The Distinct Roles of CXCR3 Variants and Their Ligands in the Tumor Microenvironment.

First thought to orchestrate exclusively leukocyte trafficking, chemokines are now acknowledged for their multiple roles in the regulation of cell proliferation, differentiation, and survival. Dysregulation of their normal functions contributes to various pathologies, including inflammatory diseases and cancer. The two chemokine receptor 3 variants CXCR3-A and CXCR3-B, together with their cognate chemokines (CXCL11, CXCL10, CXCL9, CXCL4, and CXCL4L1), are involved in the control but also in the development of many tumors. CXCR3-A drives the infiltration of leukocytes to the tumor bed to modulate tumor progression (paracrine axis). Conversely, tumor-driven changes in the expression of the CXCR3 variants and their ligands promote cancer progression (autocrine axis). This review summarizes the anti- and pro-tumoral activities of the CXCR3 variants and their associated chemokines with a focus on the understanding of their distinct biological roles in the tumor microenvironment.

Biased antagonism of CXCR4 avoids antagonist tolerance.

Repeated dosing of drugs targeting G protein-coupled receptors can stimulate antagonist tolerance, which reduces their efficacy; thus, strategies to avoid tolerance are needed. The efficacy of AMD3100, a competitive antagonist of the chemokine receptor CXCR4 that mobilizes leukemic blasts from the bone marrow into the blood to sensitize them to chemotherapy, is reduced after prolonged treatment. Tolerance to AMD3100 increases the abundance of CXCR4 on the surface of leukemic blasts, which promotes their rehoming to the bone marrow. AMD3100 inhibits both G protein signaling by CXCR4 and ß-arrestin1/2-dependent receptor endocytosis. We demonstrated that biased antagonists of G protein-dependent chemotaxis but not ß-arrestin1/2 recruitment and subsequent receptor endocytosis avoided tolerance. The peptide antagonist X4-2-6, which is derived from transmembrane helix 2 and extracellular loop 1 of CXCR4, limited chemotaxis and signaling but did not promote CXCR4 accumulation on the cell surface or cause tolerance. The activity of X4-2-6 was due to its distinct mechanism of inhibition of CXCR4. The peptide formed a ternary complex with the receptor and its ligand, the chemokine CXCL12. Within this complex, X4-2-6 released the portion of CXCL12 critical for receptor-mediated activation of G proteins but enabled the rest of the chemokine to recruit ß-arrestins to the receptor. In contrast, AMD3100 displaced all components of the chemokine responsible for CXCR4 activation. We further identified a small molecule with similar biased antagonist properties to those of X4-2-6, which may provide a viable alternative to patients when antagonist tolerance prevents drugs from reaching efficacy.

Neuraminidase 1 activates insulin receptor and reverses insulin resistance in obese mice.

OBJECTIVES: Neuraminidase 1 (NEU1) cleaves terminal sialic acids of glycoconjugates during lysosomal catabolism. It also modulates the structure and activity of cellular surface receptors affecting diverse pathways. Previously we demonstrated that NEU1 activates the insulin receptor (IR) and that NEU1-deficient CathAS190A-Neo mice (hypomorph of the NEU1 activator protein, cathepsin A/CathA) on a high-fat diet (HFD) develop hyperglycaemia and insulin resistance faster than wild-type animals. The major objective of the current work was to reveal the molecular mechanism by which NEU1 desialylation activates the IR and to test if increase of NEU1 activity in insulin target tissues reverses insulin resistance and glucose intolerance. METHODS: To test if desialylation causes a conformational change in the IR dimer we measured interaction between the receptor subunits by Bioluminescence Resonance Energy Transfer in the HEK293T cells either overexpressing NEU1 or treated with the NEU1 inhibitor. The influence of NEU1 overexpression on insulin resistance was studied in vitro in palmitate-treated HepG2 cells transduced with NEU1-expressing lentivirus and in vivo in C57Bl6 mice treated with HFD and either pharmacological inducer of NEU1, Ambroxol or NEU1-expressing adenovirus. NEU1-deficient CathAS190A-Neo mice were used as a control. RESULTS: By desialylation of IR, NEU1 induced formation of its active dimer leading to insulin signaling. Overexpression of NEU1 in palmitate-treated HepG2 cells restored insulin signaling, suggesting that increased NEU1 levels may reverse insulin resistance. Five-day treatment of glycemic C57Bl6 mice receiving HFD with the activator of the lysosomal gene network, Ambroxol, increased NEU1 expression and activity in muscle tissue, normalized fasting glucose levels, and improved physiological and molecular responses to glucose and insulin. Ambroxol did not improve insulin sensitivity in obese insulin-resistant CathAS190A-Neo mice indicating that the Ambroxol effect is mediated through NEU1 induction. Sustained increase of liver NEU1 activity through adenovirus-based gene transfer failed to attenuate insulin resistance most probably due to negative feedback regulation of IR expression. CONCLUSION: Together our results demonstrate that increase of NEU1 activity in insulin target tissues reverses insulin resistance and glucose intolerance suggesting that a pharmacological modulation of NEU1 activity may be potentially explored for restoring insulin sensitivity and resolving hyperglycemia associated with T2DM.

