Direct In Vitro Binding of Full-Length Human Immunodeficiency Virus Type 1 Nef Protein to CD4 Cytoplasmic Domain

Preußer, Andrea; Briese, Lars; Baur, Andreas S.; Willbold, Dieter

doi:10.1128/jvi.75.8.3960-3964.2001

Cited by 52 publications

(54 citation statements)

References 42 publications

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“…Since internalization of raft-associated proteins may involve a clathrin-independent route, it may be that a key role of Nef is to target the clathrin-mediated endocytosis machinery to a location normally devoid of clathrin. Although nuclear magnetic resonance studies have shown that a full-length recombinant Nef protein interacts directly with a synthetic peptide derived from the cytoplasmic tail of CD4, Nef-CD4 binding has been difficult to demonstrate (53). It may be that a combination of CD4 binding and raft association by Nef is key to the forming of a stable Nef-CD4 complex.…”

Section: Discussionmentioning

confidence: 99%

“…There is general agreement that Nef induces the internalization of CD4 via clathrin-coated pits, probably by binding directly to the cytoplasmic tail of CD4 (53), and then directly (14,49) or indirectly (26,40) linking to a cellular adaptor complex, most likely either AP-1 (7,17) or AP-2 (17,26,30). Nef has also been implicated in a second step that targets CD4 to lysosomes for degradation (33,50).…”

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confidence: 99%

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Human Immunodeficiency Virus Type 1 Nef Associates with Lipid Rafts To Downmodulate Cell Surface CD4 and Class I Major Histocompatibility Complex Expression and To Increase Viral Infectivity

Alexander

Bor

Ravichandran

et al. 2004

J Virol

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Lipid rafts are membrane microdomains that are functionally distinct from other membrane regions. We have shown that 10% of human immunodeficiency virus type 1 (HIV-1) Nef expressed in SupT1 cells is present in lipid rafts and that this represents virtually all of the membrane-associated Nef. To determine whether raft targeting, rather than simply membrane localization, has functional significance, we created a Nef fusion protein (LAT-Nef) containing the N-terminal 35 amino acids from LAT, a protein that is exclusively localized to rafts. Greater than 90% of the LAT-Nef protein was found in the raft fraction. In contrast, a mutated form, lacking two cysteine palmitoylation sites, showed less than 5% raft localization. Both proteins were equally expressed and targeted nearly exclusively to membranes. The LAT-Nef protein was more efficient than its nonraft mutant counterpart at downmodulating both cell surface CD4 and class I major histocompatibility complex (MHC) expression, as well as in enhancing first-round infectivity and being incorporated into virus particles. This demonstrates that targeting of Nef to lipid rafts is mechanistically important for all of these functions. Compared to wild-type Nef, LAT-Nef downmodulated class I MHC nearly as effectively as the wild-type Nef protein, but was only about 60% as effective for CD4 downmodulation and 30% as effective for infectivity enhancement. Since the LAT-Nef protein was found entirely in rafts while the wild-type Nef protein was distributed 10% in rafts and 90% in the soluble fraction, our results suggest that class I MHC downmodulation by Nef may be performed exclusively by raft-bound Nef. In contrast, CD4 downmodulation and infectivity enhancement may require a non-membrane-bound Nef component as well as the membrane-bound form.

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Human Immunodeficiency Virus Type 1 Nef Associates with Lipid Rafts To Downmodulate Cell Surface CD4 and Class I Major Histocompatibility Complex Expression and To Increase Viral Infectivity

Alexander

Bor

Ravichandran

et al. 2004

J Virol

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“…Instead, Nef accelerates the endocytosis of CD4 and targets the internalized molecules to the lumen of lysosomes for degradation (Aiken et al, 1994;Rhee and Marsh, 1994). To accomplish this, Nef directly binds the cytosolic tail of CD4 (Grzesiek et al, 1996;Preusser et al, 2001;Rossi et al, 1996) and the endocytic adaptor protein 2 (AP-2) (Chaudhuri et al, 2007(Chaudhuri et al, , 2009Doray et al, 2007;Lindwasser et al, 2008). This activity promotes the cooperative assembly of a tripartite Nef-AP-2-CD4 complex (Chaudhuri et al, 2009;Ren et al, 2014) that induces the endocytosis of CD4 mediated by clathrin-coated vesicles (CCVs) (Burtey et al, 2007;Greenberg et al, 1997).…”

