RESUMO
Innate immune factors may restrict hematopoietic stem cell (HSC) genetic engineering and contribute to broad individual variability in gene therapy outcomes. Here, we show that HSCs harbor an early, constitutively active innate immune block to lentiviral transduction that can be efficiently overcome by cyclosporine H (CsH). CsH potently enhances gene transfer and editing in human long-term repopulating HSCs by inhibiting interferon-induced transmembrane protein 3 (IFITM3), which potently restricts VSV glycoprotein-mediated vector entry. Importantly, individual variability in endogenous IFITM3 levels correlated with permissiveness of HSCs to lentiviral transduction, suggesting that CsH treatment will be useful for improving ex vivo gene therapy and standardizing HSC transduction across patients. Overall, our work unravels the involvement of innate pathogen recognition molecules in immune blocks to gene correction in primary human HSCs and highlights how these roadblocks can be overcome to develop innovative cell and gene therapies.
Assuntos
Ciclosporina/farmacologia , Edição de Genes , Células-Tronco Hematopoéticas/efeitos dos fármacos , Imunidade Inata/efeitos dos fármacos , Lentivirus/efeitos dos fármacos , Lentivirus/genética , Transdução Genética , Animais , Linhagem Celular , Feminino , Células HEK293 , Células-Tronco Hematopoéticas/metabolismo , Humanos , Lentivirus/imunologia , Camundongos , Camundongos Endogâmicos NOD , Camundongos KnockoutRESUMO
Alveolar macrophages (AM) are thought to have a key role in the immunopathogenesis of respiratory diseases. We sought to test the hypothesis that human AM exhibit an anti-inflammatory bias by making genome-wide comparisons with monocyte derived macrophages (MDM). Adherent AM obtained by bronchoalveolar lavage of patients under investigation for haemoptysis, but found to have no respiratory pathology, were compared to MDM from healthy volunteers by whole genome transcriptional profiling before and after innate immune stimulation. We found that freshly isolated AM exhibited a marked pro-inflammatory transcriptional signature. High levels of basal pro-inflammatory gene expression gave the impression of attenuated responses to lipopolysaccharide (LPS) and the RNA analogue, poly IC, but in rested cells pro-inflammatory gene expression declined and transcriptional responsiveness to these stimuli was restored. In comparison to MDM, both freshly isolated and rested AM showed upregulation of MHC class II molecules. In most experimental paradigms ex vivo adherent AM are used immediately after isolation. Therefore, the confounding effects of their pro-inflammatory profile at baseline need careful consideration. Moreover, despite the prevailing view that AM have an anti-inflammatory bias, our data clearly show that they can adopt a striking pro-inflammatory phenotype, and may have greater capacity for presentation of exogenous antigens than MDM.
Assuntos
Imunidade Inata/imunologia , Inflamação/imunologia , Inflamação/patologia , Macrófagos Alveolares/imunologia , Macrófagos Alveolares/patologia , Apresentação de Antígeno/efeitos dos fármacos , Apresentação de Antígeno/genética , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Separação Celular , Células Cultivadas , Perfilação da Expressão Gênica , Genoma Humano/genética , Antígenos de Histocompatibilidade Classe II/genética , Humanos , Imunidade Inata/efeitos dos fármacos , Inflamação/genética , Lipopolissacarídeos/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Fenótipo , Fatores de Tempo , Fatores de Transcrição/metabolismo , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
BACKGROUND: A new subgroup of HIV-1, designated Group P, was recently detected in two unrelated patients of Cameroonian origin. HIV-1 Group P phylogenetically clusters with SIVgor suggesting that it is the result of a cross-species transmission from gorillas. Until today, HIV-1 Group P has only been detected in two patients, and its degree of adaptation to the human host is largely unknown. Previous data have shown that pandemic HIV-1 Group M, but not non-pandemic Group O or rare Group N viruses, efficiently antagonize the human orthologue of the restriction factor tetherin (BST-2, HM1.24, CD317) suggesting that primate lentiviruses may have to gain anti-tetherin activity for efficient spread in the human population. Thus far, three SIV/HIV gene products (vpu, nef and env) are known to have the potential to counteract primate tetherin proteins, often in a species-specific manner. Here, we examined how long Group P may have been circulating in humans and determined its capability to antagonize human tetherin as an indicator of adaptation to humans. RESULTS: Our data suggest that HIV-1 Group P entered the human population between 1845 and 1989. Vpu, Env and Nef proteins from both Group P viruses failed to counteract human or gorilla tetherin to promote efficient release of HIV-1 virions, although both Group P Nef proteins moderately downmodulated gorilla tetherin from the cell surface. Notably, Vpu, Env and Nef alleles from the two HIV-1 P strains were all able to reduce CD4 cell surface expression. CONCLUSIONS: Our analyses of the two reported HIV-1 Group P viruses suggest that zoonosis occurred in the last 170 years and further support that pandemic HIV-1 Group M strains are better adapted to humans than non-pandemic or rare Group O, N and P viruses. The inability to antagonize human tetherin may potentially explain the limited spread of HIV-1 Group P in the human population.
