Co-localization of HIV-1 Nef with the AP-2 adaptor protein complex correlates with Nef-induced CD4 down-regulation.
The nef gene of human and simian immunodeficiency viruses is critical for AIDS pathogenesis. Its function in vivo is unknown, but in vitro natural isolates of Nef down-regulate expression of the cell surface CD4 molecule, a component of the T cell antigen receptor and the viral receptor, by accelerating its endocytosis. We have used chimeric proteins comprised of the natural HIV-1 NA7 Nef fused to a strongly fluorescing mutant of green fluorescent protein (GFP) to correlate Nef function with intracellular localization in human CD4-positive Jurkat T cells. The NA7-GFP fusion protein co-localizes with components of the clathrin coat, including clathrin and the beta-subunit of the AP-2 adaptor protein complex, at discrete locations that are consistent with the normal cellular distribution of clathrin coats at the plasma membrane. The NA7-GFP protein is also found in the perinuclear region of the cell, which is likely to reflect the Golgi apparatus. Evidence from a CD4-negative fibroblast cell line indicates that co-localization of NA7-GFP with components of the clathrin coat does not require expression of the CD4 molecule. Analysis of a large panel of chimeric molecules containing mutant Nef moieties demonstrated that the N-terminal membrane targeting signal cooperates with additional element(s) in the disordered loops in the Nef molecule to co-localize the Nef protein with AP-2 adaptor complexes at the cell margin. This localization of NA7-GFP correlates with, but is not sufficient for, down-regulation of surface CD4 and at least one additional function of Nef is required. In T cells co-expressing CD4 and NA7-GFP, CD4 at the cell surface is redistributed into a discrete pattern that co-localizes with that of NA7-GFP. Our observations place NA7-GFP in physical proximity to AP-2-containing clathrin coat at the plasma membrane and imply that Nef interacts, either directly or indirectly, with a component of the AP-2-containing coat at this location. This evidence supports a model whereby Nef recruits CD4 to the endocytic machinery via AP-2-containing clathrin coats at the plasma membrane.
Greenberg
M E
ME
Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.
Bronson
S
S
Lock
M
M
Neumann
M
M
Pavlakis
G N
GN
Skowronski
J
J
D033965:Adaptor Protein Complex alpha Subunits; D033966:Adaptor Protein Complex beta Subunits; D033982:Adaptor Protein Complex gamma Subunits; D033942:Adaptor Proteins, Vesicular Transport; D015704:CD4 Antigens / Q000096:biosynthesis*; D015496:CD4-Positive T-Lymphocytes; D002451:Cell Compartmentation; D002462:Cell Membrane / Q000378:metabolism; D004252:DNA Mutational Analysis; D015536:Down-Regulation; D004705:Endocytosis; D015693:Gene Products, nef / Q000235:genetics / Q000378:metabolism*; D049452:Green Fluorescent Proteins; D015497:HIV-1; D006801:Humans; D019169:Jurkat Cells; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism*; D010446:Peptide Fragments / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D054311:nef Gene Products, Human Immunodeficiency Virus
D033965:Adaptor Protein Complex alpha Subunits; D033966:Adaptor Protein Complex beta Subunits; D033982:Adaptor Protein Complex gamma Subunits; D033942:Adaptor Proteins, Vesicular Transport; D015704:CD4 Antigens; D015693:Gene Products, nef; D008164:Luminescent Proteins; D008565:Membrane Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D054311:nef Gene Products, Human Immunodeficiency Virus; D049452:Green Fluorescent Proteins