title: Phosphoinositides direct equine infectious anemia virus gag trafficking and release.
abstract: Phosphatidylinositol 4,5-biphosphate [PI(4,5)P(2) ], the predominant phosphoinositide (PI) on the plasma membrane, binds the matrix (MA) protein of human immunodeficiency virus type 1 (HIV-1) and equine infectious anemia virus (EIAV) with similar affinities in vitro. Interaction with PI(4,5)P(2) is critical for HIV-1 assembly on the plasma membrane. EIAV has been shown to localize in internal compartments; hence, the significance of its interaction with PI(4,5)P(2) is unclear. We therefore investigated the binding in vitro of other PIs to EIAV MA and whether intracellular association with compartments bearing these PIs was important for assembly and release of virus-like particles (VLPs) formed by Gag. In vitro, EIAV MA bound phosphatidylinositol 3-phosphate [PI(3)P] with higher affinity than PI(4,5)P(2) as revealed by nuclear magnetic resonance (NMR) spectra upon lipid titration. Gag was detected on the plasma membrane and in compartments enriched in phosphatidylinositol 3,5-biphosphate [PI(3,5)P(2) ]. Treatment of cells with YM201636, a kinase inhibitor that blocks production of PI(3,5)P(2) from PI(3)P, caused Gag to colocalize with aberrant compartments and inhibited VLP release. In contrast to HIV-1, release of EIAV VLPs was not significantly diminished by coexpression with 5-phosphatase IV, an enzyme that specifically depletes PI(4,5)P(2) from the plasma membrane. However, coexpression with synaptojanin 2, a phosphatase with broader specificity, diminished VLP production. PI-binding pocket mutations caused striking budding defects, as revealed by electron microscopy. One of the mutations also modified Gag-Gag interaction, as suggested by altered bimolecular fluorescence complementation. We conclude that PI-mediated targeting to peripheral and internal membranes is a critical factor in EIAV assembly and release.
authors.affiliation: Department of Molecular Genetics & Microbiology, Life Sciences Building, Room 248, Stony Brook University, Stony Brook, NY 11794, USA.
authors.forename: Fiona
authors.initials: F
authors.lastname: Fernandes
authors.forename: Kang
authors.initials: K
authors.lastname: Chen
authors.forename: Lorna S
authors.initials: LS
authors.lastname: Ehrlich
authors.forename: Jing
authors.initials: J
authors.lastname: Jin
authors.forename: Min H
authors.initials: MH
authors.lastname: Chen
authors.forename: Gisselle N
authors.initials: GN
authors.lastname: Medina
authors.forename: Marc
authors.initials: M
authors.lastname: Symons
authors.forename: Ronald
authors.initials: R
authors.lastname: Montelaro
authors.forename: Julie
authors.initials: J
authors.lastname: Donaldson
authors.forename: Nico
authors.initials: N
authors.lastname: Tjandra
authors.forename: Carol A
authors.initials: CA
authors.lastname: Carter
mesh_terms: D017766:Acid Anhydride Hydrolases / Q000378:metabolism; D000631:Aminopyridines / Q000494:pharmacology; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002522:Chlorocebus aethiops; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D006575:Heterocyclic Compounds, 3-Ring / Q000494:pharmacology; D006736:Horses; D006801:Humans; D004860:Infectious Anemia Virus, Equine / Q000235:genetics / Q000378:metabolism* / Q000502:physiology; D009154:Mutation; D018129:Phosphatidylinositol Phosphates / Q000037:antagonists & inhibitors / Q000096:biosynthesis / Q000378:metabolism*; D011485:Protein Binding / Q000502:physiology; D021381:Protein Transport; D014162:Transfection; D019065:Virus Assembly / Q000187:drug effects / Q000502:physiology
chemical_list: C540576:6-amino-N-(3-(4-(4-morpholinyl)pyrido(3',2'-4,5)furo(3,2-d)pyrimidin-2-yl)phenyl)-3-pyridinecarboxamide; D000631:Aminopyridines; D000998:Antiviral Agents; D015683:Gene Products, gag; D006575:Heterocyclic Compounds, 3-Ring; D018129:Phosphatidylinositol Phosphates; D017766:Acid Anhydride Hydrolases; C022563:endopolyphosphatase