Nef-induced CD4 downregulation: a diacidic sequence in human immunodeficiency virus type 1 Nef does not function as a protein sorting motif through direct binding to beta-COP

Abstract

The Nef protein from the human immunodeficiency virus (HIV) induces CD4 cell surface downregulation by interfering with the endocytic machinery. It has been recently proposed that binding of HIV type 1 Nef to the beta subunit of COPI coatomers participated in the Nef-induced CD4 downregulation through recognition of a novel diacidic motif found in the C-terminal disordered loop of Nef (V. Piguet, F. Gu, M. Foti, N. Demaurex, J. Gruenberg, J. L. Carpentier, and D. Trono, Cell 97:63-73, 1999). We have mutated the glutamate residues which formed this motif in order to document this observation. Surprisingly, mutation of the diacidic sequence of Nef did not significantly affect its ability (i) to interact with beta-COP, (ii) to downregulate CD4 cell surface expression, and (iii) to address an integral resident membrane protein containing Nef as the cytoplasmic domain to the endocytic pathway. Our results indicate that these acidic residues are not involved in the connection of Nef with the endocytic machinery through binding to beta-COP. Additional studies are thus required to characterize the residues of Nef involved in the binding to beta-COP and to evaluate the contribution of this interaction to the Nef-induced perturbations of membrane trafficking.

FIG. 1

Mutation of the di-Glu sequence does not affect the binding of Nef to β-COP. (A) Nef binding to β-COP in a two-hybrid assay. HF7c and SFY526 yeast strains expressing the indicated pairs of Gal4BD and Gal4AD hybrid proteins were analyzed for histidine auxotrophy and β-Gal activity, respectively. HF7c double transformants were patched on selective medium with histidine (left) and then replica plated on histidine-free medium (middle). SFY526 double transformants were patched on selective medium and then replica plated on a Whatman filter for detection of β-Gal activity (right). Growth on histidine-free medium and expression of β-Gal indicate interaction between hybrid proteins. Binding specificity was verified by the absence of interaction between β-COP and SNF1 (lane 3) and between wt or mutated Nef and SNF4 (lanes 2, 5, 7, and 9). (B) In vitro binding of Nef to β-COP. [³⁵S]-β-COP-Cter synthesized in vitro was incubated with equal amounts of GST (lane 6) or GST-Nef fusion proteins (lanes 2 to 5) immobilized on glutathione-agarose beads. Bound labeled material was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. One-fifth of the [³⁵S]-β-COP-Cter input was run on lane 1.

FIG. 2

Mutation of the di-Glu sequence does not significantly affect the trans- and cis-mediated downregulation activities of both Nef and CD8-Nef. HeLa-CD4 cells were transfected with either the Nef (A and D) or CD8-Nef (B, C, E, and F) expression vectors, along with a vector expressing GFP. The surface expression of CD4 (A, B, D, and E) or CD8-Nef (C and F) was measured by flow cytometry on GFP-positive cells with PE-conjugated anti-CD4 or anti-CD8 Mab, respectively. (A to C) CD4 or CD8 cell surface expression in cells expressing wt or mutated Nef or CD8-Nef. Data are representative of four independent experiments. (D to F) Relative downregulation activity of mutated Nef or CD8-Nef. The results are expressed as the percentages of the activity determined for each mutant relative to that of Nefwt or CD8-Nefwt. CD8Stop, which lacks a cytoplasmic tail, was used as a negative control. Values are the means of four independent experiments. Error bars represent 1 standard deviation from the mean.

FIG. 3

Subcellular distribution of the CD8-Nef chimeras and AP1 complexes. HeLa cells were transfected with the CD8-Nef expression vectors. Twenty-four hours later, cells were fixed and permeabilized. The CD8-Nef chimeras were stained with fluorescein isothiocyanate-conjugated anti-CD8 MAb (green), and AP1 was detected with anti-γ-adaptin followed by staining with a Cy3-conjugated Fab fragment recognizing mouse immunoglobulin G (red). Images were collected using a 63× oil immersion objective.