Assuntos
HIV-1/classificação , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Produtos do Gene env do Vírus da Imunodeficiência Humana/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Adaptação Biológica , Alelos , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Linfócitos T CD4-Positivos/metabolismo , Clonagem Molecular , Proteínas Ligadas por GPI/antagonistas & inibidores , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Gorilla gorilla , Células HEK293 , Infecções por HIV/metabolismo , Infecções por HIV/transmissão , Infecções por HIV/virologia , HIV-1/genética , HIV-1/patogenicidade , Interações Hospedeiro-Patógeno , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Filogenia , Especificidade da Espécie , Transfecção , Proteínas Virais Reguladoras e Acessórias/genética , Liberação de Vírus , Produtos do Gene env do Vírus da Imunodeficiência Humana/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
Zoonosis of chimpanzee simian immunodeficiency virus cpz to humans has given rise to both pandemic (M) and non-pandemic (O, N and P) groups of human immunodeficiency virus type-1 (HIV). These lentiviruses encode accessory proteins, including Vpu, which has been shown to reduce CD4 levels on the cell surface, as well as increase virion release from the cell by antagonizing tetherin (CD317, BST2). Here, we confirm that O group Vpus (Ca9 and BCF06) are unable to counteract tetherin or downregulate the protein from the cell surface, although they are still able to reduce cell-surface CD4 levels. We hypothesize that this inability to antagonize tetherin may have contributed to O group viruses failing to achieve pandemic levels of human-to-human transmission. Characterization of chimeric O/M group Vpus and Vpu mutants demonstrate that the Vpu-tetherin interaction is complex, involving several domains. We identify specific residues within the transmembrane proximal region that, along with the transmembrane domain, are crucial for tetherin counteraction and enhanced virion release. We have also shown that the critical domains are responsible for the localization of M group Vpu to the trans-Golgi network, where it relocalizes tetherin to counteract its function. This work sheds light on the acquisition of anti-tetherin activity and the molecular details of pandemic HIV infection in humans.
Assuntos
Antígenos CD/metabolismo , HIV-1/genética , Proteínas do Vírus da Imunodeficiência Humana/metabolismo , Proteínas Virais Reguladoras e Acessórias/metabolismo , Sequência de Aminoácidos , Western Blotting , Linfócitos T CD4-Positivos/metabolismo , Clonagem Molecular , Regulação para Baixo , Imunofluorescência , Proteínas Ligadas por GPI/metabolismo , Regulação Viral da Expressão Gênica , Infecções por HIV/virologia , HIV-1/metabolismo , Proteínas do Vírus da Imunodeficiência Humana/genética , Humanos , Dados de Sequência Molecular , Domínios e Motivos de Interação entre Proteínas , Proteínas Virais Reguladoras e Acessórias/genéticaRESUMO
Domestic cats endure infections by all three subfamilies of the retroviridae: lentiviruses (feline immunodeficiency virus [FIV]), gammaretroviruses (feline leukemia virus [FeLV]), and spumaretroviruses (feline foamy virus [FFV]). Thus, cats present an insight into the evolution of the host-retrovirus relationship and the development of intrinsic/innate immune mechanisms. Tetherin (BST-2) is an interferon-inducible transmembrane protein that inhibits the release of enveloped viruses from infected cells. Here, we characterize the feline homologue of tetherin and assess its effects on the replication of FIV. Tetherin was expressed in many feline cell lines, and expression was induced by interferons, including alpha interferon (IFN-α), IFN-ω, and IFN-γ. Like human tetherin, feline tetherin displayed potent inhibition of FIV and HIV-1 particle release; however, this activity resisted antagonism by either HIV-1 Vpu or the FIV Env and "OrfA" proteins. Further, as overexpression of complete FIV genomes in trans could not overcome feline tetherin, these data suggest that FIV lacks a functional tetherin antagonist. However, when expressed stably in feline cell lines, tetherin did not abrogate the replication of FIV; indeed, syncytium formation was significantly enhanced in tetherin-expressing cells infected with cell culture-adapted (CD134-independent) strains of FIV (FIV Fca-F14 and FIV Pco-CoLV). Thus, while tetherin may prevent the release of nascent viral particles, cell-to-cell spread remains efficient in the presence of abundant viral receptors and tetherin upregulation may enhance syncytium formation. Accordingly, tetherin expression in vivo may promote the selective expansion of viral variants capable of more efficient cell-to-cell spread.
