Downregulation of CD4 by Human Immunodeficiency Virus Type 1 Nef Is Dependent on Clathrin and Involves Direct Interaction of Nef with the AP2 Clathrin Adaptor

Abstract

Nef, an accessory protein of human and simian immunodeficiency viruses, is a critical determinant of pathogenesis that promotes the progression from infection to AIDS. The pathogenic effects of Nef are in large part dependent on its ability to downregulate the macrophage and T-cell coreceptor, CD4. It has been proposed that Nef induces downregulation by linking the cytosolic tail of CD4 to components of the host-cell protein trafficking machinery. To identify these components, we developed a novel Nef-CD4 downregulation system in Drosophila melanogaster S2 cells. We found that human immunodeficiency virus type 1 (HIV-1) Nef downregulates human CD4 in S2 cells and that this process is subject to the same sequence requirements as in human cells. An RNA interference screen targeting protein trafficking genes in S2 cells revealed a requirement for clathrin and the clathrin-associated, plasma membrane-localized AP2 complex in the downregulation of CD4. The requirement for AP2 was confirmed in the human cell line HeLa. We also used a yeast three-hybrid system and glutathione S-transferase pull-down analyses to demonstrate a robust, direct interaction between HIV-1 Nef and AP2. This interaction requires a dileucine motif in Nef that is also essential for downregulation of CD4. Together, these results support a model in which HIV-1 Nef downregulates CD4 by promoting its accelerated endocytosis by a clathrin/AP2 pathway.

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

“…As has been previously shown by yeast three-hybrid (Y3H) assays, the interaction of Nef with γ1-σ1A requires a conserved (D/E)xxxL(L/I) signal in Nef (i.e the ENTSLL 165 motif in HIV-1 NL4-3 Nef ), and a conserved basic residue in γ1 at position 15 (Arg 15 ) (Chaudhuri et al, 2007;Mattera et al, 2011) (Fig. 1A).…”

“…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).…”

“…These isoforms include two γ (γ1 and γ2), two μ1 (μ1A and μ1B) and three σ1 (σ1A, σ1B and σ1C) subunits for AP-1; two α subunits for AP-2; and two μ3 (μ3A and μ3B), two β3 (β3A and β3B) and two σ3 (σ3A and σ3B) subunits for AP-3 (Boehm and Bonifacino, 2002). Nef binds to APs through a conserved 'dileucine-based' (D/E)xxxL(L/I) consensus motif in the viral protein (Craig et al, 1998;Greenberg et al, 1998), which is recognized by hemi-complexes comprising the γ1-σ1, α-σ2 and δ-σ3 subunits of the respective AP (Chaudhuri et al, 2007;Coleman et al, 2005;Doray et al, 2007;Janvier et al, 2003a,b). Subsequently, it has been shown that γ2 is also capable of mediating a σ1-dependent interaction with Nef (Mattera et al, 2011).…”

“…In addition to AP-2, formed by α, β2, μ2 and σ2 subunits (Table S1), two other AP complexes, AP-1 (γ1, β1, μ1 and σ1) and AP-3 (δ, β3, μ3 and σ3), are binding partners of Nef (Bresnahan et al, 1998;Chaudhuri et al, 2007;Coleman et al, 2005;Doray et al, 2007;Greenberg et al, 1998;Janvier et al, 2003a,b;Mattera et al, 2011). However, the role of the Nef interaction with AP-1 or AP-3 in CD4 downregulation is poorly understood.…”

Localization to Mature Melanosomes by Virtue of Cytoplasmic Dileucine Motifs Is Required for Human OCA2 Function

“…Previous studies have shown that acidic dileucine signals bind to members of the clathrin-associated heterotetrameric adaptor protein family (Bonifacino and Traub, 2003;Janvier et al, 2003;Chaudhuri et al, 2007;Doray et al, 2007). The acidic dileucine signal in Tyr mediates an interaction with both AP-3 and AP-1 (Honing et al, 1998;Theos et al, 2005), whereas the corresponding signal in Tyrp1 has been shown to interact with AP-1 only (Theos et al, 2005).…”