Zhang, Fan, (Researcher in life sciences) (2012) The role of NSP1 in rotavirus pathogenesis. PhD thesis, University of Warwick.

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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.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QR Microbiology > QR355 Virology
Library of Congress Subject Headings (LCSH):	Rotaviruses -- Pathogenesis
Official Date:	September 2012
Dates:	Date Event September 2012 Submitted
Institution:	University of Warwick
Theses Department:	School of Life Sciences
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Leppard, Keith; McCrae, Malcolm
Extent:	xii, 222 leaves : illustrations, charts.
Language:	eng

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