Assuntos
Antígenos CD/farmacologia , Proteínas Ligadas por GPI/farmacologia , Vírus da Imunodeficiência Felina/fisiologia , Vírus da Imunodeficiência Felina/patogenicidade , Liberação de Vírus/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/efeitos dos fármacos , Antígenos CD/genética , Antígenos CD/metabolismo , Gatos , Linhagem Celular , Cães , Fibroblastos/virologia , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/efeitos dos fármacos , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Células Gigantes/fisiologia , Humanos , Vírus da Imunodeficiência Felina/efeitos dos fármacos , Interferons/farmacologia , Camundongos , Dados de Sequência Molecular , Receptores CXCR4/metabolismo , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Replicação ViralRESUMO
OBJECTIVE: The chemokine CXCL16 serves as a scavenger receptor for oxidized low-density lipoprotein and as an adhesion molecule and chemoattractant for cells expressing the receptor CXCR6. A commonly occurring CXCL16 allele has been described containing 2 nonsynonymous single-nucleotide polymorphisms in complete linkage disequilibrium, although the effects on CXCL16 function are unknown. Here, we examined the effect of the single-nucleotide polymorphisms on CXCL16 function and assessed the association of the mutant allele with coronary heart disease (CHD). METHODS AND RESULTS: Both wild-type and mutant T123V181-CXCL16 were readily expressed in vitro and were similarly functional in assays of oxidized low-density lipoprotein scavenging and chemotaxis. However, unlike wild-type CXCL16, T123V181-CXCL16 was unable to promote adhesion of CXCR6(+) cells. Findings were confirmed ex vivo, with monocytes from donors homozygous for the T123V181 allele unable to facilitate adhesion of CXCR6 transfectants. In the London Life Sciences Prospective Population cohort (n = 2797), we found that the T123V181 allele was not associated with protection or susceptibility to CHD (adjusted odds ratio, 1.01; 95% CI, 0.95 to 1.10; P = 0.74). CONCLUSIONS: CXCL16-mediated cell adhesion plays at best a modest role in CHD, and the scavenging and chemotactic properties of the chemokine are more likely to be more important in disease pathogenesis.
