COLOCALIZATION OF HIV-1 NEF WITH THE AP-2 ADAPTER 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 n atural isolates of Nef down-regulate expression of the cell surface CD 4 molecule, a component of the T cell antigen receptor and the viral r eceptor, by accelerating its endocytosis, We have used chimeric protei ns comprised of the natural HIV-1 NA7 Nef fused to a strongly fluoresc ing mutant of green fluorescent protein (GFP) to correlate Nef functio n with intracellular localization in human CD4-positive Jurkat T cells , The NA7-GFP fusion protein co-localizes with components of the clath rin coat, including clathrin and the beta-subunit of the AP-2 adaptor protein complex, at discrete locations that are consistent with the no rmal cellular distribution of clathrin coats at the plasma membrane, T he 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-GF P with components of the clathrin coat does not require expression of the CD4 molecule, Analysis of a large panel of chimeric molecules cont aining mutant Nef moieties demonstrated that the N-terminal membrane t argeting signal cooperates with additional element(s) in the disordere d loops in the Nef molecule to co-localize the Nef protein with AP-2 a daptor complexes at the cell margin. This localization of NA7-GFP corr elates with, but is not sufficient for, down-regulation of surface CD4 and at least one additional function of Nef is required, In T cells c o-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 o bservations place NA7-GFP in physical proximity to AP-2-containing cla thrin 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 t he plasma membrane.