title: Role of the nucleocapsid domain in HIV-1 Gag oligomerization and trafficking to the plasma membrane: a fluorescence lifetime imaging microscopy investigation.
abstract: The Pr55 Gag of human immunodeficiency virus type 1 orchestrates viral particle assembly in producer cells, which requires the genomic RNA and a lipid membrane as scaffolding platforms. The nucleocapsid (NC) domain with its two invariant CCHC zinc fingers flanked by unfolded basic sequences is thought to direct genomic RNA selection, dimerization and packaging during virus assembly. To further investigate the role of NC domain, we analyzed the assembly of Gag with deletions in the NC domain in parallel with that of wild-type Gag using fluorescence lifetime imaging microscopy combined with Förster resonance energy transfer in HeLa cells. We found that, upon binding to nucleic acids, the NC domain promotes the formation of compact Gag oligomers in the cytoplasm. Moreover, the intracellular distribution of the population of oligomers further suggests that oligomers progressively assemble during their trafficking toward the plasma membrane (PM), but with no dramatic changes in their compact arrangement. This ultimately results in the accumulation at the PM of closely packed Gag oligomers that likely arrange in hexameric lattices, as revealed by the perfect match between the experimental Förster resonance energy transfer value and the one calculated from the structural model of Gag in immature viruses. The distal finger and flanking basic sequences, but not the proximal finger, appear to be essential for Gag oligomer compaction and membrane binding. Moreover, the full NC domain was found to be instrumental in the kinetics of Gag oligomerization and intracellular trafficking. These findings further highlight the key roles played by the NC domain in virus assembly.
authors: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch Cedex, France.
authors: Salah Edin
authors: SE
authors: El Meshri
authors: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch Cedex, France.
authors: Denis
authors: D
authors: Dujardin
authors: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch Cedex, France.
authors: Julien
authors: J
authors: Godet
authors: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch Cedex, France.
authors: Ludovic
authors: L
authors: Richert
authors: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch Cedex, France.
authors: Christian
authors: C
authors: Boudier
authors: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch Cedex, France.
authors: Jean Luc
authors: JL
authors: Darlix
authors: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch Cedex, France.
authors: Pascal
authors: P
authors: Didier
authors: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch Cedex, France. Electronic address: yves.mely@unistra.fr.
authors: Yves
authors: Y
authors: Mély
authors: Laboratoire de Biophotonique et Pharmacologie, UMR 7213 CNRS, Faculté de Pharmacie, Université de Strasbourg, 74, Route du Rhin, 67401 Illkirch Cedex, France. Electronic address: hderocquigny@unistra.fr.
authors: Hugues
authors: H
authors: de Rocquigny
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer; D015658:HIV Infections / Q000378:metabolism* / Q000821:virology; D015497:HIV-1 / Q000502:physiology; D006801:Humans; D008856:Microscopy, Fluorescence; D009154:Mutation / Q000235:genetics; D019251:Nucleocapsid; D011485:Protein Binding; D055503:Protein Multimerization; D021381:Protein Transport; D012367:RNA, Viral / Q000235:genetics; D019065:Virus Assembly; D016335:Zinc Fingers / Q000235:genetics*; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: D012367:RNA, Viral; D054301:gag Gene Products, Human Immunodeficiency Virus
keywords: FRET-FLIM; Gag; HIV-1; NC; oligomer