RESUMO
Gut inflammation involves contributions from immune and non-immune cells, whose interactions are shaped by the spatial organization of the healthy gut and its remodeling during inflammation. The crosstalk between fibroblasts and immune cells is an important axis in this process, but our understanding has been challenged by incomplete cell-type definition and biogeography. To address this challenge, we used multiplexed error-robust fluorescence in situ hybridization (MERFISH) to profile the expression of 940 genes in 1.35 million cells imaged across the onset and recovery from a mouse colitis model. We identified diverse cell populations, charted their spatial organization, and revealed their polarization or recruitment in inflammation. We found a staged progression of inflammation-associated tissue neighborhoods defined, in part, by multiple inflammation-associated fibroblasts, with unique expression profiles, spatial localization, cell-cell interactions, and healthy fibroblast origins. Similar signatures in ulcerative colitis suggest conserved human processes. Broadly, we provide a framework for understanding inflammation-induced remodeling in the gut and other tissues.
Assuntos
Colite Ulcerativa , Colite , Animais , Humanos , Camundongos , Colite/metabolismo , Colite/patologia , Colite Ulcerativa/metabolismo , Colite Ulcerativa/patologia , Fibroblastos/metabolismo , Fibroblastos/patologia , Hibridização in Situ Fluorescente/métodos , Inflamação/metabolismo , Inflamação/patologia , Comunicação Celular , Trato Gastrointestinal/metabolismo , Trato Gastrointestinal/patologiaRESUMO
The APOE4 allele is the strongest genetic risk factor for late-onset Alzheimer's disease (AD). The contribution of microglial APOE4 to AD pathogenesis is unknown, although APOE has the most enriched gene expression in neurodegenerative microglia (MGnD). Here, we show in mice and humans a negative role of microglial APOE4 in the induction of the MGnD response to neurodegeneration. Deletion of microglial APOE4 restores the MGnD phenotype associated with neuroprotection in P301S tau transgenic mice and decreases pathology in APP/PS1 mice. MGnD-astrocyte cross-talk associated with ß-amyloid (Aß) plaque encapsulation and clearance are mediated via LGALS3 signaling following microglial APOE4 deletion. In the brains of AD donors carrying the APOE4 allele, we found a sex-dependent reciprocal induction of AD risk factors associated with suppression of MGnD genes in females, including LGALS3, compared to individuals homozygous for the APOE3 allele. Mechanistically, APOE4-mediated induction of ITGB8-transforming growth factor-ß (TGFß) signaling impairs the MGnD response via upregulation of microglial homeostatic checkpoints, including Inpp5d, in mice. Deletion of Inpp5d in microglia restores MGnD-astrocyte cross-talk and facilitates plaque clearance in APP/PS1 mice. We identify the microglial APOE4-ITGB8-TGFß pathway as a negative regulator of microglial response to AD pathology, and restoring the MGnD phenotype via blocking ITGB8-TGFß signaling provides a promising therapeutic intervention for AD.
Assuntos
Doença de Alzheimer , Feminino , Camundongos , Humanos , Animais , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Apolipoproteína E4/genética , Apolipoproteína E4/metabolismo , Microglia/metabolismo , Galectina 3/genética , Galectina 3/metabolismo , Peptídeos beta-Amiloides/metabolismo , Camundongos Transgênicos , Modelos Animais de DoençasRESUMO
Mucosal barrier immunity is essential for the maintenance of the commensal microflora and combating invasive bacterial infection. Although immune and epithelial cells are thought to be the canonical orchestrators of this complex equilibrium, here, we show that the enteric nervous system (ENS) plays an essential and non-redundant role in governing the antimicrobial protein (AMP) response. Using confocal microscopy and single-molecule fluorescence in situ mRNA hybridization (smFISH) studies, we observed that intestinal neurons produce the pleiotropic cytokine IL-18. Strikingly, deletion of IL-18 from the enteric neurons alone, but not immune or epithelial cells, rendered mice susceptible to invasive Salmonella typhimurium (S.t.) infection. Mechanistically, unbiased RNA sequencing and single-cell sequencing revealed that enteric neuronal IL-18 is specifically required for homeostatic goblet cell AMP production. Together, we show that neuron-derived IL-18 signaling controls tissue-wide intestinal immunity and has profound consequences on the mucosal barrier and invasive bacterial killing.
