Cellular proteins involved in rotavirus infection

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dc.contributor.advisor Taylor, John en
dc.contributor.advisor Bellamy, Dick en
dc.contributor.author Xu, Aimin en
dc.date.accessioned 2007-06-28T22:39:13Z en
dc.date.available 2007-06-28T22:39:13Z en
dc.date.issued 1999 en
dc.identifier THESIS 00-222 en
dc.identifier.citation Thesis (PhD--Biological Sciences)--University of Auckland, 1999 en
dc.identifier.uri http://hdl.handle.net/2292/578 en
dc.description Full text is available to authenticated members of The University of Auckland only. en
dc.description.abstract 1. NSP4 is a rotavirus encoded ER-localised non-structural glycoprotein which is known to play a role in both assembly and pathogenesis of the virus. The aims of this research have been to investigate interactions between NSP4 and host cellular components and thus gain an insight into the molecular basis of cellular dysfunction caused by rotavirus infection. These studies have focussed both on cellular proteins that interact directly with NSP4 and on proteins whose synthesis is induced following NSP4 expression. 2. NSP4 was found to interact with microtubules in vivo and in vitro and a microtubule-binding domain was mapped to within the C-terminal 54 amino acid residues of NSP4 by mutational analysis. Dual immunofluorescent labelling and confocal laser scanning microscopy (CLSM) revealed that NSP4 expressed in Cos-7 cells was aligned with microtubule tracks radiating throughout cytoplasm. This effect was not observed when the microtubule-binding domain of NSP4 was deleted. Morphological and biochemical studies revealed that expression of NSP4 inhibited the transport of membrane proteins between the ER and Golgi complex, causing their arrest in a microtubule-bound membrane structure that probably represents a transport vesicle or vesicular tubular cluster (VTC). The NSP4-mediated arrest in ER-to-Golgi trafficking was reversed when microtubules were depolymerised with nocodazole, indicating that NSP4 probably anchors the transport vesicles to the microtubule cytoskeleton. The finding that ER-to-Golgi transport is blocked by a viral protein that binds to microtubules represents a novel pathogenic mechanism and might also be a useful for cell biologists to dissect the process of microtubule-directed membrane trafficking. 3. The role of microtubules in rotavirus replication was also investigated using nocodazole to depolymerise microtubules in SA11 rotavirus-infected MA104 cells. Depolymerisation of microtubules prior to rotavirus infection completely blocked the expression of virus proteins, suggesting that the transport of incoming rotavirus particles might depend on the microtubule network. When microtubules were disrupted three hours post-infection, virus protein expression was not affected but the production of infectious virions was markedly reduced. Morphological and biochemical analysis of these cells indicates that microtubules are required for assembly of outer capsid proteins on the surface of the rotavirus double-layered particle (DLP), an event that normally occurs in the ER lumen. 4. The upregulated synthesis of eight cellular proteins in cells following expression of NSP4 was identified using comparative two dimensional gel electrophoresis. Five of these upregulated proteins (calreticulin, GRP170, endoplasmin/GRP94, BiP/GRP78 and CaBP1/ERP5) belong to a group of ER lumen molecular chaperones with Ca2+-binding activity. Increased abundance of these ER lumen proteins may contribute to the process of viral maturation by increasing Ca2+ levels in the ER store, a known requirement of rotavirus replication. A further three cellular proteins induced by NSP4 are nucleophosmin, TCTP and Drg1, whose expression has previously been related to cell death, differentiation and proliferation. The identification of these cellular proteins up-regulated by NSP4 may help to understand the mechanisms unerlying NSP4-mediated cell death. en
dc.language.iso en en
dc.publisher ResearchSpace@Auckland en
dc.relation.ispartof PhD Thesis - University of Auckland en
dc.relation.isreferencedby UoA9990727414002091 en
dc.rights Restricted Item. Available to authenticated members of The University of Auckland. en
dc.rights Items in ResearchSpace are protected by copyright, with all rights reserved, unless otherwise indicated. en
dc.rights.uri https://researchspace.auckland.ac.nz/docs/uoa-docs/rights.htm en
dc.title Cellular proteins involved in rotavirus infection en
dc.type Thesis en
thesis.degree.discipline Biological Sciences en
thesis.degree.grantor The University of Auckland en
thesis.degree.level Doctoral en
thesis.degree.name PhD en
dc.rights.holder Copyright: The author en

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