Greenberg, M. E., Bronson, S., Lock, M., Neumann, M., Pavlakis, G. N., Skowronski, J. (December 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
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: | 1 December 1997 |
| Date Deposited: | 07 May 2014 14:52 |
| Last Modified: | 07 May 2014 14:52 |
| PMCID: | PMC1170300 |
| Related URLs: | |
| URI: | https://repository.cshl.edu/id/eprint/30024 |
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