Co-localization of HIV-1 Nef with the AP-2 adaptor protein complex correlates with Nef-induced CD4 down-regulation

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Abstract
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 β‐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.