Structure-Activity Relationship Study of Cyclic Pentapeptide Ligands for Atypical Chemokine Receptor 3 (ACKR3).

The atypical chemokine receptor 3 (ACKR3)/CXC chemokine receptor 7 (CXCR7) recognizes stromal cell-derived factor 1 (SDF-1)/CXCL12 and is involved in a number of physiological and pathological processes. Here, we investigated the SAR of the component amino acids in an ACKR3-selective ligand, FC313 [ cyclo(-d-Tyr-l-Arg-l-MeArg-l-Nal(2)-l-Pro-)], for the development of highly active ACKR3 ligands. Notably, modification at the l-Pro position with a bulky hydrophobic side chain led to improved bioactivity toward ACKR3.

Different contributions of chemokine N-terminal features attest to a different ligand binding mode and a bias towards activation of ACKR3/CXCR7 compared with CXCR4 and CXCR3.

BACKGROUND AND PURPOSE: Chemokines and their receptors form an intricate interaction and signalling network that plays critical roles in various physiological and pathological cellular processes. The high promiscuity and apparent redundancy of this network makes probing individual chemokine/receptor interactions and functional effects, as well as targeting individual receptor axes for therapeutic applications, challenging. Despite poor sequence identity, the N-terminal regions of chemokines, which play a key role in their activity and selectivity, contain several conserved features. Thus far little is known regarding the molecular basis of their interactions with typical and atypical chemokine receptors or the conservation of their contributions across chemokine-receptor pairs. EXPERIMENTAL APPROACH: We used a broad panel of chemokine variants and modified peptides derived from the N-terminal region of chemokines CXCL12, CXCL11 and vCCL2, to compare the contributions of various features to binding and activation of their shared receptors, the two typical, canonical G protein-signalling receptors, CXCR4 and CXCR3, as well as the atypical scavenger receptor CXCR7/ACKR3, which shows exclusively arrestin-dependent activity. KEY RESULTS: We provide molecular insights into the plasticity of the ligand-binding pockets of these receptors, their chemokine binding modes and their activation mechanisms. Although the chemokine N-terminal region is a critical determinant, neither the most proximal residues nor the N-loop are essential for binding and activation of ACKR3, as distinct from binding and activation of CXCR4 and CXCR3. CONCLUSION AND IMPLICATIONS: These results suggest a different interaction mechanism between this atypical receptor and its ligands and illustrate its strong propensity to activation.

Ligand-specific conformational transitions and intracellular transport are required for atypical chemokine receptor 3-mediated chemokine scavenging.

