Induction of nitric oxide synthase by rotavirus enterotoxin NSP4: implication for rotavirus pathogenicity Mohamed A. Borghan, 1 3 Yoshio Mori, 2 Abu-Baker El-Mahmoudy, 3 Naoto Ito, 1 Makoto Sugiyama, 1 Tadashi Takewaki 3 and Nobuyuki Minamoto 1 Correspondence Naoto Ito naotoito@gifu-u.ac.jp 1 Laboratory of Zoonotic Diseases, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan 2 Research Institute for Microbial Diseases, Osaka University, Japan 3 Laboratory of Physiology, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan Received 4 October 2006 Accepted 6 March 2007 Rotavirus non-structural protein (NSP) 4 can induce aqueous secretion in the gastrointestinal tract of neonatal mice through activation of an age- and Ca 2+ -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 (iNOS), 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 10 7 focus-forming units of different rotavirus strains. Ileal iNOS 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 iNOS mRNA expression within 4 h. Furthermore, NSP4 was able to induce iNOS 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 iNOS induction in macrophages was mapped to the reported enterotoxin domain, aa 109–135. Thus, rotavirus infection induces ileal iNOS expression in vivo and rotavirus NSP4 also induces iNOS expression in the ileum and macrophages. Together, these findings suggest that NO plays a role in rotavirus-induced diarrhoea. INTRODUCTION Rotavirus, the most common mammalian enteric viral pathogen, exclusively infects differentiated epithelial cells of intestinal villi in infants and young animals (Kapikian & Chanock, 1990). Rotavirus infection in mammals is associated with blunting of the intestinal villi and mild infiltration of mononuclear cells in lamina propria (Greenberg et al., 1994). Rotavirus non-structural protein 4 (NSP4) and a synthetic peptide corresponding to aa 114– 135 of NSP4 (NSP4 114–135 ) have been shown to cause diarrhoea in suckling mice (Ball et al., 1996; Mori et al., 2002). NSP4 as a viral enterotoxin induces age- and Ca 2+ - dependent movement of halide ions across the plasma membrane in the initial cyclic nucleotide-independent pathway (Dong et al., 1997; Morris et al., 1999). Apart from the direct action of NSP4 on the mucosa at the onset of rotavirus diarrhoea, secondary mechanisms triggered by rotavirus replication and/or NSP4 are thought to sustain the diarrhoeal response. These mechanisms include muco- sal destruction (Davidson & Barnes, 1979; Greenberg et al., 1994), villus ischaemia (Osborne et al., 1991), alteration of paracellular permeability and of expression of digestive enzymes (Jourdan et al., 1998; Dickman et al., 2000), and activation of the enteric nervous system (ENS) (Lundgren et al., 2000). The latter may explain how comparatively few infected cells at the villus tips can induce secretion of electrolytes and water from intestinal crypts. However, it is not known how rotavirus infection activates the ENS. Nevertheless, it is now recognized that the intestinal epithelium can release a wide range of biologically active compounds such as cytokines, prostaglandins and nitric 3Present address: Center for Cancer Rersearch, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892, USA. Journal of General Virology (2007), 88, 2064–2072 DOI 10.1099/vir.0.82618-0 2064 0008-2618 G 2007 SGM Printed in Great Britain