RESUMO
The chemokine receptor CXCR4 and its ligand CXCL12 regulate leukocyte trafficking, homeostasis and functions and are potential therapeutic targets in many diseases such as HIV-1 infection and cancers. Here, we identified new CXCR4 ligands in the CERMN chemical library using a FRET-based high-throughput screening assay. These are bis-imidazoline compounds comprising two imidazole rings linked by an alkyl chain. The molecules displace CXCL12 binding with submicromolar potencies, similarly to AMD3100, the only marketed CXCR4 ligand. They also inhibit anti-CXCR4 mAb 12G5 binding, CXCL12-mediated chemotaxis and HIV-1 infection. Further studies with newly synthesized derivatives pointed out to a role of alkyl chain length on the bis-imidazoline properties, with molecules with an even number of carbons equal to 8, 10 or 12 being the most potent. Interestingly, these differ in the functions of CXCR4 that they influence. Site-directed mutagenesis and molecular docking predict that the alkyl chain folds in such a way that the two imidazole groups become lodged in the transmembrane binding cavity of CXCR4. Results also suggest that the alkyl chain length influences how the imidazole rings positions in the cavity. These results may provide a basis for the design of new CXCR4 antagonists targeting specific functions of the receptor.
Assuntos
Imidazolinas , Transdução de Sinais , Ligantes , Simulação de Acoplamento Molecular , Receptores CXCR4 , Imidazóis/farmacologiaRESUMO
G-protein-coupled receptors (GPCR) are present at the cell surface in different conformational and oligomeric states. However, how these states impact GPCRs biological function and therapeutic targeting remains incompletely known. Here, we investigated this issue in living cells for the CC chemokine receptor 5 (CCR5), a major receptor in inflammation and the principal entry co-receptor for Human Immunodeficiency Viruses type 1 (HIV-1). We used TIRF microscopy and a statistical method to track and classify the motion of different receptor subpopulations. We showed a diversity of ligand-free forms of CCR5 at the cell surface constituted of various oligomeric states and exhibiting transient Brownian and restricted motions. These forms were stabilized differently by distinct ligands. In particular, agonist stimulation restricted the mobility of CCR5 and led to its clustering, a feature depending on ß-arrestin, while inverse agonist stimulation exhibited the opposite effect. These results suggest a link between receptor activation and immobilization. Applied to HIV-1 envelope glycoproteins gp120, our quantitative analysis revealed agonist-like properties of gp120s. Distinct gp120s influenced CCR5 dynamics differently, suggesting that they stabilize different CCR5 conformations. Then, using a dimerization-compromized mutant, we showed that dimerization (i) impacts CCR5 precoupling to G proteins, (ii) is a pre-requisite for the immobilization and clustering of receptors upon activation, and (iii) regulates receptor endocytosis, thereby impacting the fate of activated receptors. This study demonstrates that tracking the dynamic behavior of a GPCR is an efficient way to link GPCR conformations to their functions, therefore improving the development of drugs targeting specific receptor conformations.
Assuntos
HIV-1 , Receptores CCR5 , Membrana Celular/metabolismo , HIV-1/fisiologia , Humanos , Ligantes , Multimerização Proteica , Receptores CCR5/metabolismo , Receptores Acoplados a Proteínas G/metabolismoRESUMO
HIV-1 infects CD4 T lymphocytes (CD4TL) through binding the chemokine receptors CCR5 or CXCR4. CXCR4-using viruses are considered more pathogenic, linked to accelerated depletion of CD4TL and progression to AIDS. However, counterexamples to this paradigm are common, suggesting heterogeneity in the virulence of CXCR4-using viruses. Here, we investigated the role of the CXCR4 chemokine CXCL12 as a driving force behind virus virulence. In vitro, CXCL12 prevents HIV-1 from binding CXCR4 and entering CD4TL, but its role in HIV-1 transmission and propagation remains speculative. Through analysis of thirty envelope glycoproteins (Envs) from patients at different stages of infection, mostly treatment-naïve, we first interrogated whether sensitivity of viruses to inhibition by CXCL12 varies over time in infection. Results show that Envs resistant (RES) to CXCL12 are frequent in patients experiencing low CD4TL levels, most often late in infection, only rarely at the time of primary infection. Sensitivity assays to soluble CD4 or broadly neutralizing antibodies further showed that RES Envs adopt a more closed conformation with distinct antigenicity, compared to CXCL12-sensitive (SENS) Envs. At the level of the host cell, our results suggest that resistance is not due to improved fusion or binding to CD4, but owes to viruses using particular CXCR4 molecules weakly accessible to CXCL12. We finally asked whether the low CD4TL levels in patients are related to increased pathogenicity of RES viruses. Resistance actually provides viruses with an enhanced capacity to enter naive CD4TL when surrounded by CXCL12, which mirrors their situation in lymphoid organs, and to deplete bystander activated effector memory cells. Therefore, RES viruses seem more likely to deregulate CD4TL homeostasis. This work improves our understanding of the pathophysiology and the transmission of HIV-1 and suggests that RES viruses' receptors could represent new therapeutic targets to help prevent CD4TL depletion in HIV+ patients on cART.