The atypical chemokine receptor ACKR3 contributes to chemotaxis by binding, internalizing, and degrading the chemokines CXCL11 and CXCL12 to shape and terminate chemotactic gradients during development and immune responses. Although unable to trigger G protein activation, both ligands activate G protein-independent ACKR3 responses and prompt arrestin recruitment. This offers a model to specifically study ligand-specific receptor conformations leading to G protein-independent signaling and to functional parameters such as receptor transport and chemokine degradation. We here show chemokine specificity in arrestin recruitment, by different effects of single amino acid substitutions in ACKR3 on arrestin in response to CXCL12 or CXCL11. Chemokine specificity in receptor transport was also observed, as CXCL11 induced faster receptor internalization, slower recycling, and longer intracellular sojourn of ACKR3 than CXCL12. Internalization and recycling rates of the ACKR3 R1423.50A substitution in response to each chemokine were similar; however, ACKR3 R1423.50A degraded only CXCL12 and not CXCL11. This suggests that ligand-specific intracellular receptor transport is required for chemokine degradation. Remarkably, the failure of ACKR3 R1423.50A to degrade CXCL11 was not caused by the lack of arrestin recruitment; rather, arrestin was entirely dispensable for scavenging of either chemokine. This suggests the involvement of another, yet unidentified, ACKR3 effector in scavenging. In summary, our study correlates ACKR3 ligand-specific conformational transitions with chemokine-dependent receptor transport dynamics and points toward unexpected ligand specificity in the mechanisms of chemokine degradation.

Structural basis for chemokine recognition by a G protein-coupled receptor and implications for receptor activation.

Chemokines orchestrate cell migration for development, immune surveillance, and disease by binding to cell surface heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptors (GPCRs). The array of interactions between the nearly 50 chemokines and their 20 GPCR targets generates an extensive signaling network to which promiscuity and biased agonism add further complexity. The receptor CXCR4 recognizes both monomeric and dimeric forms of the chemokine CXCL12, which is a distinct example of ligand bias in the chemokine family. We demonstrated that a constitutively monomeric CXCL12 variant reproduced the G protein-dependent and ß-arrestin-dependent responses that are associated with normal CXCR4 signaling and lead to cell migration. In addition, monomeric CXCL12 made specific contacts with CXCR4 that are not present in the structure of the receptor in complex with a dimeric form of CXCL12, a biased agonist that stimulates only G protein-dependent signaling. We produced an experimentally validated model of an agonist-bound chemokine receptor that merged a nuclear magnetic resonance-based structure of monomeric CXCL12 bound to the amino terminus of CXCR4 with a crystal structure of the transmembrane domains of CXCR4. The large CXCL12:CXCR4 protein-protein interface revealed by this structure identified previously uncharacterized functional interactions that fall outside of the classical "two-site model" for chemokine-receptor recognition. Our model suggests a mechanistic hypothesis for how interactions on the extracellular face of the receptor may stimulate the conformational changes required for chemokine receptor-mediated signal transduction.

Mutational Analysis of Atypical Chemokine Receptor 3 (ACKR3/CXCR7) Interaction with Its Chemokine Ligands CXCL11 and CXCL12.

Atypical chemokine receptors do not mediate chemotaxis or G protein signaling, but they recruit arrestin. They also efficiently scavenge their chemokine ligands, thereby contributing to gradient maintenance and termination. ACKR3, also known as CXCR7, binds and degrades the constitutive chemokine CXCL12, which also binds the canonical receptor CXCR4, and CXCL11, which also binds CXCR3. Here we report comprehensive mutational analysis of the ACKR3 interaction with its chemokine ligands, using 30 substitution mutants. Readouts are radioligand binding competition, arrestin recruitment, and chemokine scavenging. Our results suggest different binding modes for both chemokines. CXCL11 depends on the ACKR3 N terminus and some extracellular loop (ECL) positions for primary binding, ECL residues mediate secondary binding and arrestin recruitment potency. CXCL12 binding required key residues Asp-1794.60 and Asp-2756.58 (residue numbering follows the Ballesteros-Weinstein scheme), with no evident involvement of N-terminal residues, suggesting an uncommon mode of receptor engagement. Mutation of residues corresponding to CRS2 in CXCR4 (positions Ser-1032.63 and Gln-3017.39) increased CXCL11 binding, but reduced CXCL12 affinity. Mutant Q301E7.39 did not recruit arrestin. Mutant K118A3.26 in ECL1 showed moderate baseline arrestin recruitment with ablation of ligand-induced responses. Substitutions that affected CXCL11 binding also diminished scavenging. However, detection of reduced CXCL12 scavenging by mutants with impaired CXCL12 affinity required drastically reduced receptor expression levels, suggesting that scavenging pathways can be saturated and that CXCL12 binding exceeds scavenging at higher receptor expression levels. Arrestin recruitment did not correlate with scavenging; although Q301E7.39 degraded chemokines in the absence of arrestin, S103D2.63 had reduced CXCL11 scavenging despite intact arrestin responses.

