Stewart Craig

AIP1/ALIX Is a Binding Partner for HIV-1 p6 and EIAV p9 Functioning in Virus Budding

HIV-1 and other retroviruses exit infected cells by budding from the plasma membrane, a process requiring membrane fission. The primary late assembly (L) domain in the p6 region of HIV-1 Gag mediates the detachment of the virion by recruiting host Tsg101, a component of the class E vacuolar protein sorting (Vps) machinery. We now show that HIV Gag p6 contains a second region involved in L domain function that binds AIP1, a homolog of the yeast class E Vps protein Bro1. Further, AIP1 interacts with Tsg101 and homologs of a subunit of the yeast class E Vps protein complex ESCRT-III. AIP1 also binds to the L domain in EIAV p9, and this binding correlates perfectly with L domain function. These observations identify AIP1 as a component of the viral budding machinery, which serves to link a distinct region in the L domain of HIV-1 p6 and EIAV p9 to ESCRT-III.

β-arrestin- but not G protein-mediated signaling by the “decoy” receptor CXCR7

Ubiquitously expressed seven-transmembrane receptors (7TMRs) classically signal through heterotrimeric G proteins and are commonly referred to as G protein-coupled receptors. It is now recognized that 7TMRs also signal through β-arrestins, which act as versatile adapters controlling receptor signaling, desensitization, and trafficking. Most endogenous receptors appear to signal in a balanced fashion using both β-arrestin and G protein-mediated pathways. Some 7TMRs are thought to be nonsignaling “decoys” because of their inability to activate typical G protein signaling pathways; it has been proposed that these receptors act to scavenge ligands or function as coreceptors. Here we demonstrate that ligand binding to the decoy receptor CXCR7 does not result in activation of signaling pathways typical of G proteins but does activate MAP kinases through β-arrestins in transiently transfected cells. Furthermore, we observe that vascular smooth muscle cells that endogenously express CXCR7 migrate to its ligand interferon-inducible T-cell alpha chemoattractant (ITAC), an effect that is significantly attenuated by treatment with either a CXCR7 antagonist or β-arrestin depletion by siRNA. This example of an endogenous “β-arrestin-biased” 7TMR that signals through β-arrestin in the absence of G protein activation demonstrates that some 7TMRs encoded in the genome have evolved to signal through β-arrestin exclusively and suggests that other receptors that are currently thought to be orphans or decoys may also signal through such nonclassical pathways.

The C5a Receptor (C5aR) C5L2 Is a Modulator of C5aR-mediated Signal Transduction

The complement anaphylatoxin C5a is a proinflammatory component of host defense that functions through two identified receptors, C5a receptor (C5aR) and C5L2. C5aR is a classical G protein-coupled receptor, whereas C5L2 is structurally homologous but deficient in G protein coupling. In human neutrophils, we show C5L2 is predominantly intracellular, whereas C5aR is expressed on the plasma membrane. Confocal analysis shows internalized C5aR following ligand binding is co-localized with both C5L2 and β-arrestin. Antibody blockade of C5L2 results in a dramatic increase in C5a-mediated chemotaxis and ERK1/2 phosphorylation but does not alter C5a-mediated calcium mobilization, supporting its role in modulation of the β-arrestin pathway. Association of C5L2 with β-arrestin is confirmed by cellular co-immunoprecipitation assays. C5L2 blockade also has no effect on ligand uptake or C5aR endocytosis in human polymorphonuclear leukocytes, distinguishing its role from that of a rapid recycling or scavenging receptor in this cell type. This is thus the first example of a naturally occurring seven-transmembrane segment receptor that is both obligately uncoupled from G proteins and a negative modulator of signal transduction through the β-arrestin pathway. Physiologically, these properties provide the possibility for additional fine-tuning of host defense.

