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 prediction of the mechanism of action of this enterotoxin was that it is secreted from virus-infected cells. In this study, the media of cultured (i) insect cells infected with a recombinant baculovirus expressing NSP4, (ii) monkey kidney (MA104) cells infected with the simian (SA11) or porcine attenuated (OSU-a) rotavirus, and (iii) human intestinal (HT29) cells infected with SA11 were examined to determine if NSP4 was detectable. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis-Western blotting, immunoprecipitation and N-terminal amino acid sequencing identified, in the early media from virus-infected cells, a secreted, cleavage product of NSP4 with an apparent molecular weight of 7,000 that represented amino acids 112 to 175 (NSP4 aa112-175). The secretion of NSP4 aa112-175 was not affected by treatment of cells with brefeldin A but was abolished by treatment with nocodazole and cytochalasin D, indicating that secretion of this protein occurs via a nonclassical, Golgi apparatus-independent mechanism that utilizes the microtubule and actin microfilament network. A partial gene fragment coding for NSP4 aa112-175 was cloned and expressed using the baculovirus-insect cell system. Purified NSP4 aa112-175 increased intracellular calcium mobilization in intestinal cells when added exogenously, and in insect cells when expressed endogenously, similarly to full-length NSP4. NSP4 aa112-175 caused diarrhea in neonatal mice, as did full-length NSP4. These results indicate that NSP4 aa112-175 is a functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells.
Zhang
M
M
Division of Molecular Virology, Baylor College of Medicine, University of Texas Health Science Center, Houston, Texas 77030, USA.
Zeng
C Q
CQ
Morris
A P
AP
Estes
M K
MK
D000818:Animals; D002460:Cell Line; D006023:Glycoproteins / Q000378:metabolism*; D000882:Haplorhini; D006801:Humans; D051379:Mice; D011991:Receptors, Virus / Q000378:metabolism; D012401:Rotavirus / Q000502:physiology*; D012400:Rotavirus Infections / Q000378:metabolism* / Q000821:virology*; D013552:Swine; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins / Q000378:metabolism*; D014779:Virus Replication
D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D011991:Receptors, Virus; D014118:Toxins, Biological; D017361:Viral Nonstructural Proteins