Intracellularly expressed rotavirus NSP4 stimulates release of serotonin (5-HT) from human enterochromaffin cells
(English)Manuscript (preprint) (Other academic)
Rotavirus (RV) is associated with diarrhoea and vomiting, but the mechanisms behind these symptoms remain unresolved. While RV have been shown to infect and stimulate secretion of serotonin (5-hydroxytryptamine; 5-HT) from human enterochromaffin (EC) cells and to infect EC cells in the small intestine of mice, it remains to identify which intracellularly expressed viral protein (VP) being responsible for this novel property.
To address this issue, human EC cells were transfected with small interfering RNA (siRNA) targeting the structural (VP4, VP6 and VP7) and the non-structural protein 4 (NSP4) followed by infection with Rhesus rotavirus (RRV). siRNA specific to NSP4 (siRNANSP4) significantly attenuated secretion of 5-HT compared to siRNAVP4, siRNAVP6 , siRNAVP7 and non-targeting (Nt) siRNAnt. Intracellular calcium clamping with BABTA/AM showed that intracellularly expressed NSP4-stimulated secretion of 5-HT from EC cells was calcium-dependent. Furthermore RV down-regulated the 5-HT transporter (SERT) mRNA in ileum but not tryptophan hydroxylase 1 (TPH1) mRNA the rate-limiting enzyme for 5-HT synthesis. The unaffected expression of TPH1 mRNA in the intestinal segments suggests that release of 5- HT primarily originates from pre-made 5-HT rather than from newly synthesised 5-HT mRNA. Moreover, down-regulation of SERT mRNA in ileum presumably resulted in reduced re- uptake of 5-HT by SERT to EC cells and thus increased extracellular 5-HT in the small intestine. Moreover, 7/7 infant mice responded following intraperitoneal administration of 5-HT with rapid (<30 min) diarrhoea in dose-dependent manner. In the light of these results and the fact that both 5-HT and NSP4 can induce diarrhoea in mice, a disease mechanism to RV diarrhoea is proposed.
Microbiology Cell and Molecular Biology
IdentifiersURN: urn:nbn:se:liu:diva-117893OAI: oai:DiVA.org:liu-117893DiVA: diva2:811727