The Rotavirus NSP4 Viroporin Domain is a Calcium-conducting Ion Channel

<jats:title>Abstract</jats:title><jats:p>Viroporins are small virus-encoded ion channel proteins. Most viroporins are monovalent selective cation channels, with few showing the ability to conduct divalent cations, like calcium (Ca<jats:sup>2+</jats:sup>). Nevertheless, some viroporins are known to disrupt host cell Ca<jats:sup>2+</jats:sup> homeostasis, which is critical for virus replication and pathogenesis. Rotavirus nonstructural protein 4 (NSP4) is an endoplasmic reticulum transmembrane glycoprotein that has a viroporin domain (VPD), and NSP4 viroporin activity elevates cytosolic Ca<jats:sup>2+</jats:sup> in mammalian cells. The goal of this study was to demonstrate that the NSP4 VPD forms an ion channel and determine whether the channel can conduct Ca<jats:sup>2+</jats:sup>. Using planar lipid bilayer and liposome patch clamp electrophysiology, we show that a synthetic peptide of the NSP4 VPD has ion channel activity. The NSP4 VPD was selective for cations over anions and channel activity was observed to have both well-defined “square top” openings as well as fast current fluctuations, similar to other viroporins. Importantly, the NSP4 VPD showed similar conductance of divalent cations (Ca<jats:sup>2+</jats:sup> and Ba<jats:sup>2+</jats:sup>) as monovalent cations (K<jats:sup>+</jats:sup>), but a viroporin defective mutant lacked Ca<jats:sup>2+</jats:sup> conductivity. These data demonstrate that the NSP4 VPD is a Ca<jats:sup>2+</jats:sup>-conducting viroporin and establish the mechanism by which NSP4 disturbs host cell Ca<jats:sup>2+</jats:sup> homeostasis.</jats:p>