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postgraduate thesis: The SARS coronavirus envelope protein E targets the PALS1 tight junction factor and alters formation of tight junctions of epithelialcells

TitleThe SARS coronavirus envelope protein E targets the PALS1 tight junction factor and alters formation of tight junctions of epithelialcells
Authors
Advisors
Issue Date2011
PublisherThe University of Hong Kong (Pokfulam, Hong Kong)
Citation
Chan, W. [陳穎廉]. (2011). The SARS coronavirus envelope protein E targets the PALS1 tight junction factor and alters formation of tight junctions of epithelial cells. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4716924
AbstractTight junctions, as zones of close contact between epithelial and endothelial cells, form a physical barrier as one of the first host defense strategies that prevent the intrusion of pathogens across epithelia and endothelia. Recently, an interaction between the Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) envelope protein (E) and PALS1, a member of the CRB tight junction complex, was identified in the Virus-Host Interaction group at HKU-Pasteur Research Centre (Teoh et al, 2010). In this report, I present in vitro data which helps to better understand how this protein-protein interaction could interfere with the formation and maintenance of tight junctions at the apical domain of epithelial cells. In previous research, the interaction between E and PALS1 was identified through a yeast two-hybrid screen and confirmed in vitro. A PDZ-binding motif (PBM) was identified at the C-terminal end of E, which interacts with the PDZ domain of PALS1. The objective of my research was to further enhance the knowledge of this interaction by studying the effect of E expression on PALS1 localization and tight junction structure in epithelial cells. I have shown that expression of E is associated with a partial relocalization of PALS1 to the Golgi compartment. Also, I discovered that when wild-type E, E(wt), was expressed in the MDCKII cell model, the time required for tight junction formation was extended to 6-8 hours, while normal cells only required two hours. Interestingly, expression of the E protein with a deletion of the PBM, E(ΔPBM) did not affect the timing of tight junction formation. This finding indicates that the PBM plays a critical role in the process of alteration of tight junctions mediated by E, most likely through its interaction with PALS1. Furthermore, the localization pattern of E was altered when its PBM was deleted. In the MDCKII model, E(wt) located, as expected, at membranes of the Golgi compartment, whereas E(ΔPBM) had a diffused distribution in the cytosol. This observation suggests that the PBM acts as a localization signal for the E protein to the Golgi region, which is the assembly site of the virus. Finally, to examine the role of the PBM in the context of the whole virus, I participated in the production of SARS-CoV recombinant viruses, with mutations in the PBM of E. Though this work is still in progress, the use of these viruses should help to delineate the role of E PBM in SARS-CoV induced pathogenesis in vitro and ultimately in vivo.
DegreeMaster of Philosophy
SubjectProtein-protein interactions.
SARS (Disease) - Molecular aspects.
Coronaviruses.
Tight junctions (Cell biology)
Dept/ProgramPathology

 

DC FieldValueLanguage
dc.contributor.advisorNal-Rogier, BTM-
dc.contributor.advisorNicholls, JM-
dc.contributor.authorChan, Wing-lim.-
dc.contributor.author陳穎廉.-
dc.date.issued2011-
dc.identifier.citationChan, W. [陳穎廉]. (2011). The SARS coronavirus envelope protein E targets the PALS1 tight junction factor and alters formation of tight junctions of epithelial cells. (Thesis). University of Hong Kong, Pokfulam, Hong Kong SAR. Retrieved from http://dx.doi.org/10.5353/th_b4716924-
dc.description.abstractTight junctions, as zones of close contact between epithelial and endothelial cells, form a physical barrier as one of the first host defense strategies that prevent the intrusion of pathogens across epithelia and endothelia. Recently, an interaction between the Severe Acute Respiratory Syndrome-Coronavirus (SARS-CoV) envelope protein (E) and PALS1, a member of the CRB tight junction complex, was identified in the Virus-Host Interaction group at HKU-Pasteur Research Centre (Teoh et al, 2010). In this report, I present in vitro data which helps to better understand how this protein-protein interaction could interfere with the formation and maintenance of tight junctions at the apical domain of epithelial cells. In previous research, the interaction between E and PALS1 was identified through a yeast two-hybrid screen and confirmed in vitro. A PDZ-binding motif (PBM) was identified at the C-terminal end of E, which interacts with the PDZ domain of PALS1. The objective of my research was to further enhance the knowledge of this interaction by studying the effect of E expression on PALS1 localization and tight junction structure in epithelial cells. I have shown that expression of E is associated with a partial relocalization of PALS1 to the Golgi compartment. Also, I discovered that when wild-type E, E(wt), was expressed in the MDCKII cell model, the time required for tight junction formation was extended to 6-8 hours, while normal cells only required two hours. Interestingly, expression of the E protein with a deletion of the PBM, E(ΔPBM) did not affect the timing of tight junction formation. This finding indicates that the PBM plays a critical role in the process of alteration of tight junctions mediated by E, most likely through its interaction with PALS1. Furthermore, the localization pattern of E was altered when its PBM was deleted. In the MDCKII model, E(wt) located, as expected, at membranes of the Golgi compartment, whereas E(ΔPBM) had a diffused distribution in the cytosol. This observation suggests that the PBM acts as a localization signal for the E protein to the Golgi region, which is the assembly site of the virus. Finally, to examine the role of the PBM in the context of the whole virus, I participated in the production of SARS-CoV recombinant viruses, with mutations in the PBM of E. Though this work is still in progress, the use of these viruses should help to delineate the role of E PBM in SARS-CoV induced pathogenesis in vitro and ultimately in vivo.-
dc.languageeng-
dc.publisherThe University of Hong Kong (Pokfulam, Hong Kong)-
dc.relation.ispartofHKU Theses Online (HKUTO)-
dc.rightsThe author retains all proprietary rights, (such as patent rights) and the right to use in future works.-
dc.rightsCreative Commons: Attribution 3.0 Hong Kong License-
dc.source.urihttp://hub.hku.hk/bib/B47169242-
dc.subject.lcshProtein-protein interactions.-
dc.subject.lcshSARS (Disease) - Molecular aspects.-
dc.subject.lcshCoronaviruses.-
dc.subject.lcshTight junctions (Cell biology)-
dc.titleThe SARS coronavirus envelope protein E targets the PALS1 tight junction factor and alters formation of tight junctions of epithelialcells-
dc.typePG_Thesis-
dc.identifier.hkulb4716924-
dc.description.thesisnameMaster of Philosophy-
dc.description.thesislevelMaster-
dc.description.thesisdisciplinePathology-
dc.description.naturepublished_or_final_version-
dc.identifier.doi10.5353/th_b4716924-
dc.date.hkucongregation2012-

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