HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 NEF-INDUCED DOWN-MODULATION OF CD4 IS DUE TO RAPID INTERNALIZATION AND DEGRADATION OF SURFACE CD4

Human immunodeficiency virus type 1 (HIV-1) Nef is a myristylated prot ein with a relative molecular mass of 27 kDa, is localized to the cyto plasmic surfaces of cellular membranes, and has been reported to down- modulate CD4 in human T cells. To understand the mechanism of HIV-1 Ne f-mediated down-modulation of cell surface CD4, we expressed Nef prote in in human T-cell line VB. Expression of HIV-1 Nef protein down-modul ated surface CD4 molecules. In pulse-chase experiments, CD4 molecules in Nef expressing cells were synthesized at normal levels. However, th e bulk of newly synthesized CD4 protein was degraded with a half-life of approximately 6 h, compared with the 24-h half-life in control cell s. This Nef-induced acceleration of CD4 turnover was inhibited by lyso somotropic agents NH4Cl and chloroquine as well as by the protease inh ibitor leupeptin. Surface CD4 biotinylation experiments demonstrated t hat CD4 molecules in Nef-expressing T cells are transported to the pla sma membrane with normal kinetics but are then rapidly internalized. T herefore, HIV-1 Nef-induced down-modulation of CD4 is due to rapid int ernalization of surface CD4 and subsequent degradation by an acid-depe ndent process, potentially lysosomal. Additionally, in a Nef-expressin g cell, we find accelerated dissociation of the T-cell tyrosine kinase p56(lck) and CD4 but only after the complex reaches the plasma membra ne. This implies that HIV-1 Nef protein might play a role in triggerin g a series of T-cell activation-like events, which contribute to p56(l ck) dissociation and internalization of surface CD4 molecules.