HIV-1 Nef down-modulates C-C and C-X-C chemokine receptors via ubiquitin and ubiquitin-independent mechanism.

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.

HIV-1 Nef impairs heterotrimeric G-protein signaling by targeting Gα(i2) for degradation through ubiquitination.

The HIV Nef protein is an important pathogenic factor that modulates cell surface receptor trafficking and impairs cell motility, presumably by interfering at multiple steps with chemotactic receptor signaling. Here, we report that a dominant effect of Nef is to trigger AIP4 E3 ligase-mediated Gα(i2) ubiquitination, which leads to Gα(i2) endolysosomal sequestration and destruction. The loss of the Gα(i2) subunit was demonstrable in many cell types in the context of gene transfection, HIV infection, or Nef protein transduction. Nef directly interacts with Gα(i2) and ternary complexes containing AIP4, Nef, and Gα(i2) form. A substantial reversal of Gα(i2) loss and a partial recovery of impaired chemotaxis occurred following siRNA knockdown of AIP4 or NEDD4 or by inhibiting dynamin. The N-terminal myristoyl group, (62)EEEE(65) motif, and (72)PXXP(75) motif of Nef are critical for this effect to occur. Nef expression does not affect a Gq(i5) chimera where the five C-terminal residues of Gq are replaced with those of Gα(i2). Lysine at position 296 of Gα(i2) was identified as the critical determinant of Nef-induced degradation. By specifically degrading Gα(i2), Nef directly subverts leukocyte migration and homing. Impaired trafficking and homing of HIV Nef-expressing lymphocytes probably contributes to early immune dysfunction following HIV infection.

TLR signaling paralyzes monocyte chemotaxis through synergized effects of p38 MAPK and global Rap-1 activation.

Toll-like receptors (TLRs) that recognize pathogen associated molecular patterns and chemoattractant receptors (CKRs) that orchestrate leukocyte migration to infected tissue are two arms of host innate immunity. Although TLR signaling induces synthesis and secretion of proinflammatory cytokines and chemokines, which recruit leukocytes, many studies have reported the paradoxical observation that TLR stimulation inhibits leukocyte chemotaxis in vitro and impairs their recruitment to tissues during sepsis. There is consensus that physical loss of chemokine receptor (CKR) at the RNA or protein level or receptor usage switching are the mechanisms underlying this effect. We show here that a brief (<15 min) stimulation with LPS (lipopolysaccharide) at ~0.2 ng/ml inhibited chemotactic response from CCR2, CXCR4 and FPR receptors in monocytes without downmodulation of receptors. A 3 min LPS pre-treatment abolished the polarized accumulation of F-actin, integrins and PIP(3) (phosphatidylinositol-3,4,5-trisphosphate) in response to chemokines in monocytes, but not in polymorphonuclear neutrophils (PMNs). If chemoattractants were added before or simultaneously with LPS, chemotactic polarization was preserved. LPS did not alter the initial G-protein signaling, or endocytosis kinetics of agonist-occupied chemoattractant receptors (CKRs). The chemotaxis arrest did not result from downmodulation of receptors or from inordinate increase in adhesion. LPS induced rapid p38 MAPK activation, global redistribution of activated Rap1 (Ras-proximate-1 or Ras-related protein 1) GTPase and Rap1GEF (guanylate exchange factor) Epac1 (exchange proteins activated by cyclic AMP) and disruption of intracellular gradient. Co-inhibition of p38 MAPK and Rap1 GTPase reversed the LPS induced breakdown of chemotaxis suggesting that LPS effect requires the combined function of p38 MAPK and Rap1 GTPase.

HIV-1 Nef binds a subpopulation of MHC-I throughout its trafficking itinerary and down-regulates MHC-I by perturbing both anterograde and retrograde trafficking.

The HIV protein Nef is thought to mediate immune evasion and promote viral persistence in part by down-regulating major histocompatibility complex class I protein (MHC-I or HLA-I) from the cell surface. Two different models have been proposed to explain this phenomenon as follows: 1) stimulation of MHC-I retrograde trafficking from and aberrant recycling to the plasma membrane, and 2) inhibition of anterograde trafficking of newly synthesized HLA-I from the endoplasmic reticulum to the plasma membrane. We show here that Nef simultaneously uses both mechanisms to down-regulate HLA-I in peripheral blood mononuclear cells or HeLa cells. Consistent with this, we found by using fluorescence correlation spectroscopy that a third of diffusing HLA-I at the endoplasmic reticulum, Golgi/trans-Golgi network, and the plasma membrane (PM) was associated with Nef. The binding of Nef was similarly avid for native HLA-I and recombinant HLA-I A2 at the PM. Nef binding to HLA-I at the PM was sensitive to specific inhibition of endocytosis. It was also attenuated by cyclodextrin disruption of PM lipid micro-domain architecture, a change that also retarded lateral diffusion and induced large clusters of HLA-I. In all, our data support a model for Nef down-regulation of HLA-I that involves both major trafficking itineraries and persistent protein-protein interactions throughout the cell.

