids from UKtcNSP1 was not sufficient for these activities. Collectively, these data suggested that the sequence between amino acid position 165 and 135 from the C-terminus are required for this interaction and subsequent degradation of IRF3. Similar experiments focused on determining the interaction site for β-TrCP on NSP1 were more difficult to interpret according the data presented. Unexpectedly in the light of published data, not only OSUNSP1 was able to degrade β-TrCP but UKtcNSP1 appeared to have the similar effect, as well as two reciprocal pairs of NSP1 hybrid constructs. In summary, it appears that sequences from the C-terminal part of UKtcNSP1 can function in a heterogeneous NSP1 context to target IRF3 from human cells. Further analysis is clearly required to fulfil the understanding of the role of NSP1 in rotavirus pathogenesis, including its interaction with β-TrCP.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.582299  DOI: Not available
Keywords: QR355 Virology
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