Assuntos
Imunidade nas Mucosas/imunologia , Interleucina-18/imunologia , Mucosa Intestinal/imunologia , Animais , Citocinas/imunologia , Sistema Nervoso Entérico/imunologia , Sistema Nervoso Entérico/metabolismo , Células Epiteliais/imunologia , Feminino , Células Caliciformes/imunologia , Interleucina-18/biossíntese , Mucosa Intestinal/metabolismo , Intestino Delgado/imunologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurônios/imunologia , Ratos , Ratos Sprague-Dawley , Infecções por Salmonella/imunologia , Salmonella typhimurium/imunologia , Transdução de Sinais/imunologiaRESUMO
Gut-innervating nociceptor sensory neurons respond to noxious stimuli by initiating protective responses including pain and inflammation; however, their role in enteric infections is unclear. Here, we find that nociceptor neurons critically mediate host defense against the bacterial pathogen Salmonella enterica serovar Typhimurium (STm). Dorsal root ganglia nociceptors protect against STm colonization, invasion, and dissemination from the gut. Nociceptors regulate the density of microfold (M) cells in ileum Peyer's patch (PP) follicle-associated epithelia (FAE) to limit entry points for STm invasion. Downstream of M cells, nociceptors maintain levels of segmentous filamentous bacteria (SFB), a gut microbe residing on ileum villi and PP FAE that mediates resistance to STm infection. TRPV1+ nociceptors directly respond to STm by releasing calcitonin gene-related peptide (CGRP), a neuropeptide that modulates M cells and SFB levels to protect against Salmonella infection. These findings reveal a major role for nociceptor neurons in sensing and defending against enteric pathogens.
Assuntos
Microbioma Gastrointestinal/fisiologia , Interações entre Hospedeiro e Microrganismos/fisiologia , Nociceptores/fisiologia , Animais , Epitélio/metabolismo , Feminino , Gânglios Espinais/metabolismo , Gânglios Espinais/microbiologia , Mucosa Intestinal/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Nociceptores/metabolismo , Nódulos Linfáticos Agregados/inervação , Nódulos Linfáticos Agregados/metabolismo , Infecções por Salmonella/metabolismo , Salmonella typhimurium/metabolismo , Salmonella typhimurium/patogenicidade , Células Receptoras Sensoriais/metabolismo , Células Receptoras Sensoriais/fisiologiaRESUMO
Tissues are exposed to diverse inflammatory challenges that shape future inflammatory responses. While cellular metabolism regulates immune function, how metabolism programs and stabilizes immune states within tissues and tunes susceptibility to inflammation is poorly understood. Here, we describe an innate immune metabolic switch that programs long-term intestinal tolerance. Intestinal interleukin-18 (IL-18) stimulation elicited tolerogenic macrophages by preventing their proinflammatory glycolytic polarization via metabolic reprogramming to fatty acid oxidation (FAO). FAO reprogramming was triggered by IL-18 activation of SLC12A3 (NCC), leading to sodium influx, release of mitochondrial DNA, and activation of stimulator of interferon genes (STING). FAO was maintained in macrophages by a bistable switch that encoded memory of IL-18 stimulation and by intercellular positive feedback that sustained the production of macrophage-derived 2'3'-cyclic GMP-AMP (cGAMP) and epithelial-derived IL-18. Thus, a tissue-reinforced metabolic switch encodes durable immune tolerance in the gut and may enable reconstructing compromised immune tolerance in chronic inflammation.
RESUMO
The immune system safeguards organ integrity by employing a balancing act of inflammatory and immunosuppressive mechanisms designed to neutralize foreign invaders and resolve injury. Maintaining or restoring a state of immune homeostasis is particularly challenging at barrier sites where constant exposure to immunogenic environmental agents may induce destructive inflammation. