title: Effects of HIV-1 Nef on retrograde transport from the plasma membrane to the endoplasmic reticulum.
abstract: HIV-1 Nef protein down-regulates several important immunoreceptors through interactions with components of the intracellular sorting machinery. Nef expression is also known to induce modifications of the endocytic pathway. Here, we analyzed the effects of Nef on retrograde transport, from the plasma membrane to the endoplasmic reticulum using Shiga toxin B-subunit (STxB). Nef expression inhibited access of STxB to the endoplasmic reticulum, but did not modify the surface expression level of STxB receptor, Gb3, nor its internalization rate as measured with a newly developed assay. Mutation of the myristoylation site or of a di-leucine motif of Nef involved in the interaction with the clathrin adaptor complexes AP1 and AP2 abolished the inhibition of retrograde transport. In contrast, mutations of Nef motifs known to interact with PACS-1, beta COP or a subunit of the v-ATPase did not modify the inhibitory activity of Nef on retrograde transport. Ultrastructural analysis revealed that Nef was present in clusters located on endosomal or Golgi membranes together with internalized STxB. Furthermore, in strongly Nef-expressing cells, STxB accumulated in endosomal structures that labeled with AP1. Our observations show that Nef perturbs retrograde transport between the early endosome and the endoplasmic reticulum. The potential transport steps targeted by Nef are discussed.
authors.affiliation: CNRS UMR144. Institut Curie, 26 rue d'Ulm, F-75248 Paris Cedex 05. France. Ludger.Johannes@curie.fr
authors.forename: Ludger
authors.initials: L
authors.lastname: Johannes
authors.forename: Valérie
authors.initials: V
authors.lastname: Pezo
authors.forename: Frédéric
authors.initials: F
authors.lastname: Mallard
authors.forename: Danièle
authors.initials: D
authors.lastname: Tenza
authors.forename: Aimée
authors.initials: A
authors.lastname: Wiltz
authors.forename: Agnès
authors.initials: A
authors.lastname: Saint-Pol
authors.forename: Julie
authors.initials: J
authors.lastname: Helft
authors.forename: Claude
authors.initials: C
authors.lastname: Antony
authors.forename: Philippe
authors.initials: P
authors.lastname: Benaroch
mesh_terms: D002462:Cell Membrane / Q000378:metabolism*; D004721:Endoplasmic Reticulum / Q000378:metabolism* / Q000648:ultrastructure; D011992:Endosomes / Q000378:metabolism / Q000648:ultrastructure; D015693:Gene Products, nef / Q000378:metabolism*; D015497:HIV-1 / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D008854:Microscopy, Electron; D021381:Protein Transport / Q000502:physiology; D022603:Shiga Toxins / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus
chemical_list: D015693:Gene Products, nef; D022603:Shiga Toxins; D054311:nef Gene Products, Human Immunodeficiency Virus; C077239:stxB toxin