Design, synthesis, and biological evaluation of CXCR4 ligands.

A combination of the CXCR4 inverse agonist T140 with N-terminal CXCL12 oligopeptides has produced the first nanomolar synthetic CXCR4 agonists. In these agonists, the inverse agonistic portion provides affinity whereas the N-terminal CXCL12 sequence induces receptor activation. Several CXCR4 crystal structures exist with either CVX15, an inverse agonist closely related to T140 and IT1t, a small molecule; we therefore attempted to produce another CXCL12 oligopeptide combination with IT1t. For this purpose, a primary amino group was introduced by total synthesis into one of the methyl groups of IT1t, serving as an anchoring point for the oligopeptide graft. The introduction of the oligopeptides on this analog however yielded antagonists, one compound displaying high affinity. On the other hand, the amino-substituted analogue itself proved to be an inverse agonist with a binding affinity of 2.6 nM compared to 11.5 nM for IT1t. This IT1t-like analog is hitherto one of the most potent non-peptidic CXCR4 inverse agonists reported.

Structure-Activity Relationship and Signaling of New Chimeric CXCR4 Agonists.

The CXCR4 receptor binds with meaningful affinities only CXCL12 and synthetic antagonists/inverse agonists. We recently described high affinity synthetic agonists for this chemokine receptor, obtained by grafting the CXCL12 N-terminus onto the inverse agonist T140. While those chimeric molecules behave as agonists for CXCR4, their binding and activation mode are unknown. The present SAR of those CXCL12-oligopeptide grafts reveals the key determinants involved in CXCR4 activation. Position 3 (Val) controls affinity, whereas position 7 (Tyr) acts as an efficacy switch. Chimeric molecules bearing aromatic residues in position 3 possess high binding affinities for CXCR4 and are Gαi full agonists with robust chemotactic properties. Fine-tuning of electron-poor aromatic rings in position 7 enhances receptor activation. To rationalize these results, a homology model of a receptor-ligand complex was built using the published crystal structures of CXCR4. Molecular dynamics simulations reveal further details accounting for the observed SAR for this series.

Development of novel CXC chemokine receptor 7 (CXCR7) ligands: selectivity switch from CXCR4 antagonists with a cyclic pentapeptide scaffold.

The CXC chemokine receptor 7 (CXCR7)/ACKR3 is a chemokine receptor that recognizes stromal cell-derived factor 1 (SDF-1)/CXCL12 and interferon-inducible T-cell α chemoattractant (I-TAC)/CXCL11. Here, we report the development of novel CXCR7-selective ligands with a cyclic pentapeptide scaffold through an SAR study of CXC chemokine receptor 4 (CXCR4) selective antagonist FC131 [cyclo(-d-Tyr-l-Arg-l-Arg-l-Nal-Gly-), Nal = 3-(2-naphthyl)alanine]. Substitution of Gly with l-Pro switched the receptor preference of the peptides from CXCR4 to CXCR7. The SAR study led to the identification of a potent CXCR7 ligand, FC313 [cyclo(-d-Tyr-l-Arg-l-MeArg-l-Nal-l-Pro-)], which recruits ß-arrestin to CXCR7. Investigations via receptor mutagenesis and molecular modeling experiments suggest a possible binding mode of the cyclic pentapeptide CXCR7 agonist.

Mode of binding of the cyclic agonist peptide TC14012 to CXCR7: identification of receptor and compound determinants.

