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

Greenberg, M. E., Bronson, S., Lock, M., Neumann, M., Pavlakis, G. N., Skowronski, J. (1997) Co-localization of HIV-1 Nef with the AP-2 adaptor protein complex correlates with Nef-induced CD4 down-regulation. Embo Journal, 16 (23). pp. 6964-76. ISSN 0261-4189

URL: http://www.ncbi.nlm.nih.gov/pubmed/9384576
DOI: 10.1093/emboj/16.23.6964

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 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.

Item Type: Paper
Uncontrolled Keywords: Adaptor Protein Complex alpha Subunits Adaptor Protein Complex beta Subunits Adaptor Protein Complex gamma Subunits Adaptor Proteins, Vesicular Transport Antigens, CD4/ biosynthesis CD4-Positive T-Lymphocytes Cell Compartmentation Cell Membrane/metabolism DNA Mutational Analysis Down-Regulation Endocytosis Gene Products, nef/genetics/ metabolism Green Fluorescent Proteins Hiv-1 Humans Jurkat Cells Luminescent Proteins/genetics/metabolism Membrane Proteins/ metabolism Peptide Fragments/genetics/metabolism Recombinant Fusion Proteins/metabolism Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.
Subjects: diseases & disorders > viral diseases > HIV
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene regulation
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > genes, structure and function > gene regulation
CSHL Authors:
Communities: CSHL labs > Skowronski lab
Dolan DNA Learning Center
Depositing User: Kathleen Darby
Date Deposited: 07 May 2014 14:52
Last Modified: 07 May 2014 14:52
PMCID: PMC1170300
Related URLs:
URI: http://repository.cshl.edu/id/eprint/30024

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