title: Defect of human immunodeficiency virus type 2 Gag assembly in Saccharomyces cerevisiae.
abstract: We have previously shown that the expression of human immunodeficiency virus type 1 (HIV-1) Gag protein in Saccharomyces cerevisiae spheroplasts produces Gag virus-like particles (VLPs) at the plasma membrane, indicating that yeast has all the host factors necessary for HIV-1 Gag assembly. Here we expand the study by using diverse primate lentiviral Gags and show that yeast does not support the production of HIV-2 or simian immunodeficiency virus SIVmac Gag VLPs but allows the production of SIVagm and SIVmnd Gag VLPs. Particle budding was observed at the surfaces of cells expressing SIVagm and SIVmnd Gags, but cells expressing HIV-2 and SIVmac Gags showed only membrane-ruffling structures, although they were accompanied with electron-dense submembrane layers, suggesting arrest at an early stage of particle budding. Comparison of HIV-1 and HIV-2 Gag expression revealed broadly equivalent levels of intracellular Gag expression and Gag N-terminal myristoylation in yeast. Both Gags showed the same membrane-binding ability and were incorporated into lipid raft fractions at a physiological concentration of salt. HIV-2 Gag, however, failed to form a high-order multimer and easily dissociated from the membrane, phenomena which were not observed in higher eukaryotic cells. A series of chimeric Gags between HIV-1 and HIV-2 and Gag mutants with amino acid substitutions revealed that a defined region in helix 2 of HIV-2 MA (located on the membrane-binding surface of MA) affects higher-order Gag assembly and particle production in yeast. Together, these data suggest that yeast may lack a host factor(s) for HIV-2 and SIVmac Gag assembly.
authors.affiliation: Kitasato Institute for Life Sciences and Graduate School for Infection Control, Kitasato University, Shirokane 5-9-1, Minato-ku, Tokyo 108-8641, Japan. morikawa@lisci.kitasato-u.ac.jp
authors.forename: Yuko
authors.initials: Y
authors.lastname: Morikawa
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Goto
authors.forename: Daisuke
authors.initials: D
authors.lastname: Yasuoka
authors.forename: Fumitaka
authors.initials: F
authors.lastname: Momose
authors.forename: Tetsuro
authors.initials: T
authors.lastname: Matano
mesh_terms: D019943:Amino Acid Substitution; D015870:Gene Expression; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D015498:HIV-2 / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D006801:Humans; D021962:Membrane Microdomains / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D020125:Mutation, Missense; D019814:Myristic Acid / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D011499:Protein Processing, Post-Translational; D017433:Protein Structure, Secondary / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure / Q000821:virology; D015302:Simian Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism; D013104:Spheroplasts / Q000235:genetics / Q000378:metabolism* / Q000648:ultrastructure; D019065:Virus Assembly
chemical_list: D015683:Gene Products, gag; D011994:Recombinant Proteins; D019814:Myristic Acid