Nathalie Signoret

Chemokine receptor trafficking and viral replication

Summary: Chemokines and chemokine receptors have emerged as crucial factors controlling the development and function of leukocytes. Recent studies have indicated that, in addition to these essential roles, both chemokines and chemokine receptors play critical roles in viral infection and replication. Not only are chemokine receptors key components of the receptor/fusion complexes of primate immunodeficiency viruses, hut chemokines can also influence virus entry and infection. Many viruses, in particular herpesviruses, encode chemokines and chemokine receptors that influence the replication of both the parent virus and other unrelated viruses. The cell surface expression of the chemokine receptors is regulated through their interaction with membrane trafficking pathways, ligands induce receptor internalization and downmodulation through endocytosis, and recycling is regulated within endosomes. Pan of the mechanism through which chemokines protect cells from HIV infection is through ligand‐induced internalization of the specific chemokine receptor co‐receptors. In addition, mechanisms may exist to regulate the trafficking of newly synthesized receptors to the cell surface. Here we discuss aspects of the mechanisms through which chemokine receptors interact with membrane‐trafficking pathways and the influence of these interactions on viral replication.

Mechanisms regulating chemokine receptor activity

Co‐ordinated movement and controlled positioning of leucocytes is key to the development, maintenance and proper functioning of the immune system. Chemokines and their receptors play an essential role in these events by mediating directed cell migration, often referred to as chemotaxis. The chemotactic property of these molecules is also thought to contribute to an array of pathologies where inappropriate recruitment of specific chemokine receptor‐expressing leucocytes is observed, including cancer and inflammatory diseases. As a result, chemokine receptors have become major targets for therapeutic intervention, and during the past 15 years much research has been devoted to understanding the regulation of their biological activity. From these studies, processes which govern the availability of functional chemokine receptors at the cell surface have emerged as playing a central role. In this review, we summarize and discuss current knowledge on the molecular mechanisms contributing to the regulation of chemokine receptor surface expression, from gene transcription and protein degradation to post‐translational modifications, multimerization, intracellular transport and cross‐talk.

Innate killing of Leishmania donovani by macrophages of the splenic marginal zone requires IRF-7.

Highly phagocytic macrophages line the marginal zone (MZ) of the spleen and the lymph node subcapsular sinus. Although these macrophages have been attributed with a variety of functions, including the uptake and clearance of blood and lymph-borne pathogens, little is known about the effector mechanisms they employ after pathogen uptake. Here, we have combined gene expression profiling and RNAi using a stromal macrophage cell line with in situ analysis of the leishmanicidal activity of marginal zone macrophages (MZM) and marginal metallophilic macrophages (MMM) in wild type and gene targeted mice. Our data demonstrate a critical role for interferon regulatory factor-7 (IRF-7) in regulating the killing of intracellular Leishmania donovani by these specialised splenic macrophage sub-populations. This study, therefore, identifies a new role for IRF-7 as a regulator of innate microbicidal activity against this, and perhaps other, non-viral intracellular pathogens. This study also highlights the importance of selecting appropriate macrophage populations when studying pathogen interactions with this functionally diverse lineage of cells.

Lack of p56 lck expression correlates with CD4 endocytosis in primary lymphoid and myeloid cells

In cell lines the endocytic properties of CD4 are regulated through its association with the src ‐family tyrosine kinase p56  lck . In lymphoid cell lines expressing p56  lck , CD4 is restricted to the cell surface and undergoes only limited internalization. Phosphorylation of the cytoplasmic domain of CD4 causes p56  lck to dissociate and activates an endocytosis signal leading to the internalization of CD4 through clathrin‐coated pits. In p56  lck ‐negative transfected cell lines CD4 is constitutively internalized, but internalization is inhibited when p56  lck is expressed in these cells. We now demonstrate that these endocytic properties of CD4 determined in transfected cell lines hold true for CD4 naturally expressed on myeloid cell lines (HL‐60 and U937), as well as on primary lymphocytes, monocytes and macrophages isolated from human blood. CD4 showed limited internalization on p56  lck ‐positive lymphocytes, but was rapidly internalized in p56  lck ‐negative monocytes and macrophages. Surprisingly, rapid internalization of CD4 was seen with the lymphocytes from one unidentified donor. In these cells we failed to detect p56  lck expression by Western blotting.

Dishevelled (Dvl-2) activates canonical Wnt signalling in the absence of cytoplasmic puncta

Dishevelled family proteins are multidomain intracellular transducers of Wnt signals. Ectopically expressed mammalian Dishevelled 2 (Dvl-2) activates downstream signalling and localises to cytoplasmic puncta. It has been suggested that these Dvl-2-containing structures correspond to intracellular vesicles and may be involved in the Wnt signal transduction process. We report that cytoplasmic puncta are primarily formed in cells expressing Dvl-2 at high levels. Lower levels of expression can activate signalling without forming puncta. The structures do not localise with markers of the early or late endocytic pathway and time-lapse analysis demonstrates that Dvl-2 puncta move in a random fashion over short distances but do not originate from the plasma membrane. Based on our findings, we propose that Dvl-2 puncta are protein aggregates that are not required for signalling.

