title: HIV-1 Myristoylated Nef Treatment of Murine Microglial Cells Activates Inducible Nitric Oxide Synthase, NO2 Production and Neurotoxic Activity.
abstract: BACKGROUND
  The potential role of the human immunodeficiency virus-1 (HIV-1) accessory protein Nef in the pathogenesis of neuroAIDS is still poorly understood. Nef is a molecular adapter that influences several cellular signal transduction events and membrane trafficking. In human macrophages, Nef expression induces the production of extracellular factors (e.g. pro-inflammatory chemokines and cytokines) and the recruitment of T cells, thus favoring their infection and its own transfer to uninfected cells via exosomes, cellular protrusions or cell-to-cell contacts. Murine cells are normally not permissive for HIV-1 but, in transgenic mice, Nef is a major disease determinant. Both in human and murine macrophages, myristoylated Nef (myr+Nef) treatment has been shown to activate NF-κB, MAP kinases and interferon responsive factor 3 (IRF-3), thereby inducing tyrosine phosphorylation of signal transducers and activator of transcription (STAT)-1, STAT-2 and STAT-3 through the production of proinflammatory factors.
  
  
  RESULTS
  We report that treatment of BV-2 murine microglial cells with myr+Nef leads to STAT-1, -2 and -3 tyrosine phosphorylation and upregulates the expression of inducible nitric oxide synthase (iNOS) with production of nitric oxide. We provide evidence that extracellular Nef regulates iNOS expression through NF-κB activation and, at least in part, interferon-β (IFNβ) release that acts in concert with Nef. All of these effects require both myristoylation and a highly conserved acidic cluster in the viral protein. Finally, we report that Nef induces the release of neurotoxic factors in the supernatants of microglial cells.
  
  
  CONCLUSIONS
  These results suggest a potential role of extracellular Nef in promoting neuronal injury in the murine model. They also indicate a possible interplay between Nef and host factors in the pathogenesis of neuroAIDS through the production of reactive nitrogen species in microglial cells.
authors.affiliation: Department of Science, University Roma Tre, Rome, Italy; Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina, Italy.
authors.forename: Giorgio
authors.initials: G
authors.lastname: Mangino
authors.affiliation: Department of Science, University Roma Tre, Rome, Italy.
authors.forename: Marylinda
authors.initials: M
authors.lastname: Famiglietti
authors.affiliation: Department of Science, University Roma Tre, Rome, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Capone
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Caterina
authors.initials: C
authors.lastname: Veroni
authors.affiliation: Department of Science, University Roma Tre, Rome, Italy.
authors.forename: Zulema Antonia
authors.initials: ZA
authors.lastname: Percario
authors.affiliation: Department of Science, University Roma Tre, Rome, Italy.
authors.forename: Stefano
authors.initials: S
authors.lastname: Leone
authors.affiliation: Institute of Molecular Biology and Pathology, CNR, Rome, Italy.
authors.forename: Gianna
authors.initials: G
authors.lastname: Fiorucci
authors.affiliation: Center of Advanced European Studies and Research, Group of Physical Biochemistry, Bonn, Germany.
authors.forename: Sebastian
authors.initials: S
authors.lastname: Lülf
authors.affiliation: Department of Medico-Surgical Sciences and Biotechnologies, Sapienza University of Rome, Latina, Italy; Institute of Molecular Biology and Pathology, CNR, Rome, Italy.
authors.forename: Giovanna
authors.initials: G
authors.lastname: Romeo
authors.affiliation: Department of Cell Biology and Neuroscience, Istituto Superiore di Sanità, Rome, Italy.
authors.forename: Cristina
authors.initials: C
authors.lastname: Agresti
authors.affiliation: Department of Science, University Roma Tre, Rome, Italy.
authors.forename: Tiziana
authors.initials: T
authors.lastname: Persichini
authors.affiliation: Center of Advanced European Studies and Research, Group of Physical Biochemistry, Bonn, Germany.
authors.forename: Matthias
authors.initials: M
authors.lastname: Geyer
authors.affiliation: Department of Science, University Roma Tre, Rome, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Affabris
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D016207:Cytokines / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007124:Immunoenzyme Techniques; D007371:Interferon-gamma / Q000235:genetics / Q000378:metabolism; D008264:Macrophages / Q000378:metabolism / Q000473:pathology*; D051379:Mice; D017628:Microglia / Q000378:metabolism / Q000473:pathology*; D020928:Mitogen-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D019814:Myristic Acid / Q000378:metabolism*; D016328:NF-kappa B / Q000235:genetics / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D052247:Nitric Oxide Synthase Type II / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction; D054311:nef Gene Products, Human Immunodeficiency Virus / Q000235:genetics / Q000378:metabolism*
chemical_list: D016207:Cytokines; D016328:NF-kappa B; D012333:RNA, Messenger; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D019814:Myristic Acid; D009569:Nitric Oxide; D007371:Interferon-gamma; C496319:NOS2 protein, human; D052247:Nitric Oxide Synthase Type II; D020928:Mitogen-Activated Protein Kinases