Role of Rotavirus Enterotoxin NSP4 in the Inflammatory Response in Murine Macrophage RAW 264.7 Cells

ABSTRACT

The rotavirus nonstructural glycoprotein, NSP4, has been identified as the first viral enterotoxin capable of inducing diarrhea. To investigate the biological function of NSP4 in the inflammatory process, a cDNA from human rotavirus (Wa strain) RNA segment 10 was amplified by RT-PCR, cloned into TA vector, and subsequently subcloned into pET23b expression plasmid. The expression of NSP4 protein was determined by SDS-PAGE and Western blotting, then, the protein was purified by affinity chromatography on Ni-NTA-agarose column. The inflammatory effects of NSP4, namely, production of nitric oxide (NO), pro-inflammatory cytokines (IL-1β, IL-6, IL-10, and TNF-α), and prostaglandin E2 (PGE₂), was evaluated using NSP4-stimulated RAW 264.7 murine macrophages and compared with those observed after stimulation with lipopolysaccharide (LPS). The levels of IL-1β, IL-6, and TNF-α were significantly increased, and those of NO and PGE₂ also increased in NSP4-stimulated RAW 264.7 cells. These findings indicate that NSP4 plays an important role in the inflammatory response observed during rotavirus infection.