The chemokine receptor CXCR7 is an atypical CXCL12 receptor that, as opposed to the classical CXCL12 receptor CXCR4, signals preferentially via the ß-arrestin pathway and does not mediate chemotaxis. We previously reported that the cyclic peptide TC14012, a potent CXCR4 antagonist, also engaged CXCR7, albeit with lower potency. Surprisingly, the compound activated the CXCR7-arrestin pathway. The reason underlying the opposite effects of TC14012 on CXCR4 and CXCR7, and the mode of binding of TC14012 to CXCR7, remained unclear. The mode of binding of TC14012 to CXCR4 is known from cocrystallization of its analogue CVX15 with CXCR4. We here report the the mode of binding of TC14012 to CXCR7 by combining the use of compound analogues, receptor mutants, and molecular modeling. We find that the mode of binding of TC14012 to CXCR7 is indeed similar to that of CVX15 to CXCR4, with compound positions Arg2 and Arg14 engaging CXCR7 key residues D179(4.60) (on the tip of transmembrane domain 4) and D275(6.58) (on the tip of transmembrane domain 6), respectively. Interestingly, the TC14012 parent compound T140 is not a CXCR7 agonist, because of conformational constraints in its pharmacophore, which in TC14012 are relieved through C-terminal amidation. However, an engineered salt bridge between the CXCR7 ECL2 substitution R197D and compound residue Arg1 permitted T140 agonism by repositioning the compound in the binding pocket. In conclusion, our results show that the opposite effect of TC14012 on CXCR4 and CXCR7 is not explained by different binding modes. Rather, engagement of the interface between transmembrane domains and extracellular loops readily triggers CXCR7, but not CXCR4, activation.

The chemokine CXC4 and CC2 receptors form homo- and heterooligomers that can engage their signaling G-protein effectors and ßarrestin.

G-protein-coupled receptors have been shown to assemble at least as dimers early in the biosynthetic path, but some evidence suggests that they can also form larger oligomeric complexes. Using the human chemokine receptors CXCR4 and CCR2 as models, we directly probed the existence of higher order homo- and heterooligomers in human embryonic kidney cells. Combining bimolecular fluorescence and luminescence complementation (BiFC, BiLC) with bioluminescence resonance energy transfer (BRET) assays, we show that CXCR4 and CCR2 can assemble as homo- and heterooligomers, forming at least tetramers. Selective activation of CCR2 with the human monocyte chemotactic protein 1 (MCP-1) resulted in trans-conformational rearrangement of the CXCR4 dimer with an EC50 of 19.9 nM, compatible with a CCR2 action. Moreover, MCP-1 promoted the engagement of Gαi1, Gα13, Gαz, and ßarrestin2 to the heterooligomer, resulting in calcium signaling that was synergistically potentiated on coactivation of CCR2 and CXCR4, demonstrating that complexes larger than dimers reach the cell surface as functional units. A mutation of CXCR4 (N119K), which prevents Gi activation, also affects the CCR2-promoted engagement of Gαi1 and ßarrestin2 by the heterooligomer, supporting the occurrence of transprotomer regulation. Together, the results demonstrate that homo- and heteromultimeric CXCR4 and CCR2 can form functional signaling complexes that have unique properties.

Positive regulation of insulin signaling by neuraminidase 1.

Neuraminidases (sialidases) catalyze the removal of sialic acid residues from sialylated glycoconjugates. We now report that mammalian neuraminidase 1 (Neu1), in addition to its catabolic function in lysosomes, is transported to the cell surface where it is involved in the regulation of insulin signaling. Insulin binding to its receptor rapidly induces interaction of the receptor with Neu1, which hydrolyzes sialic acid residues in the glycan chains of the receptor and, consequently, induces its activation. Cells from sialidosis patients with a genetic deficiency of Neu1 show impairment of insulin-induced phosphorylation of downstream protein kinase AKT, and treatment of these cells with purified Neu1 restores signaling. Genetically modified mice with ∼10% of the normal Neu1 activity exposed to a high-fat diet develop hyperglycemia and insulin resistance twice as fast as their wild-type counterparts. Together, these studies identify Neu1 as a novel component of the signaling pathways of energy metabolism and glucose uptake.