Assuntos
Antivirais/metabolismo , Quimiocina CXCL12/metabolismo , Infecções por HIV/virologia , HIV-1/patogenicidade , Receptores CXCR4/metabolismo , Linfócitos T CD4-Positivos/virologia , Infecções por HIV/fisiopatologia , Infecções por HIV/transmissão , HIV-1/fisiologia , Homeostase , Humanos , Proteínas do Envelope Viral/metabolismo , VirulênciaRESUMO
BACKGROUND: The survival and proliferation of multiple myeloma (MM) cells in the bone marrow (BM) critically depend on interaction with stromal cells expressing the chemokine CXCL12. CXCL12 regulates the homing to the BM niche by mediating the transendothelial migration and adhesion/retention of the MM cells. The gamma isoform of CXCL12 (CXCL12γ) has been reported to be highly expressed in mouse BM and to show enhanced biological activity compared to the 'common' CXCL12α isoform, mediated by its unique extended C-terminal domain, which binds heparan sulfate proteoglycans (HSPGs) with an extraordinary high affinity. Here, we investigated the expression of CXCL12γ in human BM and studied its functional role in the interaction of MM cells with BM stromal cells (BMSCs). METHODS: We assessed CXCL12γ mRNA and protein expression by human BMSCs using qPCR, flow cytometry, and immunohistochemistry. CRISPR-Cas9 was employed to delete CXCL12γ and the heparan sulfate (HS) co-polymerase EXT1 in BMSCs. To study the functional roles of BMSC-derived CXCL12γ and HSPGs in the interaction of MM cells with BMSCs cells, MM cell lines and primary MM cells were co-cultured with BMSCs. RESULTS: We observed that CXCL12γ is expressed in situ by reticular stromal cells in both normal and MM BM, as well as by primary BMSC isolates and BMSC lines. Importantly, upon secretion, CXCL12γ, unlike the CXCL12α isoform, was retained on the surface of BMSCs. This membrane retention of CXCL12γ is HSPG mediated, since it was completely annulated by CRISPR-Cas9-mediated deletion of the HS co-polymerase EXT1. CXCL12γ expressed by BMSCs and membrane-retained by HSPGs supported robust adhesion of MM cells to the BMSCs. Specific genetic deletion of either CXCL12γ or EXT1 significantly attenuated the ability of BMSCs to support MM cell adhesion and, in addition, impaired their capacity to protect MM cells from bortezomib-induced cell death. CONCLUSIONS: We show that CXCL12γ is expressed by human BMSCs and upon secretion is retained on their cell surface by HSPGs. The membrane-bound CXCL12γ controls adhesion of MM cells to the stromal niche and mediates drug resistance. These findings designate CXCL12γ and associated HSPGs as partners in mediating MM-niche interaction and as potential therapeutic targets in MM.
