Abstract
Rotavirus (RV) NSP4, the first described viral enterotoxin, is a multifunctional glycoprotein that contributes to viral pathogenesis, morphogenesis, and replication. NSP4 binds both termini of caveolin-1 and is isolated from caveolae fractions that are rich in anionic phospholipids and cholesterol. These interactions indicate that cholesterol/caveolin-1 plays a role in NSP4 transport to the cell surface, which is essential to its enterotoxic activity. Synthetic peptides were utilized to identify target(s) of intervention by exploring the NSP4-caveolin-1 and -cholesterol interactions. NSP4112–140 that overlaps the caveolin-1 binding domain and a cholesterol recognition amino acid consensus (CRAC) motif and both termini of caveolin-1 (N-caveolin-12–20, 19–40 and C-caveolin-1161–180) were synthesized. Direct fluorescence-binding assays were employed to determine binding affinities of the NSP4-caveolin-1 peptides and cholesterol. Intracellular cholesterol alteration revealed a redistribution of NSP4 and disintegration of viroplasms. These data further imply interruption of NSP4112–140-N-caveolin-119–40 and cholesterol interactions may block NSP4 intracellular transport, hence enterotoxicity.
Highlights
As the leading cause of gastroenteritis in young children under the age of five, rotavirus (RV) infections annually are responsible for approximately 600,000 deaths worldwide [1, 2]
Rotavirus (RV) nonstructural protein 4 (NSP4), the first described viral enterotoxin, is a multifunctional glycoprotein that contributes to viral pathogenesis, morphogenesis, and replication
Our studies reveal the exposure of the NSP4 C-terminus and the enterotoxic region, but not the Nterminus, on the outer plasma membrane (PM) leaflet and subsequent release into culture media at a time when the membrane is intact as verified by a lack of exposure of cav-1 and other intracellular markers [34,35,36]
Summary
As the leading cause of gastroenteritis in young children under the age of five, rotavirus (RV) infections annually are responsible for approximately 600,000 deaths worldwide [1, 2]. The RV nonstructural protein 4 (NSP4) functions as a viral enterotoxin by binding an extracellular receptor and activating a signal transduction pathway which increases intracellular calcium ([Ca2+]i) levels through the release of ER calcium stores [3, 4]. This increase in [Ca2+]i induces secretory chloride currents which result in diarrhea, but only when initiated from the exofacial leaflet of the plasma membrane (PM) [3]. Contained within the Cterminal cytoplasmic tail is an amphipathic α-helix (AAH), coiled-coil domain (aa 95–137) [13, 14]. Residing within the amphipathic α-helix, coiled-coil domain is the enterotoxic peptide region (aa 114–135) as well as the caveolin-1 (cav-1) binding domain [16] and a putative cholesterol recognition amino acid consensus (CRAC) sequence [17]
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