Assuntos
Adesão Celular , Quimiocinas CXC/genética , Doença das Coronárias/genética , Monócitos/imunologia , Mutação , Polimorfismo de Nucleotídeo Único , Receptores de Quimiocinas/metabolismo , Receptores Depuradores/genética , Receptores Virais/metabolismo , Adulto , Idoso , Animais , Estudos de Casos e Controles , Quimiocina CXCL16 , Quimiocinas CXC/metabolismo , Quimiotaxia , Técnicas de Cocultura , Doença das Coronárias/imunologia , Feminino , Predisposição Genética para Doença , Células HEK293 , Homozigoto , Humanos , Lipoproteínas LDL/metabolismo , Modelos Logísticos , Londres , Masculino , Camundongos , Pessoa de Meia-Idade , Mutagênese Sítio-Dirigida , Razão de Chances , Fenótipo , Estudos Prospectivos , Receptores CXCR6 , Receptores de Quimiocinas/genética , Receptores Depuradores/metabolismo , Receptores Virais/genética , Medição de Risco , Fatores de Risco , Fatores de Tempo , TransfecçãoRESUMO
Tetherin is an IFN-inducible restriction factor that inhibits HIV-1 particle release in the absence of the HIV-1 countermeasure, viral protein U (Vpu). Although ubiquitous in HIV-1 and simian immunodeficiency viruses from chimpanzees, greater spot nosed monkeys, mustached monkeys, and Mona monkeys, other primate lentiviruses do not encode a Vpu protein. Here we demonstrate that SIV from Tantalus monkeys (SIVtan) encodes an envelope glycoprotein (SIVtan Env) able to counteract tetherin from Tantalus monkeys, rhesus monkeys, sooty mangabeys, and humans, but not from pigs. We show that sensitivity to Vpu but not SIVtan Env can be transferred with the human tetherin transmembrane region. We also identify a mutation in the tetherin extracellular domain, which almost completely abolishes sensitivity of human tetherin to SIVtan Env without compromising antiviral activity or sensitivity to Vpu. SIVtan Env expression results in a reduction of surface tetherin, as well as reduction in tetherin co-localization with mature surface-associated virus. Immuno-electron microscopy reveals co-localization of SIVtan Env with tetherin in intracellular tubulo-vesicular structures, suggesting that tetherin is sequestered away from budding virions at the cell surface. Along with HIV-1 Vpu and SIV Nef, envelope glycoprotein is the third and most broadly active lentiviral-encoded tetherin countermeasure to be described. Our observations emphasize the importance of tetherin in protecting mammals against viral infection and suggest that HIV-1 Vpu inhibitors may select active envelope mutants.
Assuntos
Antígenos CD/metabolismo , Espaço Intracelular/metabolismo , Glicoproteínas de Membrana/metabolismo , Vírus da Imunodeficiência Símia/metabolismo , Proteínas do Envelope Viral/metabolismo , Sequência de Aminoácidos , Animais , Antígenos CD/química , Antígenos CD/ultraestrutura , Membrana Celular/metabolismo , Membrana Celular/ultraestrutura , Espaço Extracelular/metabolismo , Proteínas Ligadas por GPI , Haplorrinos , Humanos , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/ultraestrutura , Dados de Sequência Molecular , Mutação Puntual/genética , Estrutura Terciária de Proteína , Transporte Proteico , Proteínas do Envelope Viral/química , Produtos do Gene gag do Vírus da Imunodeficiência Humana/metabolismoRESUMO
Chemokine receptor CXCR6 mediates the chemotaxis and adhesion of leukocytes to soluble and membrane-anchored forms of CXCL16, and is an HIV-1 co-receptor. Here, we describe the effects of mutation of acidic extracellular CXCR6 residues on receptor function. Although most CXCR6 mutants examined were expressed at levels similar to wild-type (WT) CXCR6, an N-terminal E3Q mutant was poorly expressed, which may explain previously reported protective effects of a similar single nucleotide polymorphism, with respect to late-stage HIV-1 infection. In contrast to several other chemokine receptors, mutation of the CXCR6 N terminus and inhibition of post-translational modifications of this region were without effect on receptor function. Likewise, N-terminal extension of CXCL16 resulted in a protein with decent potency and efficacy in chemotaxis and not, as anticipated, a CXCR6 antagonist. D176N and E274Q CXCR6 mutants were unable to interact with soluble CXCL16, suggesting a critical role for D176 and E274 in ligand binding. Intriguingly, although unable to interact with soluble CXCL16, the E274Q mutant could promote robust adhesion to membrane-anchored CXCL16, suggesting that soluble and membrane-bound forms of CXCL16 possess distinct conformations. Collectively, our data suggest a novel paradigm for the CXCR6:CXCL16 interaction, a finding which may impact the discovery of small-molecule antagonists of CXCR6.
