A dileucine motif in HIV-1 Nef is essential for sorting into clathrin-coated pits and for downregulation of CD4

Abstract

Nef, a approximately 200 residue multifunctional regulatory protein of human immunodeficiency virus (HIV) and simian immunodeficiency virus (SIV), interacts with components of host cell signal transduction and clathrin-dependent protein sorting pathways. The downregulation of surface CD4 molecules and major histocompatibility complex (MHC) class I antigens by Nef is believed to be important in AIDS pathogenesis [1-7]. Nef contains a globular core domain and two disordered segments--a myristylated arm at the amino terminus and a carboxy-terminal loop projecting from the globular core [8,9]. Here, we aimed to determine the sorting signals in HIV-1 Nef that were responsible for its involvement in the clathrin-mediated pathway. We found that a sequence in the carboxy-terminal disordered loop of Nef is essential for downregulation of CD4. This sequence resembles the dileucine motif, one of two well-characterized sorting signals that target membrane proteins to clathrin-coated vesicles. The dileucine-motif-containing segment of Nef bound directly and specifically to the beta-adaptin subunit of the clathrin adaptor complexes AP-1 and AP-2, which are responsible for recruiting sorted proteins into coated pits. Unlike wild-type Nef, a mutant form of Nef that lacked the dileucine motif did not localize to clathrin-coated pits and did not downregulate CD4 expression, although it could downregulate MHC class I surface expression. Thus, the dileucine motif in HIV-1 is required for CD4 downregulation and for interaction with clathrin adaptor complexes.

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.…”

Characterization of a Fourth Adaptor-related Protein Complex

“…The corresponding subunits in the three complexes generally share between 20 and 40% amino acid identity as well as a number of conserved motifs. The three complexes also all bind to similar types of sorting signals on the cytoplasmic domains of transmembrane proteins: tyrosine-based sorting signals, which have been shown to interact with the subunits of the three AP complexes (Ohno et al, 1995(Ohno et al, , 1998Owen and Evans, 1998), and dileucine signals, which may interact with their ␤ subunits (Greenberg et al, 1998).…”

Subunit H of the V-ATPase Involved in Endocytosis Shows Homology to β-Adaptins

“…It is required for the internalization of CD4 from the cell surface to endosomes and lysosomes (Craig et al, 1998;Greenberg et al, 1998). Several proteins have been proposed to direct the sorting of Nef.…”

“…Several proteins have been proposed to direct the sorting of Nef. They include the ␤-chain of AP-1 complexes (Bresnahan et al, 1998;Greenberg et al, 1998), the ␤-subunit of the COP-I coatomer (Piguet et al, 1999;Janvier et al, 2001), and the regulatory subunit H (V1H) of the vacuolar ATPase (VATPase), previously named NBP1 for Nef binding protein 1 (Lu et al, 1998;Mandic et al, 2001). Although the precise sites of these interactions varied, all protein complexes were reported to bind to the C-terminal flexible loop in Nef, which contains the di-leucine-based motif at its center (Grzesiek et al, 1996;Lee et al, 1996;).…”