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Slow Release of HIV-1 Protein Nef from Vesicle-like Structures Is Inhibited by Cytosolic Calcium Elevation in Single Human Microglia.
Stenovec, Matjaz; Lasic, Eva; Dominkus, Pia Puzar; Bobnar, Sasa Trkov; Zorec, Robert; Lenassi, Metka; Kreft, Marko.
Afiliação
  • Stenovec M; Celica Biomedical, Tehnoloski park 24, 1000, Ljubljana, Slovenia.
  • Lasic E; Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000, Ljubljana, Slovenia.
  • Dominkus PP; Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000, Ljubljana, Slovenia.
  • Bobnar ST; Institute of Biochemistry, Faculty of Medicine, University of Ljubljana, Vrazov trg 2, 1000, Ljubljana, Slovenia.
  • Zorec R; Celica Biomedical, Tehnoloski park 24, 1000, Ljubljana, Slovenia.
  • Lenassi M; Laboratory of Neuroendocrinology-Molecular Cell Physiology, Institute of Pathophysiology, Faculty of Medicine, University of Ljubljana, Zaloska 4, 1000, Ljubljana, Slovenia.
  • Kreft M; Celica Biomedical, Tehnoloski park 24, 1000, Ljubljana, Slovenia. robert.zorec@mf.uni-lj.si.
Mol Neurobiol ; 56(1): 102-118, 2019 Jan.
Article em En | MEDLINE | ID: mdl-29679260
ABSTRACT
Once infected by HIV-1, microglia abundantly produce accessory protein Nef that enhances virus production and infectivity, but little is known about its intracellular compartmentalization, trafficking mode(s), and release from microglia. Here, we transfected immortalized human microglia with a plasmid encoding Nef tagged with green fluorescent protein (Nef.GFP) to biochemically and microscopically identify Nef.GFP-associated cellular compartments and examine their mobility and Nef release from cultured cells. Immunoblotting revealed that Nef.GFP confined to subcellular fractions with a buoyant density similar to organelles positive for lysosomal-associated membrane protein 1 (LAMP1) but structurally segregated from dextran-laden and LysoTracker-laden endo-/lysosomes in live cells. As revealed by confocal microscopy, Nef.GFP-positive vesicle-like structures were smaller than dextran-laden vesicles and displayed slow and non-directional mobility, in contrast to the faster and directional mobility of dextran-laden vesicles. Ionomycin-evoked elevation in intracellular free Ca2+ concentration ([Ca2+]i) negligibly affected mobility of Nef.GFP structures but strongly and irrecoverably attenuated mobility of dextran-laden vesicles. A slow time-dependent decrease in the number of Nef.GFP-positive structures was observed in non-stimulated controls (5 ± 1 structures/min), but not in ionomycin-stimulated cells (0 ± 2 structures/min; P < 0.05), indicating that elevated [Ca2+]i inhibits the release of Nef.GFP structures. The latter significantly co-localized with membrane sites immunopositive for the tetraspanins CD9 (36 ± 4%) and CD81 (22 ± 1%). This is the first report to demonstrate that microglial CD9- and CD81-positive plasma membrane-derived compartments are associated with biogenesis and Nef release.
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Texto completo: 1 Coleções: 01-internacional Temas: Geral Base de dados: MEDLINE Assunto principal: Cálcio / Microglia / Vesículas Citoplasmáticas / Citosol / Produtos do Gene nef do Vírus da Imunodeficiência Humana Tipo de estudo: Prognostic_studies Limite: Humans Idioma: En Revista: Mol Neurobiol Assunto da revista: BIOLOGIA MOLECULAR / NEUROLOGIA Ano de publicação: 2019 Tipo de documento: Article País de afiliação: Eslovênia

Texto completo: 1 Coleções: 01-internacional Temas: Geral Base de dados: MEDLINE Assunto principal: Cálcio / Microglia / Vesículas Citoplasmáticas / Citosol / Produtos do Gene nef do Vírus da Imunodeficiência Humana Tipo de estudo: Prognostic_studies Limite: Humans Idioma: En Revista: Mol Neurobiol Assunto da revista: BIOLOGIA MOLECULAR / NEUROLOGIA Ano de publicação: 2019 Tipo de documento: Article País de afiliação: Eslovênia