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Human immunodeficiency virus type 1 Nef-induced down-modulation of CD4 is due to rapid internalization and
1Laboratory of Molecular Biology, National Institute of Mental Health, Bethesda, Maryland 20892.
Abstract:
Human immunodeficiency virus type 1 (HIV-1) Nef is a myristylated protein with a relative molecular mass of 27 kDa, is localized to the cytoplasmic surfaces of cellular membranes, and has been reported to down-modulate CD4 in human T cells. To understand the mechanism of HIV-1 Nef-mediated down-modulation of cell surface CD4, we expressed Nef protein in human T-cell line VB. Expression of HIV-1 Nef protein down-modulated surface CD4 molecules. In pulse-chase experiments, CD4 molecules in Nef-expressing cells were synthesized at normal levels. However, the 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 cells. This Nef-induced acceleration of CD4 turnover was inhibited by lysosomotropic agents NH4Cl and chloroquine as well as by the protease inhibitor leupeptin. Surface CD4 biotinylation experiments demonstrated that CD4 molecules in Nef-expressing T cells are transported to the plasma membrane with normal kinetics but are then rapidly internalized. Therefore, HIV-1 Nef-induced down-modulation of CD4 is due to rapid internalization of surface CD4 and subsequent degradation by an acid-dependent process, potentially lysosomal. Additionally, in a Nef-expressing cell, we find accelerated dissociation of the T-cell tyrosine kinase p56lck and CD4 but only after the complex reaches the plasma membrane. This implies that HIV-1 Nef protein might play a role in triggering a series of T-cell activation-like events, which contribute to p56lck dissociation and internalization of surface CD4 molecules.
Insights
Human immunodeficiency virus type 1 (HIV-1) Nef protein accelerates the degradation of CD4 molecules in T cells. This HIV-1 Nef-induced down-modulation involves rapid internalization and lysosomal degradation of CD4.
Area of Science:
- Virology
- Immunology
- Cell Biology
Background:
- Human immunodeficiency virus type 1 (HIV-1) Nef protein is known to down-modulate CD4 receptors on T cells.
- The precise mechanism behind Nef-mediated CD4 down-modulation requires further elucidation.
Purpose of the Study:
- To investigate the mechanism by which HIV-1 Nef protein causes the down-modulation of cell surface CD4 molecules in human T cells.
- To understand the role of protein degradation and cellular trafficking in this process.
Main Methods:
- Expression of HIV-1 Nef protein in the human T-cell line VB.
- Pulse-chase experiments to track CD4 protein synthesis and degradation.
- Treatment with lysosomotropic agents (NH4Cl, chloroquine) and protease inhibitors (leupeptin).
- Surface CD4 biotinylation assays to assess internalization kinetics.
Main Results:
- HIV-1 Nef expression led to a significantly reduced half-life of CD4 molecules (approx. 6 h vs. 24 h in control cells).
- Nef-induced CD4 degradation was inhibited by lysosomotropic agents and leupeptin, suggesting lysosomal involvement.
- CD4 molecules were rapidly internalized from the plasma membrane in Nef-expressing cells.
- Accelerated dissociation of the T-cell tyrosine kinase p56lck from CD4 was observed post-plasma membrane transport.
Conclusions:
- HIV-1 Nef-induced CD4 down-modulation results from rapid internalization and subsequent lysosomal degradation.
- The process appears to involve accelerated dissociation of CD4 from p56lck, potentially triggering T-cell activation-like events.