An anti-inflammatory function for the complement anaphylatoxin C5a-binding protein, C5L2

C5L2 is an enigmatic serpentine receptor that is co-expressed with the C5a receptor on many cells including polymorphonuclear neutrophils. The apparent absence of coupling of C5L2 with G proteins suggests that this receptor may modulate the biological activity of C5a, perhaps by acting as a decoy receptor. Alternatively, C5L2 may affect C5a function through formation of a heteromeric complex with the C5aR, or it may utilize a G protein-independent signaling pathway. Here we show that in mice bearing a targeted deletion of C5L2, the biological activity of C5a/C5adesArg is enhanced both in vivo and in vitro. The biological role of C5L2 thus appears to be limiting to the pro-inflammatory response to the anaphylatoxin. Accordingly, up-regulation of C5L2 may be of benefit in inflammatory states driven by C5a, including sepsis, asthma, cystic fibrosis, and chronic obstructive lung disease.

Identification of amino acid residues critical for aggregation of human CC chemokines macrophage inflammatory protein (MIP)-1alpha, MIP-1beta, and RANTES. Characterization of active disaggregated chemokine variants.

Human CC chemokines macrophage inflammatory protein (MIP)-1α, MIP-1β, and RANTES (regulated on activation normal T cell expressed) self-associate to form high-molecular mass aggregates. To explore the biological significance of chemokine aggregation, nonaggregating variants were sought. The phenotypes of 105 hMIP-1α variants generated by systematic mutagenesis and expression in yeast were determined. hMIP-1α residues Asp26and Glu66 were critical to the self-association process. Substitution at either residue resulted in the formation of essentially homogenous tetramers at 0.5 mg/ml. Substitution of identical or analogous residues in homologous positions in both hMIP-1β and RANTES demonstrated that they were also critical to aggregation. Our analysis suggests that a single charged residue at either position 26 or 66 is insufficient to support extensive aggregation and that two charged residues must be present. Solution of the three-dimensional NMR structure of hMIP-1α has enabled comparison of these residues in hMIP-1β and RANTES. Aggregated and disaggregated forms of hMIP-1α, hMIP-1β, and RANTES generally have equivalent G-protein-coupled receptor-mediated biological potencies. We have therefore generated novel reagents to evaluate the role of hMIP-1α, hMIP-1β, and RANTES aggregation in vitro and in vivo. The disaggregated chemokines retained their human immunodeficiency virus (HIV) inhibitory activities. Surprisingly, high concentrations of RANTES, but not disaggregated RANTES variants, enhanced infection of cells by both M- and T-tropic HIV isolates/strains. This observation has important implications for potential therapeutic uses of chemokines implying that disaggregated forms may be necessary for safe clinical investigation.

The Level of CD4 Expression Limits Infection of Primary Rhesus Monkey Macrophages by a T-Tropic Simian Immunodeficiency Virus and Macrophagetropic Human Immunodeficiency Viruses

ABSTRACT The entry of primate immunodeficiency viruses into cells is dependent on the interaction of the viral envelope glycoproteins with receptors, CD4, and specific members of the chemokine receptor family. Although in many cases the tropism of these viruses is explained by the qualitative pattern of coreceptor expression, several instances have been observed where the expression of a coreceptor on the cell surface is not sufficient to allow infection by a virus that successfully utilizes the coreceptor in a different context. For example, both the T-tropic simian immunodeficiency virus (SIV) SIVmac239 and the macrophagetropic (M-tropic) SIVmac316 can utilize CD4 and CCR5 as coreceptors, and both viruses can infect primary T lymphocytes, yet only SIVmac316 can efficiently infect CCR5-expressing primary macrophages from rhesus monkeys. Likewise, M-tropic strains of human immunodeficiency virus type 1 (HIV-1) do not infect primary rhesus monkey macrophages efficiently. Here we show that the basis of this restriction is the low level of CD4 on the surface of these cells. Overexpression of human or rhesus monkey CD4 in primary rhesus monkey macrophages allowed infection by both T-tropic and M-tropic SIV and by primary M-tropic HIV-1. By contrast, CCR5 overexpression did not specifically compensate for the inefficient infection of primary monkey macrophages by T-tropic SIV or M-tropic HIV-1. Apparently, the limited ability of these viruses to utilize a low density of CD4 for target cell entry accounts for the restriction of these viruses in primary rhesus monkey macrophages.

