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Pathways of Human T-Lymphotropic Virus type I for viral entry
Boulanger, F.; Rodriguez, Sabrina; Willems, L.
2011
 

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Abstract :
[en] Pathways of Human T-Lymphotropic Virus type I for viral entry Boulanger Fanny N.J., Rodriguez Sabrina and Willems Luc Cellular and Molecular Epigenetics, GIGA-Research, Liège The Human T-Lymphotropic Virus type I (HTLV-1), the first discovered human retrovirus, infects an estimated number of 20 million people worldwide. HTLV-1 is the causative agent of Adult T-cell Leukemia/Lymphoma (ATL) and a neurodegenerative disease called HTLV associated myeolopathy / tropical spastic paraparesis (HAM/TSP). We have been interested in the early steps of HTLV entry into cells. Infection requires interaction of the viral envelope glycoproteins (SU and TM) with the cell membrane. Three molecules are required for binding and entry into cells: heparan-sulfate proteoglycans (HSPGs), the VEGF-165 receptor Neuropilin 1 (NRP-1) and the ubiquitous glucose transporter GLUT-1. Consecutive steps of HTLV-1 entry are poorly characterized. These could involve phagocytosis/macropinocytosis, caveolae pathway, clathrin-mediated endocytosis, and several mechanisms independent of these two organelles but mediated by dynamin. To identify the endocytic pathways of HTLV-1 entry, we used two complementary strategies based either on pharmacological inhibition or on dominant-negative proteins expression. First, we evaluated the effect of a series of pharmacological inhibitors to interfere with main endocytic pathways. We used wortmannin and cytochalasin D to interfere with phagocytosis and macropinocytosis through their action on phosphatidyl-inositol-3 kinase and on the actin network, respectively. Clathrin-mediated endocytosis was blocked with chlorpromazine and with a polyclonal anti-clathrin antibody as a direct competitor for the formation of clathrin-coated pits. The caveolar function was disrupted with the sterol-binding drug nystatin and the protein tyrosine-kinase inhibitor genistein. Finally, dynamin was competed with a peptide (Dynamin Inhibitory Peptide) or inhibited by dynasore. Since pharmacological inhibitors may have off-target effects, we also used an alternate approach based on dominant-negative proteins expressed by lentivirus-derived vectors. An AP-2-binding site from Eps15 (Epidermal Growth Factor Receptor Substrate 15) was used to interfere with clathrin-mediated endocytosis. A N-terminally GFP-tagged caveolin-1 construct acted as an inhibitor of the caveolae pathway. A dominant-negative GTP-binding mutant of dynamin2(aa) was used to interfere with dynamin-dependant internalization pathways. Lentiviruses expressing these dominant-negative proteins were transduced into several cell lines or primary cells (T-lymphocytes and dendritic cells). These transduced cells were then infected by co-cultivation with HTLV-1-infected T-lymphocytes (MT2) or with free particles. These different approaches will thus allow identifying the pathways involved in cell entry by HTLV-1.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Boulanger, F.
Rodriguez, Sabrina ;  Université de Liège - ULiège > Chimie et bio-industries > Biologie cell. et moléc.
Willems, L.
Language :
English
Title :
Pathways of Human T-Lymphotropic Virus type I for viral entry
Publication date :
06 June 2011
Event name :
GIGA Cancer Day
Event organizer :
Groupe Interdisciplinaire de Genoproteomique Apliquee
Event place :
Liege, Belgium
Event date :
06-06-2011
Available on ORBi :
since 30 January 2013

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