Transduction of Activation Signal That Follows HIV-1 Binding to CD4 and CD4 Dimerization Involves the Immunoglobulin CDR3-like Region in Domain 1 of CD4

The role of CD4 during the human immunodeficiency virus type 1 (HIV-1) life cycle in T cells is not restricted to binding functions. HIV-1 binding to CD4 also triggers signals that lead to nuclear translocation of NF-κB and are important to the productive infection process. In addition to its cytoplasmic tail, in the ectodomain, the immunoglobulin (Ig) CDR3-like region of CD4 domain 1 seemed to play a role in this cascade of signals. We demonstrate in this work that the structural integrity of the CDR3-like loop is required for signal transduction. Substitutions of negatively charged residues by positively charged residues within the CDR3-like loop either inhibited NF-κB translocation after HIV-1 and gp120-anti-gp120 immune complexes binding to E91K,E92K mutants or induced its constitutive activation for E87K,D88K mutants. Moreover, A2.01–3B cells expressing the E91K,E92K mutant exhibited a lower HIV-1Lai replication. These cells, however, expressed p56 lck , demonstrated NF-κB translocation upon PMA stimulation, bound HIV-1Lai envelope glycoprotein with high affinity, and contained HIV-1 DNA 24 h after exposure to virus. E91K, E92K, and E87K,D88K mutant CD4 molecules were unable to bind a CD4 synthetic aromatically modified exocyclic, CDR3.AME-(82–89), that mimics the CDR3-like loop structure and binds to native cell surface CD4. This result together with molecular modeling studies indicates that the CDR3.AME-(82–89) analog binds to the CDR3-like loop of CD4 and strongly suggests that this region represents a site for CD4 dimerization. The negative charges on the CDR3-like loop thus appear critical for CD4-mediated signal transduction most likely related to CD4 dimer formation.

Human and simian immunodeficiency viruses: virus-receptor interactions.

The major cellular receptor for the primate immunodeficiency viruses is the CD4 molecule. As well as mediating virion attachment to the cell surface, CD4 is thought to activate the viral fusion pathway. CD4 is not, however, sufficient for viral entry; other molecules are probably involved, and in certain circumstances these may substitute for CD4. Viral tropism and cytopathogenicity are also influenced by receptor interactions.

pH-independent endocytic cycling of the chemokine receptor CCR5.

Following agonist activation, the chemokine receptor CCR5 is internalised through clathrin‐coated pits and delivered to recycling endosomes. Subsequently, ligand‐ free and resensitised receptors are recycled to the cell surface. Currently little is known of the mechanisms regulating resensitisation and recycling of this G‐protein coupled receptor. Here we show that raising the pH of endocytic compartments, using bafilomycin A, monensin or NH4Cl, does not significantly affect CCR5 endocytosis, recycling or dephosphorylation. By contrast, these reagents inhibited recycling of another well‐characterised G protein coupled receptor, the β2‐adrenergic receptor, following agonist‐induced internalisation. CCR5‐bound RANTES (CCL5) and MIP‐1β (CCL4) only exhibit pH‐dependent dissociation at pH < 4.0, below the values normally found in endocytic organelles. Although receptor‐agonist dissociation is not dependent on low pH, the subsequent degradation of released chemokine is inhibited in the presence of reagents that raise endosomal pH. Our data show that exposure to low pH is not required for RANTES or MIP‐1β dissociation from CCR5, or for recycling of internalised CCR5 to the cell surface.

TLR2-dependent pathway of heterologous down-modulation for the CC chemokine receptors 1, 2, and 5 in human blood monocytes

During innate immune responses, the inflammatory CC chemokine receptors CCR1, CCR2, and CCR5 mediate the recruitment of blood monocytes to infected tissues by promoting cell migration in response to chemokines CCL2-5. Toll-like receptors also play an essential role, allowing pathogen recognition by the recruited monocytes. Here, we demonstrate that Toll-like receptor 2 (TLR2) stimulation by lipoteichoic acid (LTA) from Staphylococcus aureus leads to gradual down-modulation of CCR1, CCR2, and CCR5 from the plasma membrane of human blood-isolated monocytes and inhibits chemotaxis. Interestingly, LTA does not promote rapid desensitization of chemokine-mediated calcium responses. We found that the TLR2 crosstalk with chemokine receptors is not dependent on the Toll/interleukin-1 receptor domain-containing adaptor protein, but instead involves phospholipase C, the small G protein Rac1, and is phorbol ester sensitive. Activation of this pathway by LTA lead to β-arrestin-mediated endocytosis of Ser349-phosphorylated CCR5 into recycling endosomes, as does CCL5 treatment. However, LTA-induced internalization of CCR5 is a slower process associated with phospholipase C-mediated and phorbol ester-sensitive phosphorylation. Overall, our data indicate that TLR2 negatively regulates CCR1, CCR2, and CCR5 on human blood monocytes by activating the machinery used to support chemokine-dependent down-modulation and provide a molecular mechanism for inhibiting monocyte migration after pathogen recognition.

Analysis of chemokine receptor endocytosis and intracellular trafficking.

Chemokine receptors are G protein-coupled receptors (GPCRs) that, through their ability to regulate chemotaxis by responding to small chemoattractant peptides termed chemokines, are involved in the development, maintenance, and functional activities of the immune system. In addition, members of the chemokine receptor family have been implicated in a number of other physiological and pathological processes, including human immunodeficiency virus infection and malaria. These activities are dependent on receptor expression at the cell surface and cellular events that reduce the cell-surface expression of chemokine receptors can abrogate these activities. Moreover, internalization of chemokine receptors by endocytosis is necessary for both receptor degradation and recycling, key regulatory processes that determine cell-surface expression levels. Here we provide detailed methods for the quantitative analysis of CCR5 endocytosis and recycling by flow cytometry, as well as fluorescence and electron microscopic procedures to analyze the endocytosis and intracellular trafficking of CCR5 by immunolabeling of cells or cryosections. In principle, the same approaches can be used for analyzing other chemokine receptors and other GPCR or non-GPCR cell-surface proteins.