title: Induction of nitric oxide synthase by rotavirus enterotoxin NSP4: implication for rotavirus pathogenicity.
abstract: 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.
authors.affiliation: Laboratory of Zoonotic Diseases, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Borghan
authors.affiliation: Research Institute for Microbial Diseases, Osaka University, Japan.
authors.forename: Yoshio
authors.initials: Y
authors.lastname: Mori
authors.affiliation: Laboratory of Physiology, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Abu-Baker
authors.initials: AB
authors.lastname: El-Mahmoudy
authors.affiliation: Laboratory of Zoonotic Diseases, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Naoto
authors.initials: N
authors.lastname: Ito
authors.affiliation: Laboratory of Zoonotic Diseases, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Sugiyama
authors.affiliation: Laboratory of Physiology, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Tadashi
authors.initials: T
authors.lastname: Takewaki
authors.affiliation: Laboratory of Zoonotic Diseases, Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, 1-1 Yanagido, Gifu 501-1193, Japan.
authors.forename: Nobuyuki
authors.initials: N
authors.lastname: Minamoto
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D002460:Cell Line; D018076:DNA, Complementary / Q000235:genetics; D004768:Enterotoxins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D004790:Enzyme Induction / Q000187:drug effects; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D007082:Ileum / Q000187:drug effects / Q000201:enzymology; D066298:In Vitro Techniques; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D051379:Mice; D009569:Nitric Oxide / Q000096:biosynthesis; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis* / Q000235:genetics; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000633:toxicity; D012401:Rotavirus / Q000235:genetics / Q000472:pathogenicity* / Q000502:physiology; D012400:Rotavirus Infections / Q000201:enzymology / Q000235:genetics / Q000821:virology; D014118:Toxins, Biological / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D015854:Up-Regulation / Q000187:drug effects; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000633:toxicity*; D014774:Virulence; D014779:Virus Replication
chemical_list: D018076:DNA, Complementary; D004768:Enterotoxins; D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse