Induction of nitric oxide synthase by rotavirus enterotoxin NSP4: implication for rotavirus pathogenicity

Rotavirus non-structural protein (NSP) 4 can induce aqueous secretion in the gastrointestinal tract of neonatal mice through activation of an age- and Ca2+ -dependent plasma membrane anion permeability. Accumulating evidence suggests that nitric oxide (NO) plays a role in the modulation of aqueous secretion and the barrier function of intestinal cells. This study investigated transcriptional changes in inducible NO synthase (NOS), an enzyme responsible for NO production, after rotavirus infection in mice and after treatment of intestinal cells with NSP4. Diarrhoea was observed in 5-day-old CD-1 mice from days 1 to 3 after inoculation with 107 focus-forming units of different rotavirus strains. Ileal NOS mRNA expression was induced as early as 6 h post-inoculation, before the onset of clinical diarrhoea in infected mice, and was upregulated during the course of rotavirus-induced diarrhoea. Ex vivo treatment of ilea excised from CD-1 suckling mice with NSP4 resulted in upregulation of ileal NOS mRNA expression within 4 h. Furthermore, NSP4 was able to induce NOS expression and NO production in murine peritoneal macrophages and RAW264.7 cells. The specificity of NSP4 inducibility was confirmed by the inhibitory effect of anti-NSP4 serum. Using a series of truncated NSP4s, the domain responsible for NOS induction in macrophages was mapped to the reported enterotoxin domain, aa 109-135. Thus, rotavirus infection induces ileal NOS expression in vivo and rotavirus NSP4 also induces NOS expression in the ileum and macrophages. Together, these findings suggest that NO plays a role in rotavirus-induced diarrhoea.