Dynamic Interaction of HIV-1 Nef with the Clathrin-Mediated Endocytic Pathway at the Plasma Membrane

Authors

  • Anne Burtey,

    1. Institut Cochin, Département Maladies Infectieuses, Paris F-75014, France
    2. Inserm, U567, Paris F-75014, France
    3. CNRS, UMR 8104, Paris F-75014, France
    4. Université Paris 5, Faculté de Médecine René Descartes, Paris F-75014, France
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  • Joshua Z. Rappoport,

    1. The Laboratory of Cellular Biophysics, The Rockefeller University, 1230 York Avenue, Box 304, New York, NY 10021, USA
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  • Jérôme Bouchet,

    1. Institut Cochin, Département Maladies Infectieuses, Paris F-75014, France
    2. Inserm, U567, Paris F-75014, France
    3. CNRS, UMR 8104, Paris F-75014, France
    4. Université Paris 5, Faculté de Médecine René Descartes, Paris F-75014, France
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  • Stéphane Basmaciogullari,

    1. Institut Cochin, Département Maladies Infectieuses, Paris F-75014, France
    2. Inserm, U567, Paris F-75014, France
    3. CNRS, UMR 8104, Paris F-75014, France
    4. Université Paris 5, Faculté de Médecine René Descartes, Paris F-75014, France
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  • John Guatelli,

    1. Department of Medicine University of California, San Diego, 9500 Gilman Drive, La Jolla, CA 92093, USA
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  • Sanford M. Simon,

    1. The Laboratory of Cellular Biophysics, The Rockefeller University, 1230 York Avenue, Box 304, New York, NY 10021, USA
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  • Serge Benichou,

    Corresponding author
    1. Institut Cochin, Département Maladies Infectieuses, Paris F-75014, France
    2. Inserm, U567, Paris F-75014, France
    3. CNRS, UMR 8104, Paris F-75014, France
    4. Université Paris 5, Faculté de Médecine René Descartes, Paris F-75014, France
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  • Alexandre Benmerah

    Corresponding author
    1. Institut Cochin, Département Maladies Infectieuses, Paris F-75014, France
    2. Inserm, U567, Paris F-75014, France
    3. CNRS, UMR 8104, Paris F-75014, France
    4. Université Paris 5, Faculté de Médecine René Descartes, Paris F-75014, France
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Alexandre Benmerah or Serge Benichou, benmerah@cochin.inserm.fr or benichou@cochin.inserm.fr

Abstract

The HIV-1 Nef protein perturbs the trafficking of membrane proteins such as CD4 by interacting with clathrin–adaptor complexes. We previously reported that Nef alters early/recycling endosomes, but its role at the plasma membrane is poorly documented. Here, we used total internal reflection fluorescence microscopy, which restricts the analysis to a ∼100 nm region of the adherent surface of the cells, to focus on the dynamic of Nef at the plasma membrane relative to that of clathrin. Nef colocalized both with clathrin spots (CS) that remained static at the cell surface, corresponding to clathrin-coated pits (CCPs), and with ∼50% of CS that disappeared from the cell surface, corresponding to forming clathrin-coated vesicles (CCVs). The colocalization of Nef with clathrin required the di-leucine motif essential for Nef binding to AP complexes and was independent of CD4 expression. Furthermore, analysis of Nef mutants showed that the capacity of Nef to induce internalization and downregulation of CD4 in T lymphocytes correlated with its localization into CCPs. In conclusion, this analysis shows that Nef is recruited into CCPs and into forming CCVs at the plasma membrane, in agreement with a model in which Nef uses the clathrin-mediated endocytic pathway to induce internalization of some membrane proteins from the surface of HIV-1-infected T cells.

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