Assuntos
Adesão Celular , Quimiocina CXCL12/metabolismo , Heparitina Sulfato/metabolismo , Mieloma Múltiplo/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Linhagem Celular Tumoral , Técnicas de Cocultura , Resistencia a Medicamentos Antineoplásicos , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Mieloma Múltiplo/tratamento farmacológico , Inibidores de Proteassoma/farmacologiaAssuntos
Linfócitos T CD8-Positivos/imunologia , Doenças Cardiovasculares/imunologia , Doenças Cardiovasculares/virologia , Infecções por Coronavirus/imunologia , Hipertensão/imunologia , Pneumonia Viral/imunologia , Animais , COVID-19 , Doenças Cardiovasculares/complicações , Doenças Cardiovasculares/patologia , Comorbidade , Infecções por Coronavirus/complicações , Infecções por Coronavirus/epidemiologia , Humanos , Hipertensão/complicações , Camundongos , Pandemias , Pneumonia Viral/complicações , Pneumonia Viral/epidemiologia , Receptor Tipo 2 de Angiotensina/metabolismoRESUMO
BACKGROUND: During muscle regeneration, the chemokine CXCL12 (SDF-1) and the synthesis of some specific heparan sulfates (HS) have been shown to be critical. CXCL12 activity has been shown to be heavily influenced by its binding to extracellular glycosaminoglycans (GAG) by modulating its presentation to its receptors and by generating haptotactic gradients. Although CXCL12 has been implicated in several phases of tissue repair, the influence of GAG binding under HS influencing conditions such as acute tissue destruction remains understudied. METHODS: To investigate the role of the CXCL12/HS proteoglycan interactions in the pathophysiology of muscle regeneration, we performed two models of muscle injuries (notexin and freeze injury) in mutant CXCL12Gagtm/Gagtm mice, where the CXCL12 gene having been selectively mutated in critical binding sites of CXCL12 to interact with HS. Histological, cytometric, functional transcriptomic, and ultrastructure analysis focusing on the satellite cell behavior and the vessels were conducted on muscles before and after injuries. Unless specified, statistical analysis was performed with the Mann-Whitney test. RESULTS: We showed that despite normal histology of the resting muscle and normal muscle stem cell behavior in the mutant mice, endothelial cells displayed an increase in the angiogenic response in resting muscle despite the downregulated transcriptomic changes induced by the CXCL12 mutation. The regenerative capacity of the CXCL12-mutated mice was only delayed after a notexin injury, but a severe damage by freeze injury revealed a persistent defect in the muscle regeneration of CXCL12 mutant mice associated with vascular defect and fibroadipose deposition with persistent immune cell infiltration. CONCLUSION: The present study shows that CXCL12 is crucial for proper muscle regeneration. We highlight that this homing molecule could play an important role in drastic muscle injuries and that the regeneration defect could be due to an impairment of angiogenesis, associated with a long-lasting fibro-adipogenic scar.
Assuntos
Quimiocina CXCL12/genética , Quimiocina CXCL12/fisiologia , Músculo Esquelético/irrigação sanguínea , Músculo Esquelético/fisiopatologia , Neovascularização Fisiológica/genética , Regeneração/genética , Regeneração/fisiologia , Animais , Venenos Elapídicos/toxicidade , Células Endoteliais/patologia , Células Endoteliais/fisiologia , Congelamento/efeitos adversos , Perfilação da Expressão Gênica , Proteoglicanas de Heparan Sulfato/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Camundongos Transgênicos , Músculo Esquelético/lesões , Regeneração/efeitos dos fármacos , Células Satélites de Músculo Esquelético/patologia , Células Satélites de Músculo Esquelético/fisiologiaRESUMO
CCR5 plays immune functions and is the coreceptor for R5 HIV-1 strains. It exists in diverse conformations and oligomerization states. We interrogated the significance of the CCR5 structural diversity on HIV-1 infection. We show that envelope glycoproteins (gp120s) from different HIV-1 strains exhibit divergent binding levels to CCR5 on cell lines and primary cells, but not to CD4 or the CD4i monoclonal antibody E51. This owed to differential binding of the gp120s to different CCR5 populations, which exist in varying quantities at the cell surface and are differentially expressed between different cell types. Some, but not all, of these populations are antigenically distinct conformations of the coreceptor. The different binding levels of gp120s also correspond to differences in their capacity to bind CCR5 dimers/oligomers. Mutating the CCR5 dimerization interface changed conformation of the CCR5 homodimers and modulated differentially the binding of distinct gp120s. Env-pseudotyped viruses also use particular CCR5 conformations for entry, which may differ between different viruses and represent a subset of those binding gp120s. In particular, even if gp120s can bind both CCR5 monomers and oligomers, impairment of CCR5 oligomerization improved viral entry, suggesting that HIV-1 prefers monomers for entry. From a functional standpoint, we illustrate that the nature of the CCR5 molecules to which gp120/HIV-1 binds shapes sensitivity to inhibition by CCR5 ligands and cellular tropism. Differences exist in the CCR5 populations between T-cells and macrophages, and this is associated with differential capacity to bind gp120s and to support viral entry. In macrophages, CCR5 structural plasticity is critical for entry of blood-derived R5 isolates, which, in contrast to prototypical M-tropic strains from brain tissues, cannot benefit from enhanced affinity for CD4. Collectively, our results support a role for CCR5 heterogeneity in diversifying the phenotypic properties of HIV-1 isolates and provide new clues for development of CCR5-targeting drugs.
