Norovirus non-structural protein p20 leads to impaired restitution of epithelial defects by inhibition of actin cytoskeleton remodelling

Abstract

Objective. Norovirus is the most common cause of acute gastroenteritis in humans worldwide. Typical symptoms are vomiting, nausea and severe watery diarrhea. Because of the lack of cell lines susceptible to human norovirus infection, pathomechanisms and replication cycle are largely unknown. Here, we address the issue of how norovirus infection could lead to epithelial barrier dysfunction. Material and methods. Expression of the non-structural norovirus protein p20 in the epithelial cell line HT-29/B6 was activated through a tetracycline sensitive promoter. Tight junction proteins were studied by Western blot and confocal laser scanning microscopy. Apoptoses were detected in TUNEL stainings. Epithelial restitution was monitored by conductance scanning after induction of single cell lesions. Results. Changes in the expression or localization of the tight junction proteins occludin and/or claudin-1, -2,- 3, -4, -5, -7 and -8 could be ruled out to mediate epithelial barrier modulation. Cell motility was also unaltered by p20. Investigation of epithelial apoptosis revealed an accumulation of apoptic cells in epithelial monolayers after induction of p20 expression. In epithelial cell restitution assays, an arrest was identified in p20 expressing cells. Fluorescence microscopy revealed an inability for condensation and redistribution of cellular actin, which led to a reduced transepithelial electrical resistance. Conclusions. Functional data for norovirus protein p20 suggest a role in modulation of the actin cytoskeleton leading to barrier dysfunction through impairment of restitution of epithelial defects.

Key Words::

• Actin
• apoptosis
• cytoskeleton
• epithelial barrier
• norovirus
• tight junction

Acknowledgements

This work was supported by grant KFO 104 from the Deutsche Forschungsgemeinschaft and by the Sonnenfeld Stiftung. We gratefully acknowledge the technical assistance of Susanna Schön and Detlef Sorgenfrei.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.