A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells

Previous studies have shown that the nonstructural glycoprotein NSP4 plays a role in rotavirus pathogenesis by functioning as an enterotoxin. One pred iction of the mechanism of action of this enterotoxin was that it is secret ed from virus-infected cells. In this study, the media of cultured (i) inse ct cells infected with a recombinant baculovirus expressing NSP4, (ii) monk ey kidney (MA104) cells infected with the simian (SA11) or porcine attenuat ed (OSU-a) rotavirus, and (iii) human intestinal (HT29) cells infected with SA11 were examined to determine if NSP4 was detectable. Sodium dodecyl sul fate-polyacrylamide gel electrophoresis-Western blotting, immunoprecipitati on and N-terminal amino acid sequencing identified, in the early media from virus-infected cells, a secreted, cleavage product of NSP4 with an apparen t molecular weight of 7,000 that represented amino acids 112 to 175 (NSP4 a a112-175). The secretion of NSP4 aa112-175 was riot affected by treatment o f cells with brefeldin A but was abolished by treatment with nocodazole and cytochalasin D, indicating that secretion of this protein occurs via a non classical, Golgi apparatus-independent mechanism that utilizes the microtub ule and actin microfilament network. A partial gene fragment coding for NSP 4 aa112-175 was cloned and expressed using the baculovirus-insect cell syst em. Purified NSP4 aa112-175 increased intracellular calcium mobilization in intestinal cells when added exogenously, and in insect cells when expresse d endogenously, similarly to full-length NSP4. NSP4 aa112-175 caused diarrh ea in neonatal mice, as did full-length NSP4. These results indicate that N SP4 aa112-175 is a functional NSP4 enterotoxin peptide secreted from rotavi rus-infected cells.