The Nef Protein of Human Immunodeficiency Virus Is a Broad-Spectrum Modulator of Chemokine Receptor Cell Surface Levels That Acts Independently of Classical Motifs for Receptor Endocytosis and Gα_iSignaling

Abstract

Chemokine receptors (CKRs) are important physiological mediators of immune defense, inflammatory responses, and angiogenesis, and they have also been implicated in a number of viral disease processes. Here, we report that the Nef protein of human immunodeficiency virus (HIV) reduces cell surface levels of eight different members of the CC-and CXC-family of CKRs by up to 92%. This broad-range activity required specific elements in HIV SF2 Nef, including the proline-rich motif P 73 P 76 P 79 P 82 as well as the acidic cluster motif E 66 E 67 E 68 E 69 , and Nef expression induced a marked perinuclear accumulation of CKRs. Surprisingly, receptor mutagenesis demonstrated that the cytoplasmic tail of CCR5 and CXCR4, which is critical for basal and ligand-mediated endocytosis, was completely dispensable for this Nef activity. In contrast, triple-mutation of the highly conserved DRY motif in the second intracellular CKR loop abolished the Nef-mediated down-regulation of CXCR4 independently of this motif's role in CKR binding to heterotrimeric G proteins and signaling via the G␣ i subunit. Thus, we identify the lentiviral pathogenicity factor Nef as a unique and broad-range modulator of CKR cell surface levels. Nef uses a mechanism that is distinct from well-established pathways orchestrating CKR metabolism and offers an interesting tool to study the multifaceted biology of CKRs. INTRODUCTIONChemokine receptors (CKRs) are a specialized subset of seven-transmembrane (7-TM) G protein-coupled receptors (GPCRs) that is broadly grouped into CC, CXC, CX 3 C, and C classes based on the structure of their cognate agonists (Murphy et al., 2000). All CKRs are composed of an extracellular amino-terminal domain, seven hydrophobic transmembrane domains, and a cytoplasmic carboxy-terminal tail that harbors important motifs for basal and ligand-induced signaling, desensitization, and endocytosis. CKRs transduce signals via multiple mediators, i.e., heterotrimeric G proteins, ␤-arrestin, and GPCR kinases. Signal transduction after ligand binding is initiated by stabilizing the CKR in an active conformation that enables the binding and activation of heterotrimeric G proteins (Scheer et al., 1997;Rasmussen et al., 1999;Scheer et al., 2000;Seifert and Wenzel-Seifert, 2003). A highly conserved sequence starting with Asp-ArgTyr (DRY) in the second intracellular loop of all CKRs plays a critical role in mediating the binding and signaling via heterotrimeric G proteins. After agonist engagement, desensitization of CKRs rapidly occurs by the interaction of ␤-arrestin with phosphorylated Ser/Thr residues in the cytoplasmic tail of CKRs. Phosphorylation abolishes the signaling via heterotrimeric G proteins, and ␤-arrestin binding lowers the receptor cell surface expression by targeting the molecule for endocytosis (Krupnick and Benovic, 1998). In addition, some CKRs contain a dileucine-based element in their cytoplasmic tail that provides a second independent motif for receptor endocytosis. Receptor internalization, recycling, and/or degradation...

Lentiviral Nef Proteins Utilize PAK2-Mediated Deregulation of Cofilin as a General Strategy To Interfere with Actin Remodeling

“…In parallel, the ability of these nef alleles to downregulate CXCR4 from the cell surface was analyzed in order to test whether downregulation of this chemokine receptor determines the ability of Nef to block the response to chemokine treatment. In line with our previous findings (23,24), comparably moderate reduction of CXCR4 cell surface exposure was induced by all nef alleles but not by the SF2 Nef AxxA (prolines 76 and 79 mutated to alanine) negative control (Fig. 1D).…”

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“…The dye-loaded cells were transferred to a SpectraMax M2 e (Molecular Devices) and, after stimulation, the intracellular calcium release was recorded for 60 s at room temperature monitoring fluorescence emission at 520 nm (λ ex = 485 nm) in the presence of the analogues at the indicated concentrations. Maximum fluorescence was measured after addition of 25 μmol/L ionomycin (31,38).…”

Viral Modulation of T-Cell Receptor Signaling

“…Nef promotes HIV replication (26,100,143) and infectivity (2,22,100,131). It downregulates several cell surface molecules relevant to T-cell biology, including CD4 (44), MHC class I (132), MHC class II (144), and multiple chemokine receptors (99). In addition, Nef modulates a number of intracellular signaling pathways.…”