title: Urokinase plasminogen activator inhibits HIV virion release from macrophage-differentiated chronically infected cells via activation of RhoA and PKCε.
abstract: BACKGROUND
  HIV replication in mononuclear phagocytes is a multi-step process regulated by viral and cellular proteins with the peculiar feature of virion budding and accumulation in intra-cytoplasmic vesicles. Interaction of urokinase-type plasminogen activator (uPA) with its cell surface receptor (uPAR) has been shown to favor virion accumulation in such sub-cellular compartment in primary monocyte-derived macrophages and chronically infected promonocytic U1 cells differentiated into macrophage-like cells by stimulation with phorbol myristate acetate (PMA). By adopting this latter model system, we have here investigated which intracellular signaling pathways were triggered by uPA/uPAR interaction leading the redirection of virion accumulation in intra-cytoplasmic vesicles.
  
  
  RESULTS
  uPA induced activation of RhoA, PKCδ and PKCε in PMA-differentiated U1 cells. In the same conditions, RhoA, PKCδ and PKCε modulated uPA-induced cell adhesion and polarization, whereas only RhoA and PKCε were also responsible for the redirection of virions in intracellular vesicles. Distribution of G and F actin revealed that uPA reorganized the cytoskeleton in both adherent and polarized cells. The role of G and F actin isoforms was unveiled by the use of cytochalasin D, a cell-permeable fungal toxin that prevents F actin polymerization. Receptor-independent cytoskeleton remodeling by Cytochalasin D resulted in cell adhesion, polarization and intracellular accumulation of HIV virions similar to the effects gained with uPA.
  
  
  CONCLUSIONS
  These findings illustrate the potential contribution of the uPA/uPAR system in the generation and/or maintenance of intra-cytoplasmic vesicles that actively accumulate virions, thus sustaining the presence of HIV reservoirs of macrophage origin. In addition, our observations also provide evidences that pathways controlling cytoskeleton remodeling and activation of PKCε bear relevance for the design of new antiviral strategies aimed at interfering with the partitioning of virion budding between intra-cytoplasmic vesicles and plasma membrane in infected human macrophages.
authors.affiliation: AIDS Immunophatogenesis Unit, Division of Immunology, Transplantation and Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Graziano
authors.forename: Chiara
authors.initials: C
authors.lastname: Elia
authors.forename: Carlo
authors.initials: C
authors.lastname: Laudanna
authors.forename: Guido
authors.initials: G
authors.lastname: Poli
authors.forename: Massimo
authors.initials: M
authors.lastname: Alfano
mesh_terms: D002448:Cell Adhesion / Q000187:drug effects; D002454:Cell Differentiation; D016764:Cell Polarity / Q000187:drug effects; D015638:Cytochalasin D / Q000494:pharmacology; D022162:Cytoplasmic Vesicles / Q000187:drug effects / Q000648:ultrastructure / Q000821:virology; D004789:Enzyme Activation / Q000187:drug effects; D015497:HIV-1 / Q000187:drug effects / Q000502:physiology; D054884:Host-Pathogen Interactions / Q000187:drug effects; D006801:Humans; D008264:Macrophages / Q000378:metabolism* / Q000473:pathology / Q000821:virology; D018613:Microscopy, Confocal; D008854:Microscopy, Electron; D019384:Nucleic Acid Synthesis Inhibitors / Q000494:pharmacology; D051745:Protein Kinase C-delta / Q000378:metabolism; D051744:Protein Kinase C-epsilon / Q000378:metabolism*; D055293:Receptors, Urokinase Plasminogen Activator / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020298:U937 Cells; D014568:Urokinase-Type Plasminogen Activator / Q000378:metabolism / Q000494:pharmacology*; D014771:Virion / Q000187:drug effects* / Q000502:physiology; D057074:Virus Release / Q000187:drug effects*; D020742:rhoA GTP-Binding Protein / Q000378:metabolism*
chemical_list: D019384:Nucleic Acid Synthesis Inhibitors; D055293:Receptors, Urokinase Plasminogen Activator; D015638:Cytochalasin D; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D014568:Urokinase-Type Plasminogen Activator; D020742:rhoA GTP-Binding Protein; D013755:Tetradecanoylphorbol Acetate