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. 1. Introduction 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]. During infection, 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]. The initiated increases in intracellular, calcium levels fail to induce the chloride secretory response [4–6]. Traditionally defined as an ER glycoprotein, NSP4 contains a single transmembrane domain that serves to anchor the protein into the ER membrane, such that a short N-terminal domain (amino acids [aa] 1–24) remains within the lumen of the ER while the longer C-terminus (aa 45–175) extends into the cytoplasm [7]. Interactions with numerous viral and cellular proteins occur within the extended C-terminal tail of NSP4 [8–12]. Also contained within the C-terminal cytoplasmic tail is an amphipathic α-helix (AAH), coiled-coil domain (aa 95–137) [13, 14]. Cross-linking and crystallographic experiments reveal that this region of NSP4
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