Cholesterol is obligatory for polarization and chemotaxis but not for endocytosis and associated signaling from chemoattractant receptors in human neutrophils.

Plasma membrane cholesterol is critical for neutrophil chemotaxis, although how cholesterol affects chemotactic signaling pathway has not been clearly delineated. Here we demonstrate that cholesterol was absolutely required for polarized redistribution of key chemotactic mediators in human neutrophils in response to all chemoattractants tested (fMet-Leu-Phe, and the chemokines CXCL1, CXCL8 and CXCL12). In particular, PI3K and phosphatidylinositol-3,4,5 triphosphate (PIP(3)) failed to accumulate at the front and phosphatase and tensin homolog (PTEN) at the back of chemoattractant-stimulated neutrophils after cholesterol depletion. Cholesterol depletion did not affect early chemoattractant signaling events such as G-protein activation, intracellular calcium flux or G-protein-independent endocytosis-linked signaling, including the activation of mitogen-activated protein kinase (MAPK), Hck and Fgr transduced by beta-arrestin. During cell polarization, F-actin assemblies redistributed the cholesterol-rich microdomains and cytoskeleton-anchored proteins, including CD16 and CD44 from the leading edge. These data suggest that spatial polarization of chemotactic mediators is orchestrated by protein:protein interactions that organize cholesterol-rich domains of the plasma membrane.

CD4 down-regulation by HIV-1 and simian immunodeficiency virus (SIV) Nef proteins involves both internalization and intracellular retention mechanisms.

Among the pleiotropic effects of Nef proteins of HIV and simian immunodeficiency virus (SIV), down-modulation of cell surface expression of CD4 is a prominent phenotype. It has been presumed that Nef proteins accelerate endocytosis of CD4 by linking the receptor to the AP-2 clathrin adaptor. However, the related AP-1 and AP-3 adaptors have also been shown to interact with Nef, hinting at role(s) for these complexes in the intracellular retention of CD4. By using genetic inhibitors of endocytosis and small interfering RNA-induced knockdown of AP-2, we show that accelerated CD4 endocytosis is not a dominant mechanism of HIV-1 (NL4-3 strain) Nef in epithelial cells, T lymphocyte cell lines, or peripheral blood lymphocytes. Furthermore, we show that both the CD4 recycling from the plasma membrane and the nascent CD4 in transit to the plasma membrane are susceptible to intracellular retention in HIV-1 Nef-expressing cells. In contrast, AP-2-mediated enhanced endocytosis constitutes the predominant mechanism for SIV (MAC-239 strain) Nef-induced down-regulation of human CD4 in human cells.

On the mechanism and significance of ligand-induced internalization of human neutrophil chemokine receptors CXCR1 and CXCR2.

It is well established that leukocyte chemotactic receptors, a subset of G protein-coupled receptors, undergo endocytosis after stimulation by ligand. However, the significance of this phenomenon to cell motility and other important leukocyte functions induced by chemoattractants has not been clearly defined. Here we show that in primary human neutrophils, the threshold levels of agonist required for endocytosis of the chemotactic receptors CXCR1 and CXCR2 were approximately 10-fold or higher than those needed for maximal chemotactic and calcium flux responses. Moreover, when stimulated by agonists at concentrations that are high enough for chemotaxis but too low for receptor endocytosis, neutrophil CXCR1 and CXCR2 could be reactivated in response to repeated application of the same agonist. Both receptors were excluded from Triton X-100-insoluble lipid rafts, and at high agonist concentrations were rapidly endocytosed by a clathrin/rab5/dynamin-dependent pathway. These data support the conclusion that neutrophil migration in response to CXCR1 or CXCR2 agonists is not dependent on endocytosis of CXCR1 or CXCR2. Rather than being integral to the process of cell migration, receptor endocytosis may be a terminal stop signal when cells reach the focus of inflammation where the chemoattractant concentrations are the highest.

Recognition of dileucine-based sorting signals from HIV-1 Nef and LIMP-II by the AP-1 gamma-sigma1 and AP-3 delta-sigma3 hemicomplexes.

The sorting of transmembrane proteins to endosomes and lysosomes is mediated by signals present in the cytosolic tails of the proteins. A subset of these signals conform to the [DE]XXXL[LI] consensus motif and mediate sorting via interactions with heterotetrameric adaptor protein (AP) complexes. However, the identity of the AP subunits that recognize these signals remains controversial. We have used a yeast three-hybrid assay to demonstrate that [DE]XXXL[LI]-type signals from the human immunodeficiency virus negative factor protein and the lysosomal integral membrane protein II interact with combinations of the gamma and sigma1 subunits of AP-1 and the delta and sigma3 subunits of AP-3, but not the analogous combinations of AP-2 and AP-4 subunits. The sequence requirements for these interactions are similar to those for binding to the whole AP complexes in vitro and for function of the signals in vivo. These observations reveal a novel mode of recognition of sorting signals involving the gamma/delta and sigma subunits of AP-1 and AP-3.

