title: The Rotavirus NSP4 Viroporin Domain is a Calcium-conducting Ion Channel.
abstract: 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 (Ca2+). Nevertheless, some viroporins are known to disrupt host cell Ca2+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 Ca2+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 Ca2+. 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 (Ca2+and Ba2+) as monovalent cations (K+), but a viroporin defective mutant lacked Ca2+conductivity. These data demonstrate that the NSP4 VPD is a Ca2+-conducting viroporin and establish the mechanism by which NSP4 disturbs host cell Ca2+homeostasis.
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, TX, USA.
authors.forename: Thieng
authors.initials: T
authors.lastname: Pham
authors.affiliation: Alkek Center for Metagenomic and Microbiome Research, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Jacob L
authors.initials: JL
authors.lastname: Perry
authors.affiliation: Verna and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Timothy L
authors.initials: TL
authors.lastname: Dosey
authors.affiliation: Department of Biology and Biochemistry, University of Houston, Houston, TX, USA.
authors.forename: Anne H
authors.initials: AH
authors.lastname: Delcour
authors.affiliation: Alkek Center for Metagenomic and Microbiome Research, Department of Molecular Virology and Microbiology, Baylor College of Medicine, Houston, TX, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Hyser
mesh_terms: D000595:Amino Acid Sequence; D001464:Barium / Q000737:chemistry / Q000378:metabolism*; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002784:Cholesterol / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017136:Ion Transport; D008564:Membrane Potentials / Q000502:physiology; D009154:Mutation; D018408:Patch-Clamp Techniques; D010455:Peptides / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010713:Phosphatidylcholines / Q000737:chemistry / Q000378:metabolism; D011510:Proteolipids / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012401:Rotavirus / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D014118:Toxins, Biological / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D053835:Unilamellar Liposomes / Q000737:chemistry / Q000378:metabolism; D017361:Viral Nonstructural Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D015220:Calcium Channels; D006023:Glycoproteins; C081369:NS28 protein, rotavirus; D010455:Peptides; D010713:Phosphatidylcholines; D011510:Proteolipids; D011994:Recombinant Proteins; D014118:Toxins, Biological; D053835:Unilamellar Liposomes; D017361:Viral Nonstructural Proteins; C020485:proteoliposomes; D001464:Barium; C021016:1,2-diphytanoylphosphatidylcholine; C024990:asolectin; D002784:Cholesterol; D002118:Calcium