An interdomain binding site on HIV-1 Nef interacts with PACS-1 and PACS-2 on endosomes to down-regulate MHC-I

Abstract

<jats:p>The human immunodeficiency virus type 1 (HIV-1) accessory protein Nef directs virus escape from immune surveillance by subverting host cell intracellular signaling and membrane traffic to down-regulate cell-surface major histocompatibility complex class I (MHC-I). The interaction of Nef with the sorting proteins PACS-1 and PACS-2 mediates key signaling and trafficking steps required for Nef-mediated MHC-I down-regulation. Little is known, however, about the molecular basis underlying the Nef–PACS interaction. Here we identify the sites on Nef and the PACS proteins required for their interaction and describe the consequences of disrupting this interaction for Nef action. A previously unidentified cargo subsite on PACS-1 and PACS-2 interacted with a bipartite site on Nef formed by the EEEE<jats:sub>65</jats:sub>acidic cluster on the N-terminal domain and W<jats:sub>113</jats:sub>in the core domain. Mutation of these sites prevented the interaction between Nef and the PACS proteins on Rab5 (PACS-2 and PACS-1)- or Rab7 (PACS-1)-positive endosomes as determined by bimolecular fluorescence complementation and caused a Nef mutant defective in PACS binding to localize to distorted endosomal compartments. Consequently, disruption of the Nef–PACS interaction repressed Nef-induced MHC-I down-regulation in peripheral blood mononuclear cells. Our results provide insight into the molecular basis of Nef action and suggest new strategies to combat HIV-1.</jats:p>

PACS-2 mediates the ATM and NF-κB-dependent induction of anti-apoptotic Bcl-xL in response to DNA damage

“…53 The PACS genes appeared first in metazoans where they have early-and conserved roles in secretory pathway traffic. 22,23,[54][55][56][57] Sequence alignment and functional analyses reveal that the vertebrate PACS proteins underwent evolutionary adaptation with the acquisition of nuclear-trafficking motifs and, in the case of PACS-2, an Akt phosphorylation site at Ser 437 . 57 The acquisition of nucleartrafficking functions in PACS-2 coincided with the ability of vertebrate p53 to direct cytoprotective p21-dependent cell cycle arrest in addition to its evolutionarily conserved proapoptotic functions.…”

“…PACS-2 was initially identified by its role in mediating secretory pathway traffic and formation of contacts between the ER and mitochondria to regulate interorganellar communication and autophagy. [20][21][22][23][24][25][26][27] In response to the death ligand TRAIL (TNF-related apoptosis-inducing ligand), PACS-2 becomes dephosphorylated at Ser 437 , switching PACS-2 to a proapoptotic effector that drives permeabilization of mitochondria and lysosomes, which promotes activation of executioner caspases. 20 Recent studies show that PACS-2 responds to DNA damage by promoting cell cycle arrest and cell survival.…”

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

“…To investigate whether interaction of Nef with the AP-1 complex during CD4 downregulation occurs in subcellular sites where γ2 is present, we used bimolecular fluorescence complementation (BiFC). This technique has been broadly used to study Nef dimerization (Poe and Smithgall, 2009;Poe et al, 2014) and the interaction of Nef with host cell proteins (Amorim et al, 2014;Dikeakos et al, 2012;Dirk et al, 2015). To this end, we generated constructs encoding Nef and μ1A fused to the N-or C-terminal halves of the Venus fluorescent protein, respectively (Nef-VNt and μ1A-VCt).…”

Caught in the act – protein adaptation and the expanding roles of the PACS proteins in tissue homeostasis and disease

“…The PACS-1 FBR binds to acidic clusters that can be phosphorylated by casein kinase 2 (CK2), as well as α-helices on a large number of client proteins (Wan et al, 1998;Youker et al, 2009;Dikeakos et al, 2012). Thus, PACS-1, which interacts with the clathrin adaptors AP-1 and AP-3, as well as the monomeric adaptor GGA3, mediates localization of furin and other client proteins to the trans-Golgi network (TGN) and also targets a subset of client proteins to the primary cilium, including the adaptor protein nephrocystin (also known as NPHP1) and the olfactory cyclic-nucleotide-gated ion channel (CNG), the latter by binding to the β1 subunit (CNGB1) (Fig.…”

“…This interaction enables KSHV to downregulate the cell adhesion molecule CD31 (also known as PECAM1), which may contribute to KSHV-induced cancer (Mansouri et al, 2006). The HIV-1 accessory protein Nef uses a bipartite motif composed of a short acidic cluster and the αB helix to interact with both PACS-1 and PACS-2 (Piguet et al, 2000;Atkins et al, 2008;Dikeakos et al, 2012). This bipartite binding enables HIV-1 Nef to downregulate major histocompatibility complex class I (MHC-I) from the cell surface, which allows the virus to escape immune detection (Pawlak and Dikeakos, 2015) (Fig.…”