Assuntos
Infecções por HIV/metabolismo , HIV-1/fisiologia , Receptores CCR5/química , Receptores CCR5/metabolismo , Internalização do Vírus , Proteína gp120 do Envelope de HIV/metabolismo , Humanos , Fenótipo , Ligação ProteicaRESUMO
Antibody-secreting plasma cells (PCs) arise rapidly during adaptive immunity to control infections. The early PCs are retained within the reactive lymphoid organ where their localization and homeostasis rely on extrinsic factors, presumably produced by local niche cells. While myeloid cells have been proposed to form those niches, the contribution by colocalizing stromal cells has remained unclear. Here, we characterized a subset of fibroblastic reticular cells (FRCs) that forms a dense meshwork throughout medullary cords of lymph nodes (LNs) where PCs reside. This medullary FRC type is shown to be anatomically, phenotypically, and functionally distinct from T zone FRCs, both in mice and humans. By using static and dynamic imaging approaches, we provide evidence that medullary FRCs are the main cell type in contact with PCs guiding them in their migration. Medullary FRCs also represent a major local source of the PC survival factors IL-6, BAFF, and CXCL12, besides also producing APRIL. In vitro, medullary FRCs alone or in combination with macrophages promote PC survival while other LN cell types do not have this property. Thus, we propose that this FRC subset, together with medullary macrophages, forms PC survival niches within the LN medulla, and thereby helps in promoting the rapid development of humoral immunity, which is critical in limiting early pathogen spread.
Assuntos
Formação de Anticorpos , Homeostase/imunologia , Linfonodos/imunologia , Plasmócitos/imunologia , Animais , Fator Ativador de Células B/imunologia , Quimiocina CXCL12/imunologia , Interleucina-6/imunologia , Linfonodos/citologia , Masculino , Camundongos , Plasmócitos/citologia , Células Estromais/citologia , Células Estromais/imunologiaRESUMO
Biophysical methods and x-ray crystallography have revealed that class A G protein-coupled receptors (GPCRs) can form homodimers. We combined computational approaches with receptor cross-linking, energy transfer, and a newly developed functional export assay to characterize the residues involved in the dimerization interfaces of the chemokine receptor CCR5, the major co-receptor for HIV-1 entry into cells. We provide evidence of three distinct CCR5 dimeric organizations, involving residues of transmembrane helix 5. Two dimeric states corresponded to unliganded receptors, whereas the binding of the inverse agonist maraviroc stabilized a third state. We found that CCR5 dimerization was required for targeting the receptor to the plasma membrane. These data suggest that dimerization contributes to the conformational diversity of inactive class A GPCRs and may provide new opportunities to investigate the cellular entry of HIV-1 and mechanisms for its inhibition.
