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

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). Nevertheless, some viroporins are known to disrupt host cell Ca 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 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. 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 and Ba) as monovalent cations (K), but a viroporin defective mutant lacked Ca conductivity. These data demonstrate that the NSP4 VPD is a Ca-conducting viroporin and establish the mechanism by which NSP4 disturbs host cell Ca homeostasis.