The Sars Coronavirus Envelope Protein E Targets the Pals1 Tight Junction Factor and Alters Formation of Tight Junctions of Epithelial Cells

This dissertation, "The SARS Coronavirus Envelope Protein E Targets the PALS1 Tight Junction Factor and Alters Formation of Tight Junctions of Epithelial Cells" by Wing-lim, Chan, 陳穎廉, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Tight 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. DOI: 10.5353/th_b4716924 Subjects: Tight junctions (Cell biology) Protein-protein interactions SARS (Disease) - Molecular aspects Coronaviruses