Assuntos
Membrana Celular/metabolismo , HIV-1/fisiologia , Maraviroc/metabolismo , Multimerização Proteica , Receptores CCR5/química , Receptores CCR5/metabolismo , Sequência de Aminoácidos , Antagonistas dos Receptores CCR5/metabolismo , Cristalografia por Raios X , Células HEK293 , Humanos , Modelos Moleculares , Conformação Proteica , Receptores CCR5/genéticaRESUMO
Chemokines control the migration of a large array of cells by binding to specific receptors on cell surfaces. The biological function of chemokines also depends on interactions between nonreceptor binding domains and proteoglycans, which mediate chemokine immobilization on cellular or extracellular surfaces and formation of fixed gradients. Chemokine gradients regulate synchronous cell motility and integrin-dependent cell adhesion. Of the various chemokines, CXCL12 has a unique structure because its receptor-binding domain is distinct and does not overlap with the immobilization domains. Although CXCL12 is known to be essential for the germinal center (GC) response, the role of its immobilization in biological functions has never been addressed. In this work, we investigated the unexplored paradigm of CXCL12 immobilization during the germinal center reaction, a fundamental process where cellular traffic is crucial for the quality of humoral immune responses. We show that the structure of murine germinal centers and the localization of GC B cells are impaired when CXCL12 is unable to bind to cellular or extracellular surfaces. In such mice, B cells carry fewer somatic mutations in Ig genes and are impaired in affinity maturation. Therefore, immobilization of CXCL12 is necessary for proper trafficking of B cells during GC reaction and for optimal humoral immune responses.
Assuntos
Linfócitos B/imunologia , Quimiocina CXCL12/imunologia , Centro Germinativo/imunologia , Proteínas Imobilizadas/imunologia , Imunidade Humoral , Imunoglobulinas/genética , Animais , Antígenos CD/genética , Antígenos CD/imunologia , Linfócitos B/citologia , Movimento Celular , Quimiocina CXCL12/genética , Eritrócitos/química , Eritrócitos/imunologia , Expressão Gênica , Regulação da Expressão Gênica , Centro Germinativo/citologia , Proteínas Imobilizadas/genética , Imunização , Imunoglobulinas/metabolismo , Camundongos , Camundongos Transgênicos , Ovinos , Hipermutação Somática de ImunoglobulinaRESUMO
Suramin was previously shown to bind to the EV-A71 capsid through its naphthalenetrisulfonic acid groups, thereby reducing virus-cell binding and inhibiting viral replication. Here, we identify VP1-145 as the critical amino acid that accounts for the differential sensitivity of EVA-71 viruses to suramin. A single Q or G to E substitution at VP1-145 results in an approximately 30-fold shift of IC50 or IC90 values reproducing the inhibition profile observed with field isolates expressing either the 145Q or E mutation. Our data support the conclusion that suramin binds to the positively charged region surrounding the 5-fold axis of the capsid and consequently blocks the virus attachment and entry into host cells. In order to assess the antiviral-spectrum of suramin, we analyzed 18 representative enteroviruses: A (n = 7), B (n = 5), C (n = 5) and D (n = 1). We show that suramin potency is restricted to enterovirus A species. Clinical development of suramin is further supported by pharmacokinetic data demonstrating bioactive plasma levels after a single dose intramuscular administration in macaques. Altogether, our findings support the clinical development of suramin as a novel entry inhibitor for the treatment of enterovirus A infections.
Assuntos
Proteínas do Capsídeo/metabolismo , Enterovirus/fisiologia , Suramina/metabolismo , Animais , Proteínas do Capsídeo/química , Proteínas do Capsídeo/genética , Chlorocebus aethiops , Enterovirus/classificação , Enterovirus/genética , Genótipo , Meia-Vida , Humanos , Macaca fascicularis , Mutagênese , Filogenia , Suramina/química , Suramina/farmacologia , Células Vero , Replicação Viral/efeitos dos fármacosRESUMO
Chemokines stimulate signals in cells by binding to G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors. These chemoattractant cytokines also interact with heparan sulfate (HS), which provides positional information within tissues in the form of haptotactic gradients along which cells can migrate directionally. To investigate the mechanism by which HS modulates chemokine functions, we used the CXC chemokine CXCL12, which exists in different isoforms that all signal through CXCR4 but have distinct HS-binding domains. In experiments with both cell-associated and solubilized CXCR4, we found that although CXCL12γ bound to CXCR4 with a higher affinity than did CXCL12α, CXCL12γ displayed reduced signaling and chemotactic activities. These properties were caused by the specific carboxyl-terminal region of CXCL12γ, which, by interacting with CXCR4 sulfotyrosines, mediated high-affinity, but nonproductive, binding to CXCR4. HS prevented CXCL12γ from interacting with the CXCR4 sulfotyrosines, thereby functionally presenting the chemokine to its receptor such that its activity was similar to that of CXCL12α. HS had no effects on the binding of CXCL12α to CXCR4 or its biological activity, suggesting that this polysaccharide controls CXCL12 in an isoform-specific manner. These data suggest that the HS-dependent regulation of chemokine functions extends beyond the simple process of immobilization and directly modulates receptor ligation and activation.