Restoration of renal function by a novel prostaglandin EP4 receptor-derived peptide in models of acute renal failure.

Acute renal failure (ARF) is a serious medical complication characterized by an abrupt and sustained decline in renal function. Despite significant advances in supportive care, there is currently no effective treatment to restore renal function. PGE(2) is a lipid hormone mediator abundantly produced in the kidney, where it acts locally to regulate renal function; several studies suggest that modulating EP(4) receptor activity could improve renal function following kidney injury. An optimized peptidomimetic ligand of EP(4) receptor, THG213.29, was tested for its efficacy to improve renal function (glomerular filtration rate, renal plasma flow, and urine output) and histological changes in a model of ARF induced by either cisplatin or renal artery occlusion in Sprague-Dawley rats. THG213.29 modulated PGE(2)-binding dissociation kinetics, indicative of an allosteric binding mode. Consistently, THG213.29 antagonized EP(4)-mediated relaxation of piglet saphenous vein rings, partially inhibited EP(4)-mediated cAMP production, but did not affect Gα(i) activation or ß-arrestin recruitment. In vivo, THG213.29 significantly improved renal function and histological changes in cisplatin- and renal artery occlusion-induced ARF models. THG213.29 increased mRNA expression of heme-oxygenase 1, Bcl2, and FGF-2 in renal cortex; correspondingly, in EP(4)-transfected HEK293 cells, THG213.29 augmented FGF-2 and abrogated EP(4)-dependent overexpression of inflammatory IL-6 and of apoptotic death domain-associated protein and BCL2-associated agonist of cell death. Our results demonstrate that THG213.29 represents a novel class of diuretic agent with noncompetitive allosteric modulator effects on EP(4) receptor, resulting in improved renal function and integrity following acute renal failure.

Structure-based ligand discovery for the protein-protein interface of chemokine receptor CXCR4.

G-protein-coupled receptors (GPCRs) are key signaling molecules and are intensely studied. Whereas GPCRs recognizing small-molecules have been successfully targeted for drug discovery, protein-recognizing GPCRs, such as the chemokine receptors, claim few drugs or even useful small molecule reagents. This reflects both the difficulties that attend protein-protein interface inhibitor discovery, and the lack of structures for these targets. Imminent structure determination of chemokine receptor CXCR4 motivated docking screens for new ligands against a homology model and subsequently the crystal structure. More than 3 million molecules were docked against the model and then against the crystal structure; 24 and 23 high-scoring compounds from the respective screens were tested experimentally. Docking against the model yielded only one antagonist, which resembled known ligands and lacked specificity, whereas the crystal structure docking yielded four that were dissimilar to previously known scaffolds and apparently specific. Intriguingly, several were potent and relatively small, with IC(50) values as low as 306 nM, ligand efficiencies as high as 0.36, and with efficacy in cellular chemotaxis. The potency and efficiency of these molecules has few precedents among protein-protein interface inhibitors, and supports structure-based efforts to discover leads for chemokine GPCRs.

Monomeric and dimeric CXCL12 inhibit metastasis through distinct CXCR4 interactions and signaling pathways.

Chemokines and chemokine receptors are extensively and broadly involved in cancer metastasis. Previously, we demonstrated that epigenetic silencing of the chemokine CXCL12 sensitizes breast and colon cancer cells to endocrine signaling and metastasis to distant tissues. Yet, the precise mechanism whereby CXCL12 production by tumor cells regulates dissemination remains unclear. Here, we show that administration of CXCL12 extended survival of tumor-bearing mice by potently limiting metastasis of colorectal carcinoma or murine melanoma. Because secreted CXCL12 is a mixture of monomeric and dimeric species in equilibrium, oligomeric variants that either promote (monomer) or halt (dimer) chemotaxis were used to dissect the mechanisms interrupting carcinoma metastasis. Monomeric CXCL12 mobilized intracellular calcium, inhibited cAMP signaling, recruited ß-arrestin-2, and stimulated filamentous-actin accumulation and cell migration. Dimeric CXCL12 activated G-protein-dependent calcium flux, adenylyl cyclase inhibition, and the rapid activation of ERK1/2, but only weakly, if at all, recruited arrestin, stimulated actin polymerization, or promoted chemotaxis. NMR analyses illustrated that CXCL12 monomers made specific contacts with CXCR4 that were lost following dimerization. Our results establish the potential for inhibiting CXCR4-mediated metastasis by administration of CXCL12. Chemokine-mediated migration and ß-arrestin responses did not dictate the antitumor effect of CXCL12. We conclude that cellular migration is tightly regulated by selective CXCR4 signaling evoked by unique interactions with distinct ligand quaternary structures.

