title: HIV-1 Gag-RNA interaction occurs at a perinuclear/centrosomal site; analysis by confocal microscopy and FRET.
abstract: The Gag polyprotein is the major structural protein of human immunodeficiency virus-1 (HIV-1) constituting the viral core. Between translation on cytoplasmic polysomes and assembly into viral particles at the plasma membrane, it specifically captures the RNA genome of the virus through binding RNA structural motifs (packaging signals -Psi) in the RNA. RNA is believed to be a structural facilitator of Gag assembly. Using a combined approach of immunofluorescence detection of Gag protein and in situ hybridisation detection of viral genomic RNA, we demonstrate that Gag protein colocalises early after expression with Psi+ RNA in the perinuclear region and also colocalises with centrioles. Colocalised RNA and protein subsequently traffic through the cytoplasm to the plasma membrane of the cell. Gag expressed from Psi- RNA diffuses throughout the cell. It is not found at centrioles and shows delayed cytoplasmic colocalisation with the RNA genome. RNA capture through Psi does not influence binding of Gag to microfilaments. Gag does not bind to tubulin during export. The presence of the packaging signal may coordinate capture of Psi+ RNA by Gag protein at the centrosome followed by their combined transport to the site of budding. HIV-1 Psi thus acts as a subcellular localisation signal as well as a high-affinity-binding site for Gag.
authors.affiliation: Department of Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, CB2 2QQ, UK.
authors.forename: Emma
authors.initials: E
authors.lastname: Poole
authors.forename: Padraig
authors.initials: P
authors.lastname: Strappe
authors.forename: Hoi-Ping
authors.initials: HP
authors.lastname: Mok
authors.forename: Ray
authors.initials: R
authors.lastname: Hicks
authors.forename: Andrew M L
authors.initials: AM
authors.lastname: Lever
mesh_terms: D000818:Animals; D001665:Binding Sites; D019556:COS Cells; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism*; D018385:Centrosome / Q000378:metabolism*; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer; D015683:Gene Products, gag / Q000235:genetics / Q000378:metabolism*; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D018613:Microscopy, Confocal; D011485:Protein Binding
chemical_list: D015683:Gene Products, gag