Assuntos
Movimento Celular , Quimiocina CXCL12/metabolismo , Heparitina Sulfato/metabolismo , Receptores CXCR4/metabolismo , Linfócitos T/metabolismo , Humanos , Isoformas de Proteínas/metabolismo , Linfócitos T/citologiaRESUMO
The trimeric HIV-1 envelope (Env) glycoproteins gp120 and gp41 mediate virus entry into target cells by engaging CD4 and the coreceptors CCR5 or CXCR4 at the cell surface and driving membrane fusion. Receptor/gp120 interactions regulate the virus life cycle, HIV infection transmission and pathogenesis. Env is also the target of neutralizing antibodies. Efforts have thus been made to produce soluble HIV-1 glycoproteins to develop vaccines and study the role and mechanisms of HIV/receptor interactions. However, production and purification of Env glycoproteins and their functional assessment has to cope with multiple obstacles. These include difficulties in amplifying and cloning env sequences and setting up receptor binding assays that are suitable for studies on large collections of glycoproteins, flexible enough to adapt to Env and receptor structural heterogeneities, and allow recapitulating the receptor binding properties of virion-associated Env trimers. Here we identify these difficulties and present protocols to produce primary gp120 and determination of their binding properties to receptors. The receptor binding assays confirmed that the produced glycoproteins are competent for binding CD4 and undergo proper CD4-induced conformational changes required for interaction with CCR5. These assays may help elucidate the role of gp120/receptor interactions in the pathophysiology of HIV infection and develop HIV-1 entry inhibitors.
Assuntos
Clonagem Molecular , Expressão Gênica , Biologia Molecular/métodos , Proteínas Recombinantes/isolamento & purificação , Produtos do Gene env do Vírus da Imunodeficiência Humana/isolamento & purificação , Guias como Assunto , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismoRESUMO
The rare patients who are able to spontaneously control HIV replication in the absence of therapy show signs of a particularly efficient cellular immune response. To identify the molecular determinants that underlie this response, we characterized the T cell receptor (TCR) repertoire directed at Gag293, the most immunoprevalent CD4 epitope in the HIV-1 capsid. HIV controllers from the ANRS CODEX cohort showed a highly skewed TCR repertoire that was characterized by a predominance of TRAV24 and TRBV2 variable genes, shared CDR3 motifs, and a high frequency of public clonotypes. The most prevalent public clonotypes generated TCRs with affinities at the higher end of values reported for naturally occurring TCRs. The high-affinity Gag293-specific TCRs were cross-restricted by up to 5 distinct HLA-DR alleles, accounting for the expression of these TCRs in HIV controllers of diverse genetic backgrounds. Transfer of these TCRs to healthy donor CD4+ T cells conferred high antigen sensitivity and polyfunctionality, thus recapitulating key features of the controller CD4 response. Transfer of a high-affinity Gag293-specific TCR also redirected CD8+ T cells to target HIV-1 capsid via nonconventional MHC II restriction. Together, these findings indicate that TCR clonotypes with superior functions are associated with HIV control. Amplification or transfer of such clonotypes may contribute to immunotherapeutic approaches aiming at a functional HIV cure.
