The family Hantaviridae, of the Bunyavirales order, contains many important human pathogens of which Puumala virus (PUUV) is the most widely distributed member in Europe. It causes nephropathia epidemica, a milder form of haemorrhagic fever with renal syndrome and mortality rates of up to 1% have been reported. They are enveloped viruses, with a tripartite single-stranded negative sense RNA genome, that replicate solely in the cell cytoplasm. Several factors have been proposed to play a role in hantavirus pathogenicity, including regulation of innate immune responses, cell signalling and enhancement of endothelial cell permeability. The work presented in this thesis describes biological and molecular characterisation of the mechanisms behind a hantavirus infection. Transcriptome analysis was a valuable tool that allowed the investigation of the broader picture of the effect of PUUV on the host cell. 549 and as many as 7,283 genes were differentially expressed at 24 and 48 hours post infection, respectively, in PUUV-infected cells, revealing extensive transcriptional change. By 48 hours normal cellular function appeared severely disrupted. Most genes involved in mitochondrial functioning were down-regulated, suggesting a reduced cellular energy level. Dysregulation of an important signalling hub such as mitochondria might have a more global impact on cellular functions, consistent with findings in this study. Intrinsic apoptosis pathway, which is mediated by mitochondria, appeared inhibited. Whereas, death receptor signalling was activated. Pathways associated with actin formation, organisation and signalling also appeared inhibited. Members of Rho family of GTPases, which are key regulators of actin dynamics, were down-regulated overall. Furthermore, integrin signalling, which mediates Rho GTPase activity, was also inhibited. Immunofluorescence studies revealed marked morphological changes in mitochondria and substantial remodelling of the actin cytoskeleton. Further analysis revealed a direct interaction between PUUV N protein and anillin, a scaffolding protein that mediates formation and organisation of actin filaments, suggesting a potential novel mechanism behind actin cytoskeleton reorganisation. Biological interferon (IFN) assays enabled the identification of two IFN antagonists encoded by PUUV, the cytoplasmic tail of the Gn glycoprotein and the non-structural protein, NSs. The Gn tail inhibited type I IFN induction at the level of TRAF3-TBK, in agreement with previous studies on other hantaviruses. Whereas, NSs was found to block IFN induction downstream of IRF3, suggesting it was able to disrupt transcription or translation. Utilising immunofluorescence and chromatin immunoprecipitation methods, it was found that PUUV NSs possessed a potential mechanism to inhibit transcription by blocking serine 2 phosphorylation at the C-terminal domain of RNA polymerase II in a similar manner to the previously described Bunyamwera virus NSs. The data presented in this thesis illustrates the broad range of mechanisms employed by PUUV to alter cell function to aid virus replication and subvert innate immune responses.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:754388 |
| Date | January 2018 |
| Creators | Koudriakova, Elina |
| Publisher | University of Glasgow |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://theses.gla.ac.uk/30771/ |
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