Identification of a Novel Binding Site Between HIV Type 1 Nef C-Terminal Flexible Loop and AP2 Required for Nef-Mediated CD4 Downregulation

Abstract

HIV-1 Nef is an accessory protein necessary for HIV-1 virulence and rapid AIDS development. Nef promotes viral replication and infection by connecting CD4 and several other cell surface receptors to the clathrin adaptor protein AP2, resulting in the internalization and degradation of the receptors interacting with Nef. We investigated how Nef can mediate constitutive receptor endocytosis through the interaction of the dileucine motif in its C-terminal flexible loop (C-loop) with AP2, whereas AP2 binding of the transmembrane receptors usually results in an equilibrated (recycled) endocytosis. Our results indicated that in addition to the dileucine motif, there is a second motif in the Nef C-loop involved in the Nef-AP2 interaction. Nef-mediated CD4 downregulation was impaired when the residue in the hydrophobic region in the Nef C-loop (LL165HPMSLHGM173) was mutated to a basic residue K/R or an acidic residue E/D or to the rigid residue P, or when M168L170, L170H171, or G172M173 was mutated to AA. A pull-down assay indicated that AP2 was not coprecipitated with Nef mutants that did not downregulate CD4. Molecular modeling of the Nef C-terminal flexible loop in complex with AP2 suggests that M168L170 occupies a pocket in the AP2 σ2 subunit. Our data suggest a new model in the Nef-AP2 interaction in which the hydrophobic region in the Nef C-loop with the dileucine (L164L165) motif and M168L170 motif binds to AP2(σ2), while the acidic motif E174 and D175 binds to AP2(α), which explains how Nef through the flexible loop connects CD4 to AP2 for constitutive CD4 downregulation.

How HIV-1 Nef hijacks the AP-2 clathrin adaptor to downregulate CD4

“…The Nef-AP-2 interaction is so central to CD4 downregulation that it has inspired exhaustive mutational analyses ( Aiken et al, 1996 ; Hua et al, 1997 ; Craig et al, 1998 ; Janvier et al, 2003 ; Lindwasser et al, 2008 ; Chaudhuri et al, 2009 ; Mattera et al, 2011 ; Jin et al, 2013 ). These results can now be mapped onto the structure ( Figure 4 ).…”

“…CD4 downregulation depends on both Nef myristoylation ( Aiken et al, 1994 ) and specific residues in the loop, including Leu164 and Leu165 ( Bresnahan et al, 1998 ; Craig et al, 1998 ; Greenberg et al, 1998 ; Janvier et al, 2003 ), which are in a sequence context fitting the [DE]XXXL[LI] motif for dileucine-based sorting signals ( Bonifacino and Traub, 2003 ), and the diacidic motif, Asp174-Asp175 ( Aiken et al, 1996 ; Lindwasser et al, 2008 ). Myristoylation allows recruitment of Nef from the cytosol to the inner leaflet of the plasma membrane ( Yu and Felsted, 1992 ) while the loop engages the clathrin-associated adaptor protein 2 (AP-2) complex ( Jin et al, 2005 ; Chaudhuri et al, 2007 ; Doray et al, 2007 ; Lindwasser et al, 2008 ; Mattera et al, 2011 ; Jin et al, 2013 ). The Nef:AP-2 complex interacts with the cytosolic tail of CD4, leading to the cooperative assembly of a tripartite Nef:AP-2:CD4 complex ( Chaudhuri et al, 2009 ).…”

Cargo Recognition in Clathrin-Mediated Endocytosis

“…In between the dileucine and diacidic motifs there are additional hydrophobic residues that also bind to AP-2 likely via s2 (Fig. 2B) (Jin et al 2012). The Nef loop thus exemplifies how a long sequence containing both canonical and noncanonical elements can interact with AP-2.…”

HIV‐1 Nef: Taking Control of Protein Trafficking

“…Three domains within the conserved 27‐amino acid central loop of Nef were implicated as crucial in mediating interaction with AP‐2. The dileucine motif EXXXLL 160–165 and the novel hydrophobic motif M 168 /L 170 were predicted to bind to the σ2 subunit, while an acidic motif DD 174,175 was suggested to bind to a basic patch in the α subunit .…”