Assuntos
Antígenos CD4/imunologia , Infecções por HIV/imunologia , HIV-1 , Receptores de Antígenos de Linfócitos T/imunologia , Transferência Adotiva , Adulto , Animais , Terapia Antirretroviral de Alta Atividade , Linfócitos T CD4-Positivos/imunologia , Genes Codificadores da Cadeia alfa de Receptores de Linfócitos T , Genes Codificadores da Cadeia beta de Receptores de Linfócitos T , Infecções por HIV/tratamento farmacológico , Infecções por HIV/genética , Sobreviventes de Longo Prazo ao HIV , Antígenos HLA-DR/genética , Humanos , Imunidade Celular , Células L , Camundongos , Pessoa de Meia-Idade , Produtos do Gene gag do Vírus da Imunodeficiência Humana/imunologiaRESUMO
UNLABELLED: The live attenuated yellow fever virus (YFV) vaccine 17D stands as a "gold standard" for a successful vaccine. 17D was developed empirically by passaging the wild-type Asibi strain in mouse and chicken embryo tissues. Despite its immense success, the molecular determinants for virulence attenuation and immunogenicity of the 17D vaccine are poorly understood. 17D evolved several mutations in its genome, most of which lie within the envelope (E) protein. Given the major role played by the YFV E protein during virus entry, it has been hypothesized that the residues that diverge between the Asibi and 17D E proteins may be key determinants of attenuation. In this study, we define the process of YFV entry into target cells and investigate its implication in the activation of the antiviral cytokine response. We found that Asibi infects host cells exclusively via the classical clathrin-mediated endocytosis, while 17D exploits a clathrin-independent pathway for infectious entry. We demonstrate that the mutations in the 17D E protein acquired during the attenuation process are sufficient to explain the differential entry of Asibi versus 17D. Interestingly, we show that 17D binds to and infects host cells more efficiently than Asibi, which culminates in increased delivery of viral RNA into the cytosol and robust activation of the cytokine-mediated antiviral response. Overall, our study reveals that 17D vaccine and Asibi enter target cells through distinct mechanisms and highlights a link between 17D attenuation, virus entry, and immune activation. IMPORTANCE: The yellow fever virus (YFV) vaccine 17D is one of the safest and most effective live virus vaccines ever developed. The molecular determinants for virulence attenuation and immunogenicity of 17D are poorly understood. 17D was generated by serially passaging the virulent Asibi strain in vertebrate tissues. Here we examined the entry mechanisms engaged by YFV Asibi and the 17D vaccine. We found the two viruses use different entry pathways. We show that the mutations differentiating the Asibi envelope (E) protein from the 17D E protein, which arose during attenuation, are key determinants for the use of these distinct entry routes. Finally, we demonstrate that 17D binds and enters host cells more efficiently than Asibi. This results in a higher uptake of viral RNA into the cytoplasm and consequently a greater cytokine-mediated antiviral response. Overall, our data provide new insights into the biology of YFV infection and the mechanisms of viral attenuation.
Assuntos
Imunidade Inata , Internalização do Vírus , Vacina contra Febre Amarela/imunologia , Vírus da Febre Amarela/imunologia , Vírus da Febre Amarela/fisiologia , Linhagem Celular , Endocitose , HumanosRESUMO
Glycosaminoglycans are polysaccharides that occur both at the cell surface and within extracellular matrices. Through their ability to bind to a large array of proteins, almost 500 of which have been identified to date, including most chemokines, these molecules regulate key biologic processes at the cell-tissue interface. To do so, glycosaminoglycans can provide scaffolds to ensure that proteins mediating specific functions will be presented at the correct site and time and can also directly contribute to biologic activities or signaling processes. The binding of chemokines to glycosaminoglycans, which, at the biochemical level, has been mostly studied using heparin, has traditionally been thought of as a mechanism for maintaining haptotactic gradients within tissues along which cells can migrate directionally. Many aspects of chemokine-glycosaminoglycan interactions, however, also suggest that the formation of these complexes could serve additional purposes that go well beyond a simple immobilization process. In addition, progress in glycobiology has revealed that glycosaminoglycan structures, in term of length, sulfation, and epimerization pattern, are specific for cell, tissue, and developmental stage. Glycosaminoglycan regulation and glycosaminoglycan diversity, which cannot be replicated using heparin, thus suggests that these molecules may fine-tune the immune response by selectively recruiting specific chemokines to cell surfaces. In this context, the aim of the present text is to review the chemokine-glycosaminoglycan complexes described to date and provide a critical analysis of the tools, molecules, and strategies that can be used to structurally and functionally investigate the formation of these complexes.