Section: Introductionmentioning

confidence: 99%

CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking

Tavares

Silva

Silva-Januário

et al. 2017

Journal of Cell Science

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The HIV accessory protein Nef is a major determinant of viral pathogenesis that facilitates viral particle release, prevents viral antigen presentation and increases infectivity of new virus particles. These functions of Nef involve its ability to remove specific host proteins from the surface of infected cells, including the CD4 receptor. Nef binds to the adaptor protein 2 (AP-2) and CD4 in clathrin-coated pits, forcing CD4 internalization and its subsequent targeting to lysosomes. Herein, we report that this lysosomal targeting requires a variant of AP-1 containing isoform 2 of γ-adaptin (AP1G2, hereafter γ2). Depletion of the γ2 or μ1A (AP1M1) subunits of AP-1, but not of γ1 (AP1G1), precludes Nef-mediated lysosomal degradation of CD4. In γ2-depleted cells, CD4 internalized by Nef accumulates in early endosomes and this alleviates CD4 removal from the cell surface. Depletion of γ2 also hinders EGFR-EGF-complex targeting to lysosomes, an effect that is not observed upon γ1 depletion. Taken together, our data provide evidence that the presence of γ1 or γ2 subunits delineates two distinct variants of AP-1 complexes, with different functions in protein sorting.

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“…Replacing the CD4 tail with Nef results in a chimeric protein that down-regulates CD4 in trans as well as the chimeric protein itself in cis (23). Several studies have shown that CD4 interacts directly with Nef in vitro and in insect cells and that the dileucine motif in the CD4 tail is essential for the interaction (25,26,28,29). However, the direct interaction between CD4 and Nef in mammalian cells has yet to be demonstrated (24).…”

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confidence: 99%

CD4 Phosphorylation Partially Reverses Nef Down-Regulation of CD4

Jin

Zhang

Boursiquot

et al. 2004

The Journal of Immunology

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HIV Nef down-regulates CD4 from the cell surface in the absence of CD4 phosphorylation, whereas PMA down-regulates CD4 through a phosphorylation-dependent pathway. In this study we show that the down-regulation of CD4 in human Jurkat T cells expressing Nef was nearly complete (∼95%), whereas that induced by PMA was partial (∼40%). Unexpectedly, treating T cells expressing Nef with PMA restored the surface CD4 up to 35% of the steady state level. Both mutating the phosphorylation sites in the CD4 cytoplasmic tail (Ser408 and Ser415) and the use of a protein kinase C inhibitor, bisindolylmaleimide1, abolished the restoration of surface CD4, suggesting that the restoration required CD4 phosphorylation. CD4 and Nef could be cross-linked by a chemical cross-linker, 3,3-dithiobis[sulfosuccinimidyl-propionate], in control T cell membranes, but not in PMA-treated T cell membrane, suggesting that CD4 and Nef interacted with each other in T cells, and the phosphorylation disrupted the CD4-Nef interaction. We propose that this dissociation switches CD4 internalization from the Nef-mediated, nearly complete down-regulation to a phosphorylation-dependent, partial down-regulation, resulting in a net gain of CD4 on the T cell surface.

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Direct In Vitro Binding of Full-Length Human Immunodeficiency Virus Type 1 Nef Protein to CD4 Cytoplasmic Domain

Cited by 52 publications

References 42 publications

Human Immunodeficiency Virus Type 1 Nef Associates with Lipid Rafts To Downmodulate Cell Surface CD4 and Class I Major Histocompatibility Complex Expression and To Increase Viral Infectivity

Human Immunodeficiency Virus Type 1 Nef Associates with Lipid Rafts To Downmodulate Cell Surface CD4 and Class I Major Histocompatibility Complex Expression and To Increase Viral Infectivity

CD4 downregulation by the HIV-1 protein Nef reveals distinct roles for the γ1 and γ2 subunits of the AP-1 complex in protein trafficking

CD4 Phosphorylation Partially Reverses Nef Down-Regulation of CD4

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