HIV-1 Nef-induced Down-Regulation of MHC Class I Requires AP-1 and Clathrin but Not PACS-1 and Is Impeded by AP-2

Abstract

Major histocompatibility complex class I is down-regulated from the surface of human immunodeficiency virus (HIV)-1-infected cells by Nef, a virally encoded protein that is thought to reroute MHC-I to the trans-Golgi network (TGN) in a phosphofurin acidic cluster sorting protein (PACS) 1, adaptor protein (AP)-1, and clathrin-dependent manner. More recently, an alternative model has been proposed, in which Nef uses AP-1 to direct MHC-I to endosomes and lysosomes. Here, we show that knocking down either AP-1 or clathrin with small interfering RNA inhibits the down-regulation of HLA-A2 (an MHC-I isotype) by Nef in HeLa cells. However, knocking down PACS-1 has no effect, not only on Nef-induced down-regulation of HLA-A2 but also on the localization of other proteins containing acidic cluster motifs. Surprisingly, knocking down AP-2 actually enhances Nef activity. Immuno-electron microscopy labeling of Nef-expressing cells indicates that HLA-A2 is rerouted not to the TGN, but to endosomes. In AP-2-depleted cells, more of the HLA-A2 localizes to the inner vesicles of multivesicular bodies. We propose that depleting AP-2 potentiates Nef activity by altering the membrane composition and dynamics of endosomes and causing increased delivery of HLA-A2 to a prelysosomal compartment. INTRODUCTIONLike many viruses, human immunodeficiency virus (HIV)-1 has evolved strategies to avoid detection and destruction by the host. One such strategy is the down-regulation of major histocompatability complex (MHC) class I from the plasma membrane of HIV-1-infected cells, which prevents the cells from being attacked by cytotoxic T lymphocytes. MHC-I down-regulation has been shown to be a function of a virally encoded protein called Nef (Schwartz et al., 1996;Collins et al., 1998). Nef is one of the so-called "accessory proteins" of both human and simian immunodeficiency viruses: although not required for viral replication in vitro, Nef plays a key role in the development of acquired immunodeficiency syndrome. Nef is a small protein, only ϳ200 amino acids, but it has been reported to interact with a large number of host cell proteins, including several proteins that are involved in membrane traffic. These interactions are thought to be responsible for the ability of Nef to modulate the surface expression of MHC-I and other molecules (for review, see Collins and Baltimore, 1999;Piguet et al., 1999;Doms and Trono, 2000;Roeth and Collins, 2006).Among the binding partners that have been identified for Nef are the adaptor protein (AP) complexes. There are four AP complexes in mammalian cells, two of which, AP-1 and AP-2, are highly enriched in clathrin-coated vesicles (CCVs). AP-1 facilitates clathrin-mediated trafficking between the trans-Golgi network (TGN) and endosomes (although there is still some question about directionality), and AP-2 facilitates clathrin-mediated endocytosis (Robinson, 2004). AP-3, which seems to be able to act both in a clathrin-dependent and in a clathrin-independent manner (Dell'Angelica et al., 1998;Peden et al., 2...

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

“…This weakened effect could be the consequence of co-depletion of AP-1 complexes comprising γ1, as the absence of both AP-1 variants might favor pathways mediated by other adaptor proteins that utilize common co-factors, such as AP-2. Specifically, it has been previously proposed that AP-2 depletion enhances AP-1-dependent pathways (Lubben et al, 2007). Therefore, γ2 is most likely part of an AP-1 complex variant involved in Nef-mediated targeting of CD4 to lysosomes, a notion that is further confirmed by BiFC assays showing that γ2 labeling greatly overlaps with signals for Nef interaction with AP-1 (μ1A) in CD4-positive structures ( Fig.…”

The AP-1 Complex Regulates Intracellular Localization of Insulin Receptor Substrate 1, Which Is Required for Insulin-Like Growth Factor I-Dependent Cell Proliferation

“…The fact that we detected little or no overlap between endogenous IRS-1 and conventional endosome markers (e.g., EEA1, Rab11, and transferrin receptor) (data not shown) indicates that IRS-1 is localized to a unique vesicular compartment. The sorting directionality of AP-1 is reported to be both anterograde (TGN/post-TGN compartment to peripheral endosome) and retrograde (endosome to TGN) and seems to depend on the type of cargo protein (20)(21)(22)(23)36). Thus, defining the intracellular localization of IRS-1 will help us understand the more detailed AP-1 sorting pathway of IRS-1.…”

“…In spite of numerous studies, there is still some uncertainty about the directionality of AP-1 trafficking. For example, some studies have observed accumulation of cargo proteins in endosome or postendosome compartments in AP-1-deficient cells (20,21), while others showed that they accumulated in the Golgi region or at the plasma membrane (22,23). Irrespective of directionality, AP-1 is generally believed to sort cargo proteins between endosomes and the trans-Golgi network (TGN).…”

TE-domestication and horizontal transfer in a putative Nef-AP1mu mimic of HLA-A cytoplasmic domain re-trafficking

“…3C) for how the Loc-103275158 protein might combat Nef downregulation of MHC-I from the surface of tarsier cells infected by an appropriate primate lentivirus, i.e., after the mechanism of HIV-1 Nef in humans. 5,7,[19][20][21] Theoretically, a phosphorylation/dephosphorylation event within the CD loop would occur when the protein is in a "closed" conformation; based upon our modeled contacts, we prefer the notion that the MHC-I CD initially associates with some affinity to this closed conformation (left cartoon, Fig. 3C).…”