Distinct mechanisms of agonist-induced endocytosis for human chemokine receptors CCR5 and CXCR4.

Desensitization of the chemokine receptors, a large class of G protein-coupled receptors, is mediated in part by agonist-driven receptor endocytosis. However, the exact pathways have not been fully defined. Here we demonstrate that the rate of ligand-induced endocytosis of CCR5 in leukocytes and expression systems is significantly slower than that of CXCR4 and requires prolonged agonist treatment, suggesting that these two receptors use distinct mechanisms. We show that the C-terminal domain of CCR5 is the determinant of its slow endocytosis phenotype. When the C-tail of CXCR4 was exchanged for that of CCR5, the resulting CXCR4-CCR5 (X4-R5) chimera displayed a CCR5-like trafficking phenotype. We found that the palmitoylated cysteine residues in this domain anchor CCR5 to plasma membrane rafts. CXCR4 and a C-terminally truncated CCR5 mutant (CCR5-KRFX) lacking these cysteines are not raft associated and are endocytosed by a clathrin-dependent pathway. Genetic inhibition of clathrin-mediated endocytosis demonstrated that a significant fraction of ligand-occupied CCR5 trafficked by clathrin-independent routes into caveolin-containing vesicular structures. Thus, the palmitoylated C-tail of CCR5 is the major determinant of its raft association and endocytic itineraries, differentiating it from CXCR4 and other chemokine receptors. This novel feature of CCR5 may modulate its signaling potential and could explain its preferential use by HIV for person-to-person transmission of disease.

Effects of HIV-1 Nef on cellular gene expression profiles.

The early human immunodeficiency virus (HIV) accessory protein Nef makes an important contribution to virulence, but the mechanisms by which Nef influences pathogenesis remain unclear. Many well-studied effects of Nef, like CD4 and class I MHC downregulation, occur posttranslationally. However, Nef has the potential to affect gene expression by interfering with cell signaling pathways and by virtue of structural features such as the Pro-X-X-Pro motif, which may interact with src homology region-3 domains of src-like kinases. We used a cDNA microarray screening strategy to identify cellular genes whose steady state transcriptional levels may be affected by Nef. We generated HeLa cell lines expressing wild-type or mutant HIV-1 nef protein sequences. Using cDNA microarray technology, we compared the patterns of cellular gene expression in the various cell lines to the pattern in non-Nef-expressing HeLa cells. By matching the patterns of cellular gene expression in HeLa cell lines expressing various Nefs with that of parental HeLa cells, we identified several cellular genes whose expression was modulated differentially by Nef and its mutants. We confirmed the differential expression of selected genes by RNA filter blotting. Genes expressed at higher levels included proteases, transcription factors, protein kinases, nuclear import/export proteins, adaptor molecules and cyclins, some of which have previously been implicated as being important for HIV replication and pathogenesis. The results indicate that Nef expression can alter the expression of cellular genes and suggest that this alteration in cellular gene expression may serve to optimize the cell to support the subsequent stages of viral replication.

Reduced cell surface expression of CCR5 in CCR5Delta 32 heterozygotes is mediated by gene dosage, rather than by receptor sequestration.

Macrophage tropic (M-tropic) human immunodeficiency virus (HIV) infection of primary human T cells and macrophages requires optimal cell surface expression of the chemokine receptor CCR5 in addition to CD4. Natural mutations of CCR5 that impair surface expression bestow in the homozygous state complete resistance to M-tropic HIV infection. ccr5Delta32 is the major prototype of such mutants. ccr5Delta32 heterozygosity is associated with delayed onset of AIDS and reduced risk of initial transmission, and this correlates with reduced levels of CCR5 and reduced infectability of CD4+ cells. In addition to gene dosage, sequestration of wild type (WT) CCR5 by mutant protein has been proposed as a mechanism to explain reduced surface expression of CCR5 in cells from ccr5Delta32 and CCR5-893(-) heterozygotes. However, here we demonstrate that a molar excess of ccr5Delta32 or related deletion mutants does not significantly impair the cell surface density of co-expressed WT receptor either in human epithelial cells or Jurkat T cells. Further, ligand-dependent signaling and M-tropic HIV usage of WT receptor are also unaffected. Nascent WT receptor does associate with ccr5Delta32 and related mutant proteins and with other unrelated CC and CXC chemokine receptors under transient labeling conditions. However, using confocal microscopy, we demonstrate that in the steady state, WT and truncated CCR5 proteins segregate into nonoverlapping subcellular compartments. These findings together with the observed and known variability in the cell surface density of CCR5 on quiescent PBLs lead us to conclude that reduced CCR5 gene dosage rather than receptor sequestration is the major determinant of reduced CCR5 expression in cells from ccr5Delta32 heterozygotes.