Assuntos
Movimento Celular , Quimiocinas/metabolismo , Glicosaminoglicanos/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Quimiocinas/química , Humanos , Ligantes , Transdução de SinaisRESUMO
BACKGROUND: Maraviroc (MVC) is an allosteric CCR5 inhibitor used against HIV-1 infection. While MVC-resistant viruses have been identified in patients, it still remains incompletely known how they adjust their CD4 and CCR5 binding properties to resist MVC inhibition while preserving their replicative capacity. It is thought that they maintain high efficiency of receptor binding. To date however, information about the binding affinities to receptors for inhibitor-resistant HIV-1 remains limited. RESULTS: Here, we show by means of viral envelope (gp120) binding experiments and virus-cell fusion kinetics that a MVC-resistant virus (MVC-Res) that had emerged as a dominant viral quasispecies in a patient displays reduced affinities for CD4 and CCR5 either free or bound to MVC, as compared to its MVC-sensitive counterpart isolated before MVC therapy. An alanine insertion within the GPG motif (G310_P311insA) of the MVC-resistant gp120 V3 loop is responsible for the decreased CCR5 binding affinity, while impaired binding to CD4 is due to sequence changes outside V3. Molecular dynamics simulations of gp120 binding to CCR5 further emphasize that the Ala insertion alters the structure of the V3 tip and weakens interaction with CCR5 ECL2. Paradoxically, infection experiments on cells expressing high levels of CCR5 also showed that Ala allows MVC-Res to use CCR5 efficiently, thereby improving viral fusion and replication efficiencies. Actually, although we found that the V3 loop of MVC-Res is required for high levels of MVC resistance, other regions outside V3 are sufficient to confer a moderate level of resistance. These sequence changes outside V3, however, come with a replication cost, which is compensated for by the Ala insertion in V3. CONCLUSION: These results indicate that changes in the V3 loop of MVC-resistant viruses can augment the efficiency of CCR5-dependent steps of viral entry other than gp120 binding, thereby compensating for their decreased affinity for entry receptors and improving their fusion and replication efficiencies. This study thus sheds light on unsuspected mechanisms whereby MVC-resistant HIV-1 could emerge and grow in treated patients.
Assuntos
Fármacos Anti-HIV/farmacologia , Cicloexanos/farmacologia , Farmacorresistência Viral , Proteína gp120 do Envelope de HIV/metabolismo , HIV-1/efeitos dos fármacos , Mutação de Sentido Incorreto , Receptores CCR5/metabolismo , Triazóis/farmacologia , Proteína gp120 do Envelope de HIV/genética , HIV-1/genética , Humanos , Maraviroc , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Ligação Proteica , Receptores de HIV/metabolismo , Internalização do Vírus , Replicação ViralRESUMO
Dengue disease is caused by four different flavivirus serotypes, which infect 390 million people yearly with 25% symptomatic cases and for which no licensed vaccine is available. Recent phase III vaccine trials showed partial protection, and in particular no protection for dengue virus serotype 2 (refs 3, 4). Structural studies so far have characterized only epitopes recognized by serotype-specific human antibodies. We recently isolated human antibodies potently neutralizing all four dengue virus serotypes. Here we describe the X-ray structures of four of these broadly neutralizing antibodies in complex with the envelope glycoprotein E from dengue virus serotype 2, revealing that the recognition determinants are at a serotype-invariant site at the E-dimer interface, including the exposed main chain of the E fusion loop and the two conserved glycan chains. This 'E-dimer-dependent epitope' is also the binding site for the viral glycoprotein prM during virus maturation in the secretory pathway of the infected cell, explaining its conservation across serotypes and highlighting an Achilles' heel of the virus with respect to antibody neutralization. These findings will be instrumental for devising novel immunogens to protect simultaneously against all four serotypes of dengue virus.