Assuntos
Quimiocina CXCL6/fisiologia , Receptores CXCR/fisiologia , Sequência de Aminoácidos , Animais , Aterosclerose/etiologia , Sequência de Bases , Células CHO , Quimiocina CXCL16 , Quimiocina CXCL6/química , Cricetinae , Cricetulus , Camundongos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Receptores CXCR/química , Receptores CXCR6RESUMO
The chemokine receptor CXCR3 is expressed on the surface of both resting and activated T lymphocytes. We describe in this study the endocytosis of CXCR3 using T lymphocytes and CXCR3 transfectants. Chemokine-induced CXCR3 down-regulation occurred in a rapid, dose-dependent manner, with CXCL11 the most potent and efficacious ligand. Endocytosis was mediated in part by arrestins, but appeared to occur independently of clathrin and caveolae. In contrast to other chemokine receptors, which are largely recycled to the cell surface within an hour, cell surface replenishment of CXCR3 occurred over several hours and was dependent upon mRNA transcription, de novo protein synthesis, and transport through the endoplasmic reticulum and Golgi. Confocal microscopy and Western blotting confirmed the fate of endocytosed CXCR3 to be degradation, mediated in part by lysosomes and proteosomes. Site-directed mutagenesis of the CXCR3 C terminus revealed that internalization and degradation were independent of phosphorylation, ubiquitination, or a conserved LL motif. CXCR3 was found to be efficiently internalized in the absence of ligand, a process involving a YXXL motif at the extreme of the C terminus. Although freshly isolated T lymphocytes expressed moderate cell surface levels of CXCR3, they were only responsive to CXCL11 with CXCL9 and CXCL10 only having significant activity on activated T lymphocytes. Thus, the activities of CXCR3 are tightly controlled following mRNA translation. Because CXCR3(+) cells are themselves a source of IFN-gamma, which potently induces the expression of CXCR3 ligands, such tight regulation of CXCR3 may serve as a control to avoid the unnecessary amplification of activated T lymphocyte recruitment.
Assuntos
Membrana Celular/metabolismo , Receptores CXCR3/metabolismo , Linfócitos T/metabolismo , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Western Blotting , Linhagem Celular , Eletroforese em Gel de Poliacrilamida , Endocitose/fisiologia , Citometria de Fluxo , Expressão Gênica , Humanos , Camundongos , Microscopia Confocal , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Biossíntese de Proteínas , Estrutura Terciária de Proteína , Transporte Proteico/fisiologia , Receptores CXCR3/química , Receptores CXCR3/genética , TransfecçãoRESUMO
The chemokine CXCL4/platelet factor-4 is released by activated platelets in micromolar concentrations and is a chemoattractant for leukocytes via an unidentified receptor. Recently, a variant of the human chemokine receptor CXCR3 (CXCR3-B) was described, which transduced apoptotic but not chemotactic signals in microvascular endothelial cells following exposure to high concentrations of CXCL4. Here, we show that CXCL4 can induce intracellular calcium release and the migration of activated human T lymphocytes. CXCL4-induced chemotaxis of T lymphocytes was inhibited by a CXCR3 antagonist and pretreatment of cells with pertussis toxin (PTX), suggestive of CXCR3-mediated G-protein signaling via Galphai-sensitive subunits. Specific binding by T lymphocytes of the CXCR3 ligand CXCL10 was not effectively competed by CXCL4, suggesting that the two are allotopic ligands. We subsequently used expression systems to dissect the potential roles of each CXCR3 isoform in mediating CXCL4 function. Transient expression of the CXCR3-A and CXCR3-B isoforms in the murine pre-B cell L1.2 produced cells that migrated in response to CXCL4 in a manner sensitive to PTX and a CXCR3 antagonist. Binding of radiolabeled CXCL4 to L1.2 CXCR3 transfectants was of low affinity and appeared to be mediated chiefly by glycosaminoglycans (GAGs), as no specific CXCL4 binding was observed in GAG-deficient 745-Chinese hamster ovary cells stably expressing CXCR3. We suggest that following platelet activation, the CXCR3/CXCL4 axis may play a role in T lymphocyte recruitment and the subsequent amplification of inflammation observed in diseases such as atherosclerosis. In such a setting, antagonism of the CXCR3/CXCL4 axis may represent a useful, therapeutic intervention.