Dynamic interaction of HIV-1 Nef with the clathrin-mediated endocytic pathway at the plasma membrane.
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 approximately 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 approximately 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.
Burtey
Anne
A
Institut Cochin, Département Maladies Infectieuses, Paris F-75014, France.
Rappoport
Joshua Z
JZ
Bouchet
Jérôme
J
Basmaciogullari
Stéphane
S
Guatelli
John
J
Simon
Sanford M
SM
Benichou
Serge
S
Benmerah
Alexandre
A
D020816:Amino Acid Motifs; D001665:Binding Sites; D001693:Biological Transport, Active; D015704:CD4 Antigens / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology / Q000378:metabolism / Q000821:virology; D002462:Cell Membrane / Q000378:metabolism / Q000821:virology; D002966:Clathrin / Q000378:metabolism*; D003034:Coated Pits, Cell-Membrane / Q000378:metabolism / Q000821:virology; D004705:Endocytosis; D015693:Gene Products, nef / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus
D015704:CD4 Antigens; D002966:Clathrin; D015693:Gene Products, nef; D011993:Recombinant Fusion Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; C514837:nef protein, Human immunodeficiency virus 1; D049452:Green Fluorescent Proteins