title: Involvement of the Rac1-IRSp53-Wave2-Arp2/3 Signaling Pathway in HIV-1 Gag Particle Release in CD4 T Cells.
abstract: During HIV-1 assembly, the Gag viral proteins are targeted and assemble at the inner leaflet of the cell plasma membrane. This process could modulate the cortical actin cytoskeleton, located underneath the plasma membrane, since actin dynamics are able to promote localized membrane reorganization. In addition, activated small Rho GTPases are known for regulating actin dynamics and membrane remodeling. Therefore, the modulation of such Rho GTPase activity and of F-actin by the Gag protein during virus particle formation was considered. Here, we studied the implication of the main Rac1, Cdc42, and RhoA small GTPases, and some of their effectors, in this process. The effect of small interfering RNA (siRNA)-mediated Rho GTPases and silencing of their effectors on Gag localization, Gag membrane attachment, and virus-like particle production was analyzed by immunofluorescence coupled to confocal microscopy, membrane flotation assays, and immunoblot assays, respectively. In parallel, the effect of Gag expression on the Rac1 activation level was monitored by G-LISA, and the intracellular F-actin content in T cells was monitored by flow cytometry and fluorescence microscopy. Our results revealed the involvement of activated Rac1 and of the IRSp53-Wave2-Arp2/3 signaling pathway in HIV-1 Gag membrane localization and particle release in T cells as well as a role for actin branching and polymerization, and this was solely dependent on the Gag viral protein. In conclusion, our results highlight a new role for the Rac1-IRSp53-Wave2-Arp2/3 signaling pathway in the late steps of HIV-1 replication in CD4 T lymphocytes.
  
  
  OBJECTIVE
  During HIV-1 assembly, the Gag proteins are targeted and assembled at the inner leaflet of the host cell plasma membrane. Gag interacts with specific membrane phospholipids that can also modulate the regulation of cortical actin cytoskeleton dynamics. Actin dynamics can promote localized membrane reorganization and thus can be involved in facilitating Gag assembly and particle formation. Activated small Rho GTPases and effectors are regulators of actin dynamics and membrane remodeling. We thus studied the effects of the Rac1, Cdc42, and RhoA GTPases and their specific effectors on HIV-1 Gag membrane localization and viral particle release in T cells. Our results show that activated Rac1 and the IRSp53-Wave2-Arp2/3 signaling pathway are involved in Gag plasma membrane localization and viral particle production. This work uncovers a role for cortical actin through the activation of Rac1 and the IRSp53/Wave2 signaling pathway in HIV-1 particle formation in CD4 T lymphocytes.
authors.affiliation: Centre d'Études d'Agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRS UMR5236, Montpellier, France ENS de Lyon, Lyon, France.
authors.forename: Audrey
authors.initials: A
authors.lastname: Thomas
authors.affiliation: Centre d'Études d'Agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRS UMR5236, Montpellier, France.
authors.forename: Charlotte
authors.initials: C
authors.lastname: Mariani-Floderer
authors.affiliation: Unidad de Inmunopatología del SIDA, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain ENS de Lyon, Lyon, France.
authors.forename: Maria Rosa
authors.initials: MR
authors.lastname: López-Huertas
authors.affiliation: Centre d'Études d'Agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRS UMR5236, Montpellier, France.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Gros
authors.affiliation: ENS de Lyon, Lyon, France.
authors.forename: Elise
authors.initials: E
authors.lastname: Hamard-Péron
authors.affiliation: Centre d'Études d'Agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRS UMR5236, Montpellier, France.
authors.forename: Cyril
authors.initials: C
authors.lastname: Favard
authors.affiliation: CIRI, INSERM U1111, Lyon, France ENS de Lyon, Lyon, France.
authors.forename: Theophile
authors.initials: T
authors.lastname: Ohlmann
authors.affiliation: Unidad de Inmunopatología del SIDA, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Madrid, Spain.
authors.forename: José
authors.initials: J
authors.lastname: Alcamí
authors.affiliation: Centre d'Études d'Agents Pathogènes et Biotechnologies pour la Santé (CPBS), CNRS UMR5236, Montpellier, France ENS de Lyon, Lyon, France delphine.muriaux@cpbs.cnrs.fr.
authors.forename: Delphine
authors.initials: D
authors.lastname: Muriaux
mesh_terms: D051376:Actin-Related Protein 2-3 Complex / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000378:metabolism*; D015683:Gene Products, gag / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006801:Humans; D019169:Jurkat Cells; D009419:Nerve Tissue Proteins / Q000378:metabolism; D015398:Signal Transduction; D051300:Wiskott-Aldrich Syndrome Protein Family / Q000378:metabolism; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: D051376:Actin-Related Protein 2-3 Complex; C119539:BAIAP2 protein, human; D015683:Gene Products, gag; D009419:Nerve Tissue Proteins; C107953:RAC1 protein, human; C120152:WASF2 protein, human; D051300:Wiskott-Aldrich Syndrome Protein Family; D020830:rac1 GTP-Binding Protein