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Impact of interferon β and interferon stimulated gene induction on Bunyamwera virus replication

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CharlesCarlton-SmithPhDThesis.pdf (32.09Mb)
Date
20/06/2012
Author
Carlton-Smith, Charles
Supervisor
Elliott, Richard Michael
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Abstract
The first line of defence against viral infection is the interferon (IFN) response, which must be overcome by a virus for successful replication. Pattern recognition receptors detect virus which triggers induction of IFNβ. Secreted IFNβ stimulates the JAK/STAT signal transduction pathway and the upregulation of IFN stimulated genes (ISGs) culminating with expression of hundreds of antiviral proteins. Bunyamwera virus (BUNV) is the prototype virus for the genus Orthobunyavirus and the family Bunyaviridae. BUNV is a trisegmented single stranded negative sense RNA virus whose genome comprises the Large (L), Medium (M) and Small (S) RNA segments. The L segment encodes the RNA polymerase, the M segment the two glycoproteins Gn and Gc and a non-structural protein NSm, and the S segment the nucleoprotein and a non-structural protein NSs in overlapping reading frames. The NSs protein interferes with RNA polymerase II mediated transcription thereby inhibiting cellular mRNA production, including IFN mRNA, and hence it is the primary IFN antagonist. A recombinant virus, rBUNdelNSs, that is unable to express the NSs protein, does not inhibit cellular transcription and is thus a strong IFN inducer. The aim of this thesis was to understand how IFN inhibits BUNV replication. Cells stimulated into the antiviral state by IFN treatment were protected against BUNV infection but addition of IFN 6 hours (or later) post infection had little effect on the replication cycle. However, addition of IFN immediately following infection conferred restriction on BUNV replication by initially increasing viral protein synthesis and then by blocking translation of positive sense viral RNA. To identify ISGs with anti-BUNV activity, I screened a panel of 26 cell lines that inducibly express individual ISGs. To aid screening, recombinant BUNV that expressed green fluorescent protein (GFP) were employed, including an NSs deletion virus with GFP fused to the Gc, rBUNGceGFPdelNSs, that I created and characterised. By a combination of virus yield assays, Western blotting and fluorescence techniques, three cell lines that inducibly express PKR, viperin or MTAP44 were shown to restrict BUNV replication. More detailed studies revealed PKR to restrict BUNV RNA and protein synthesis, but when PKR was knocked-down in IFN competent A549 cells viral replication was not blocked in cells pre-treated with IFN. Viperin inhibited viral protein synthesis and virally-induced host cell protein synthesis shut-off. Additionally, viral RNA synthesis was restricted by viperin and this was dependent on the CX₃CX₂C motif 1 of viperin. Taken together, these data show that the restriction of BUNV replication mediated by IFN is an accumulated effect of several different ISGs acting on different stages of the viral life cycle.
Type
Thesis, PhD Doctor of Philosophy
Collections
  • Biology Theses
URI
http://hdl.handle.net/10023/3179

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