Leucine-Specific, Functional Interactions between Human Immunodeficiency Virus Type 1 Nef and Adaptor Protein Complexes

Abstract

The human immunodeficiency virus type 1 virulence protein Nef interacts with the endosomal sorting machinery via a leucine-based motif. Similar sequences within the cytoplasmic domains of cellular transmembrane proteins bind to the adaptor protein (AP) complexes of coated vesicles to modulate protein traffic, but the molecular basis of the interactions between these motifs and the heterotetrameric complexes is controversial. To identify the target of the Nef leucine motif, the native sequence was replaced with either leucine-or tyrosine-based AP-binding sequences from cellular proteins, and the interactions with AP subunits were correlated with function. Tyrosine motifs predictably modulated the interactions between Nef and the subunits of AP-1, AP-2, and AP-3; heterologous leucine motifs caused little change in these interactions. Conversely, leucine motifs mediated a ternary interaction between Nef and hemicomplexes containing the 1 plus ␥ subunits of AP-1 or the 3 plus ␦ subunits of AP-3, whereas tyrosine motifs did not. Similarly, only leucine motifs supported the Nef-mediated association of AP-1 and AP-3 with endosomal membranes in cells treated with brefeldin A. Functionally, Nef proteins containing leucine motifs down-regulated CD4 from the cell surface and enhanced viral replication, whereas those containing tyrosine motifs were inactive. Apparently, the interaction of Nef with the subunits of AP complexes is insufficient for function. A leucine-specific mode of interaction that likely involves AP hemicomplexes is further required for Nef activity. The and hemicomplex interactions may cooperate to yield high avidity binding of AP complexes to Nef. This binding likely underlies the unusual ability of Nef to induce the stabilization of these complexes on endosomal membranes, an activity that correlates with enhancement of viral replication.

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

“…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.…”

“…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).…”

AP-2-dependent Internalization of Potassium Channel Kir2.3 Is Driven by a Novel Di-hydrophobic Signal

“…In addition, the type of internalization motif (i.e. tyrosine-or di-leucine-based) present on a particular protein can have functional consequences for trafficking (38). Our effort to elucidate the mechanism by which Kir2.3 is controlled by endocytosis expands the repertoire of signals known to interact with AP complexes.…”

Estudo da função de AP1y2 e Alix no direcionamento de proteínas para degradação em lisossomos ou liberação em vesículas extracelulares

“…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;. However, the role of the Nef interaction with AP-1 or AP-3 in CD4 downregulation is poorly understood.…”

“…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 .…”