Characterisation Of Intracellular Replication And Pathogenesis of Murine Norovirus 1

Jennifer Hyde (2010). Characterisation Of Intracellular Replication And Pathogenesis of Murine Norovirus 1 PhD Thesis, School of Chemistry and Molecular Biosciences, The University of Queensland.

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Author Jennifer Hyde
Thesis Title Characterisation Of Intracellular Replication And Pathogenesis of Murine Norovirus 1
School, Centre or Institute School of Chemistry and Molecular Biosciences
Institution The University of Queensland
Publication date 2010-11
Thesis type PhD Thesis
Supervisor Associate Professor Jason Mackenzie
Professor Alexander Khromykh
Total pages 205
Total colour pages 39
Total black and white pages 166
Subjects 06 Biological Sciences
Abstract/Summary Human Noroviruses (HuNoVs; family Caliciviridae) are the leading cause acute of non-bacterial gastroenteritis worldwide. Despite the prevalence of these viruses within the community there are no specific antiviral therapies or vaccines available to treat or prevent HuNoV infection. As there are no reliable HuNoV culture systems in existence, study of NoV replication, pathogenesis, and immunity has been largely hindered, and relatively little is known about the intracellular events associated with NoV replication. In 2003, isolation of a novel mouse NoV (MNV-1) from immunodeficient laboratory mice provided the first opportunity to study NoV pathogenesis and immunity in a small animal model. This study highlighted the importance of the signal transducer and activator of transcription 1 (STAT1)-mediated innate immune response in protection against lethal MNV-1 infection in immunocompromised mice. Subsequent to this discovery, the identification of the tropism of MNV-1 for macrophage and dendritic cells provided the first reliable tissue culture system for the study of NoV replication, pathogenesis, and immunity. Using MNV-1 as a model for the study of NoV replication, key events associated with MNV-1 replication and pathogenesis were identified and characterised. Specifically, the subcellular localisation of the viral replication complex (RC) as well as the individual open reading frame 1 (ORF1) proteins was identified. It was observed that MNV-1 RNA replication is closely associated with virus-induced vesicular clusters that accumulate in the cytoplasm of infected cells. It was subsequently demonstrated that these virus-induced membranes structures were derived from components of the host secretory pathway, namely the endoplasmic reticulum, the Golgi apparatus, endosomes, and lipids. Individually expressed ORF1 proteins were also shown to exhibit unique subcellular localisation profiles, with NS1-2 localising with markers for the endoplasmic reticulum, NS3 with the Golgi apparatus and intracellular lipids, NS4 with endosomes and the Golgi apparatus, and NS6 with mitochondria. Based on the subcellular localisation patterns observed, potential roles for the individual viral ORF1 proteins in membrane association and proliferation, host cell trafficking, and immune signalling and apoptosis have been proposed. These studies were subsequently extended to examine the role of other cellular components in MNV-1 replication, particularly the role of the cytoskeleton, as it was observed that the MNV-1 RC was closely aligned with the centrosome. It was demonstrated that the microtubule and actin networks play an important role in efficient MNV-1 replication and that the virus also interacts with and recruits acetylated tubulin to the RC. It was subsequently demonstrated that NS3, NS4, and the major structural protein VP1 interact with acetylated tubulin and therefore likely mediate this interaction between MNV-1 and acetylated microtubules. In addition to exploring the role of host components during MNV-1 replication potential strategies used by MNV-1 to evade the host innate immune response was also examined. MNV-1 replication was shown to prevent the phosphorylation of STAT1 in the presence of interferon (IFN) thereby inhibiting the IFN response. It was subsequently demonstrated that the individual N-terminal ORF1 proteins (NS1-2, NS3, and NS4) were able to prevent the translocation of STAT1 and phosphorylated STAT1 to the cell nucleus in the presence of IFN. Additionally, the ability of MNV-1 to modulate other arms of the innate immune response was examined, principally the production of key innate immune cytokines. Here it was shown that during infection MNV-1 was able to inhibit the transcriptional upregulation and production of tumour necrosis factor alpha (TNF-α), interleukin-6 (IL-6), Regulated upon activation, Normal T-cell Expressed, and Secreted (RANTES), and IFN-β, cytokines which play key roles in activating and regulating the innate and adaptive immune responses. Overall, this study illuminates some of the key intracellular events associated with MNV-1 replication and pathogenesis which were previously unknown, including the intracellular localisation of the MNV-1 RC, the potential role of the ORF1 proteins in replication, the exploitation of specific cellular components during replication, and the perturbation of the innate immune response. In the absence of a viable HuNoV culture system, MNV-1 provides an ideal model to study many aspects of NoV biology and replication, and the findings outlined in this study provide important preliminary insights into key events associated with NoV replication and pathogenesis. By better understanding the replication events associated with NoV infection, effective treatments, vaccines, and containment strategies can be employed to control the incidence and spread of this group of medically important viruses.
Keyword norovirus
Additional Notes 29, 37-38, 41, 43, 53, 80, 82-86, 90, 93, 102, 105-107, 109-110, 112-115, 120, 125-127, 129-130, 132-133, 135, 137, 150-151, 203-205

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Created: Tue, 21 Jun 2011, 14:51:17 EST by Ms Jennifer Hyde on behalf of Library - Information Access Service