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


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.
Hillenbrand
Bernd
B
Department of Gastroenterology, Infectious Diseases and Rheumatology, Charité Universitätsmedizin Berlin, Berlin, Germany.
Günzel
Dorothee
D
Richter
Jan F
JF
Höhne
Marina
M
Schreier
Eckart
E
Schulzke
Jörg-Dieter
JD
Mankertz
Joachim
J
D000199:Actins / Q000378:metabolism; D017209:Apoptosis; D015153:Blotting, Western; D017250:Caliciviridae Infections / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D003599:Cytoskeleton / Q000378:metabolism* / Q000821:virology; D015967:Gene Expression Regulation, Viral; D019073:HT29 Cells; D006801:Humans; D020287:In Situ Nick-End Labeling; D018613:Microscopy, Confocal; D008856:Microscopy, Fluorescence; D029322:Norovirus / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics*; D014758:Viral Core Proteins / Q000096:biosynthesis / Q000235:genetics*; D017361:Viral Nonstructural Proteins / Q000378:metabolism*
D000199:Actins; D012333:RNA, Messenger; D014758:Viral Core Proteins; D017361:Viral Nonstructural Proteins