Breakwell, Lucy

2018-05-14T10:16:10Z

2018-05-14T10:16:10Z

2006

The type I interferon (IFN) system is a potent anti-viral innate immune response. It is primed by IFN-p and IFN-0C4, which are the immediately expressed IFNs following detection of virus infection. IFN-P establishes the anti-viral immune response within the infected cell, augments further IFN production through the induced expression of IRF-7, a transcription factor for other IFN-as, and promotes the adaptive immune response. Induction of IFN-P requires the activation of multiple transcription factors, including NF-kB; some of these are maintained in an inactive state within the cytoplasm of the resting cell. PKR is an IFN-induced, dsRNA-activated kinase capable of phosphorylating and activating the IkK, which ultimately releases NF-kB enabling its nuclear translocation. Within the nucleus NF-kB associates with IRF-3 and AP-1 on the IFN-P promoter to induce IFN-P expression. Delineation of the pathways that result in IFN-P expression has revealed viral proteins which target components of these signalling networks. To date no anti-IFN mechanisms have been observed for Semliki Forest virus (SFV), an alphavirus of the Togaviridae. The SFV genome is 11 kb in length and encodes two open reading frames; the non-structural proteins (nsP 1-4), which encode the replicase complex and the structural proteins. Studies with Sindbis virus, a closely related alphavirus have suggested that nsP2 may play a role in IFN suppression. Previous studies with SFV nsP2 observed that 50 % of nsP2 was translocated to the nucleus. When nsP2 nuclear translocation is prevented, the infection has reduced neuropathology.`

This thesis explores the importance of IFN in SFV encephalitis. A quantitative PCR assay for IFN-P and IFN-a transcripts and a quantitative IFN bioassay were developed to determine differences in IFN expression under different infection conditions. Mouse models and primary cell lines were used to establish the importance of PKR for IFN-P expression during SFV infection and to determine whether SFV nsP2 has a role in modulating IFN responses. In the absence of PKR, at early times post-infection, cultured cells reproducibly produced significantly lower levels of IFN-P transcripts. Reduced levels of functional IFN were also demonstrated xii by bioassay. Previous data has shown that PKR is not required for IFN-P induction. The sensitivity of the qPCR assay has allowed the demonstration that PKR, although not critical for IFN induction, is involved in IFN-P induction and is particularly important at early time points post-infection.

SFV-nsP2 has been postulated to be involved in IFN interference. Comparing SFV4 to SFV4-nsP2-RDR (a mutant virus with a single amino acid change within the nuclear localisation signal of nsP2, which prevents its translocation into the nucleus) demonstrated that relative to the number of infected cells, the SFV4nsP2-RDR mutant induced over ten-fold more IFN-P transcripts than the wildtype SFV4 strain; this upregulation was specific to IFN-p. The IFN bioassay results supported this data; SFV4-nsP2-RDR induced higher functional IFN levels in comparison to wt SFV4. Both viruses grew to similar titres and at similar rates. In the mutant and wt infections both NF-kB and IRF-3 translocated into the nucleus; however, preliminary EMSA data has suggested that the amount ofNF-kB bound to the IFN-P promoter is reduced during a wt infection. This suggests a possible mechanism for the differential IFN expression and represents the first IFN evasion mechanism described for Semliki Forest virus.

http://hdl.handle.net/1842/29993

The University of Edinburgh

Annexe Thesis Digitisation Project 2018 Block 18

Already catalogued

Role of interferon in Semliki Forest virus encephalitis

Thesis or Dissertation

Doctoral

PhD Doctor of Philosophy