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Assays for the Specific Growth Rate and Cell-binding Ability of Rotavirus
Published on: January 28, 2019
A functional NSP4 enterotoxin peptide secreted from rotavirus-infected cells
M Zhang1, C Q Zeng, A P Morris
1Division of Molecular Virology, Baylor College of Medicine, University of Texas Health Science Center, Houston, Texas 77030, USA.
Journal of Virology
|November 23, 2000
Summary
Rotavirus nonstructural glycoprotein NSP4, a known enterotoxin, is secreted from infected cells as a functional peptide (NSP4 aa112-175). This peptide causes diarrhea and calcium mobilization, indicating its role in rotavirus pathogenesis.
Area of Science:
- Virology
- Molecular Biology
- Cell Biology
Background:
- Rotavirus nonstructural glycoprotein 4 (NSP4) is implicated in pathogenesis as an enterotoxin.
- The mechanism of NSP4 enterotoxin activity suggests it is secreted from infected cells.
Purpose of the Study:
- To investigate the secretion of NSP4 from rotavirus-infected cells.
- To identify and characterize the secreted form of NSP4 and its functional role.
Main Methods:
- Detection of NSP4 in culture media of infected insect, monkey kidney (MA104), and human intestinal (HT29) cells using SDS-PAGE Western blotting and immunoprecipitation.
- N-terminal amino acid sequencing to identify the secreted peptide.
- Treatment with brefeldin A, nocodazole, and cytochalasin D to elucidate the secretion pathway.
- Cloning and expression of the NSP4 aa112-175 fragment in a baculovirus-insect cell system.
- Assessment of intracellular calcium mobilization and induction of diarrhea in neonatal mice.
Main Results:
- A secreted, cleaved fragment of NSP4 (NSP4 aa112-175) was identified in the media of infected cells.
- Secretion of NSP4 aa112-175 utilizes a nonclassical, Golgi-independent pathway involving microtubules and actin filaments.
- Exogenously added or endogenously expressed NSP4 aa112-175 induced intracellular calcium mobilization.
- NSP4 aa112-175 caused diarrhea in neonatal mice, similar to full-length NSP4.
Conclusions:
- NSP4 aa112-175 is a functional enterotoxin peptide.
- This peptide is secreted from rotavirus-infected cells via a nonclassical pathway.
- The findings elucidate a key mechanism of rotavirus pathogenesis involving secreted NSP4.
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