Agonists for the Chemokine Receptor CXCR4.

The development of agonists for the chemokine receptor CXCR4 could provide promising therapeutic candidates. On the basis of previously forwarded two site model of chemokine-receptor interactions, we hypothesized that linking the agonistic N-terminus of SDF-1 to the T140 backbone would yield new high-affinity agonists of CXCR4. We developed chimeras with the agonistic SDF-1 N-terminus grafted to a T140 side chain and tested their binding affinity and chemotactic agonist activity. While chimeras with the peptide grafted onto position 12 of T140 remained high-affinity antagonists, those bearing the peptide on position 14 were in part agonists. One chimera was a full CXCR4 agonist with 25 nM affinity, and several chimeras showed low nanomolar affinities with partial agonist activity. Our results confirmed that we have developed high-affinity agonists of CXCR4.

Herpes simplex virus type 1-induced FasL expression in human monocytic cells and its implications for cell death, viral replication, and immune evasion.

Herpes simplex virus type 1 (HSV-1) is a ubiquitously occurring pathogen that infects humans early in childhood. The virus persists as a latent infection in dorsal root ganglia, especially of the trigeminal nerve, and frequently becomes reactivated in humans under conditions of stress. Monocytic cells constitute an important component of the innate and adaptive immune responses. We show here for the first time that HSV-1 stimulates human FasL promoter and induces de novo expression of FasL on the surface of human monocytic cells, including monocytes and macrophages. This virus-induced FasL expression causes death of monocytic cells growing in suspension, but not in monolayers (e.g., macrophages). The addition of a broad-spectrum caspase inhibitor, as well as anti-FasL antibodies, reduced cell death but increased viral replication in the virus-infected cell cultures. We also show here for the first time that the virus-induced de novo expression of FasL on the cell surface acts as an immune evasion mechanism by causing the death of interacting human CD4+ T cells, CD8+ T cells, and natural killer (NK) cells. Our study provides novel insights on FasL expression and cell death in HSV-infected human monocytic cells and their impact on interacting immune cells.

The activity of the HIV-1 IRES is stimulated by oxidative stress and controlled by a negative regulatory element.

Initiation of translation of the full-length messenger RNA of HIV-1, which generates the viral structural proteins and enzymes, is cap-dependent but can also use an internal ribosome entry site (IRES) located in the 5' untranslated region. Our aim was to define, through a mutational analysis, regions of HIV-1 IRES that are important for its activity. A dual-luciferase reporter construct where the Renilla luciferase (Rluc) translation is cap-dependent while the firefly luciferase (Fluc) translation depends on HIV-1 IRES was used. The Fluc/Rluc ratio was measured in lysates of Jurkat T cells transfected with the dual-luciferase plasmid bearing either the wild-type or a mutated IRES. Deletions or mutations in three regions decreased the IRES activity but deletion or mutations of a stem-loop preceding the primer binding site increased the IRES activity. The wild-type IRES activity, but not that of an IRES with a mutated stem-loop, was increased when cells were treated with agents that induce oxidative stress. Such stress is known to be caused by HIV-1 infection and we propose that this stem-loop is involved in a switch that stimulates the IRES activity in cells infected with HIV-1, supporting the suggestion that the IRES activity is up-regulated in the course of HIV-1 replication cycle.