title: Murine leukemia virus particle assembly quantitated by fluorescence microscopy: role of Gag-Gag interactions and membrane association.
abstract: In order to track the assembly of murine leukemia virus (MLV), we used fluorescence microscopy to visualize particles containing Gag molecules fused to fluorescent proteins (FPs). Gag-FP chimeras budded from cells to produce fluorescent spots, which passed through the same pore-size filters and sedimented at the same velocity as authentic MLV. N-terminal myristylation of Gag-FPs was necessary for particle formation unless wild-type Gag was coexpressed. By labeling nonmyristylated Gag with yellow FP and wild-type Gag with cyan FP, we could quantitate the coincorporation of two proteins into single particles. This experiment showed that nonmyristylated Gag was incorporated into mixed particles at approximately 50% the efficiency of wild-type Gag. Mutations that inhibit Gag-Gag interactions (K. Alin and S. P. Goff, Virology 216:418-424, 1996; K. Alin and S. P. Goff, Virology 222:339-351, 1996) were then introduced into the capsid (CA) region of Gag-FPs. The mutations P150L and R119C/P133L inhibited fluorescent particle formation by these Gag-FPs, but Gag-FPs containing these mutations could be efficiently incorporated into particles when coexpressed with wild-type Gag. When these mutations were introduced into nonmyristylated Gag-FPs, no incorporation into particles in the presence of wild-type Gag was detected. These data suggest that two independent mechanisms, CA interactions and membrane association following myristylation, cooperate in MLV Gag assembly and budding.
authors.affiliation: Department of Immunology and Molecular Pathology, Windeyer Institute of Medical Science, University College London, London, United Kingdom.
authors.forename: Mariam
authors.initials: M
authors.lastname: Andrawiss
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Takeuchi
authors.forename: Lindsay
authors.initials: L
authors.lastname: Hewlett
authors.forename: Mary
authors.initials: M
authors.lastname: Collins
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D019556:COS Cells; D002213:Capsid / Q000737:chemistry / Q000378:metabolism; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D015683:Gene Products, gag / Q000378:metabolism*; D006801:Humans; D009052:Leukemia Virus, Murine / Q000235:genetics / Q000378:metabolism*; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D009154:Mutation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D018411:Spodoptera; D014771:Virion / Q000378:metabolism*; D019065:Virus Assembly
chemical_list: D001426:Bacterial Proteins; D015683:Gene Products, gag; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; C054562:yellow fluorescent protein, Bacteria