HIV-1 Nef Down-Modulates C-C and C-X-C Chemokine Receptors via Ubiquitin and Ubiquitin-Independent Mechanism

Authors:
Prabha Chandrasekaran, Victoria Moore, Monica Buckley, Joshua Spurrier, John H. Kehrl, Sundararajan Venkatesan
In:
Source: PLoS ONE
Publication Date: (2014)
Issue: 9(1): e86998
Research Area:
Immunotherapy / Hematology
Cells used in publication:
Jurkat
Species: human
Tissue Origin: blood
Monocyte, human
Species: human
Tissue Origin: blood
T cell, human cord blood unstim.
Species: human
Tissue Origin: blood
Platform:
Nucleofector® I/II/2b
Experiment
Nef is an HIV protein that is involved in the pathogenesis of HIV infec¬tions by dysregulating the trafficking of immune cell receptors. In this comprehensive study, the authors examined how Nef modulates various chemokine receptors, e.g. CXCR4. For this purpose they transfected various primary cell types (human T cells and monocytes) and T cell lines (CEM, Jurkat, K562) using Nucleofection™ with plasmids or siRNA to overexpress or down-regulate potentially involved components of the suggested signaling pathway. For easy-to-transfect cell lines (e.g. HEK293) a lipid reagent was used.
Abstract
Human and Simian Immunodeficiency virus (HIV-1, HIV-2, and SIV) encode an accessory protein, Nef, which is a pathogenesis and virulence factor. Nef is a multivalent adapter that dysregulates the trafficking of many immune cell receptors, including chemokine receptors (CKRs). Physiological endocytic itinerary of agonist occupied CXCR4 involves ubiquitinylation of the phosphorylated receptor at three critical lysine residues and dynamin-dependent trafficking through the ESCRT pathway into lysosomes for degradation. Likewise, Nef induced CXCR4 degradation was critically dependent on the three lysines in the C-terminal -SSLKILSKGK- motif. Nef directly recruits the HECT domain E3 ligases AIP4 or NEDD4 to CXCR4 in the resting state. This mechanism was confirmed by ternary interactions of Nef, CXCR4 and AIP4 or NEDD4; by reversal of Nef effect by expression of catalytically inactive AIP4-C830A mutant; and siRNA knockdown of AIP4, NEDD4 or some ESCRT-0 adapters. However, ubiquitinylation dependent lysosomal degradation was not the only mechanism by which Nef downregulated CKRs. Agonist and Nef mediated CXCR2 (and CXCR1) degradation was ubiquitinylation independent. Nef also profoundly downregulated the naturally truncated CXCR4 associated with WHIM syndrome and engineered variants of CXCR4 that resist CXCL12 induced internalization via an ubiquitinylation independent mechanism.