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. INTRODUCTIONRotavirus, 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 ) 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 mucosal 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 fro...

Activation of COX-2/PGE 2 Promotes Sapovirus Replication via the Inhibition of Nitric Oxide Production

“…NO secretion can be triggered in vitro and in vivo by rotavirus nonstructural protein 4 (NSP4) (28–30), which in turn may cause diarrhea by elevating intestinal permeability (31, 32) and regulating intestinal motility (33) and intestinal ion transport (34, 35). In contrast to rotavirus infection (28–30), little NO production was observed during PSaV replication in LLC-PK cells, possibly due to the synthesis of PGE 2 , suggesting that NO may not be involved in sapovirus-induced diarrhea. However, it is important to note that we cannot fully rule out a potential role for NO in PSaV pathogenesis in vivo , where the cellular response to infection is influenced by numerous cell types and may not be entirely reproduced in immortalized cells in vitro .…”

Activation and function of murine primary microglia in the absence of the prion protein

“…To this end, we stimulated the cells with LPS and quantified the number of cells expressing the inducible NO synthase (Borghan et al, 2007). Treatment with LPS induced increased number of iNOS expressing cells and fluorescence intensity, but the quantity of iNOS- positive cells and the intensity of the staining did not differ between cells from both genotypes (Fig.…”

Human Astroviruses

“…Although it has not yet been proved whether iNOS activity is also relevant in controlling the infection in mice as happens in avian species and which cell type would produce it, it has been demonstrated that it plays a role in protecting mice from norovirus infection (143), suggesting that it might play a role against astroviruses as well. On the other hand, however, since NO has also been shown to regulate intestinal mucosal permeability and ion transport, a role of NO as a mediator of diarrhea caused by other viral infections such as rotaviruses has also been demonstrated (144,145).…”