CD4-Induced T-20 Binding to Human Immunodeficiency Virus Type 1 gp120 Blocks Interaction with the CXCR4 Coreceptor

ABSTRACT The synthetic peptide T-20, which corresponds to a sequence within the C-terminal heptad repeat region (HR2) of the human immunodeficiency virus type 1 (HIV-1) gp41 envelope glycoprotein, potently inhibits viral membrane fusion and entry. Although T-20 is thought to bind the N-terminal heptad repeat region (HR1) of gp41 and interfere with gp41 conformational changes required for membrane fusion, coreceptor specificity determined by the V3 loop of gp120 strongly influences the sensitivity of HIV-1 variants to T-20. Here, we show that T-20 binds to the gp120 glycoproteins of HIV-1 isolates that utilize CXCR4 as a coreceptor in a manner determined by the sequences of the gp120 V3 loop. T-20 binding to gp120 was enhanced in the presence of soluble CD4. Analysis of T-20 binding to gp120 mutants with variable loop deletions and the reciprocal competition of T-20 and particular anti-gp120 antibodies suggested that T-20 interacts with a gp120 region near the base of the V3 loop. Consistent with the involvement of this region in coreceptor binding, T-20 was able to block the interaction of gp120-CD4 complexes with the CXCR4 coreceptor. These results help to explain the increased sensitivity of CXCR4-specific HIV-1 isolates to the T-20 peptide. Interactions between the gp41 HR2 region and coreceptor-binding regions of gp120 may also play a role in the function of the HIV-1 envelope glycoproteins.

A paradoxical protective role for the proinflammatory peptide substance P receptor (NK1R) in acute hyperoxic lung injury

The neuropeptide substance P manifests its biological functions through ligation of a G protein-coupled receptor, the NK1R. Mice with targeted deletion of this receptor reveal a preponderance of proinflammatory properties resulting from ligand activation, demonstrating a neurogenic component to multiple forms of inflammation and injury. We hypothesized that NK1R deficiency would afford a similar protection from inflammation associated with hyperoxia. Counter to our expectations, however, NK1R-/- animals suffered significantly worse lung injury compared with wild-type mice following exposure to 90% oxygen. Median survival was shortened to 84 h for NK1R-/- mice from 120 h for wild-type animals. Infiltration of inflammatory cells into the lungs was significantly increased; NK1R-/- animals also exhibited increased pulmonary edema, hemorrhage, and bronchoalveolar lavage fluid protein levels. TdT-mediated dUTP nick end labeling (TUNEL) staining was significantly elevated in NK1R-/- animals following hyperoxia. Furthermore, induction of metallothionein and Na(+)-K(+)-ATPase was accelerated in NK1R-/- compared with wild-type mice, consistent with increased oxidative injury and edema. In cultured mouse lung epithelial cells in 95% O(2), however, addition of substance P promoted cell death, suggesting the neurogenic component of hyperoxic lung injury is mediated by additional mechanisms in vivo. Release of bioactive constituents including substance P from sensory neurons results from activation of the vanilloid receptor, TRPV1. In mice with targeted deletion of the TRPV1 gene, acute hyperoxic injury is attenuated relative to NK1R-/- animals. Our findings thus reveal a major neurogenic mechanism in acute hyperoxic lung injury and demonstrate concerted actions of sensory neurotransmitters revealing significant protection for NK1R-mediated functions.

Assays for macrophage inflammatory proteins

Publisher Summary This chapter describes methodology to provide the basic experimental grounding in two clinically relevant areas of chemokine research: the regulation of hematopoietic progenitor cell proliferation and the mechanism of human immunodeficiency virus (HIV) infection. Preclinical studies using the in vivo and in vitro assays described have supported progression of macrophage inflammatory protein-lα (MIP-lα) as a candidate biopharmaceutical with potential application in cancer and HIV therapy. As the list of chemokines continues to grow, these assays will remain central to elucidation of the basic biology and the clinical potential of this fascinating molecular family. The chapter discusses other methods used for the characterization of chemokines, and further background information on the structure, function, and clinical utility of the chemokine family in general.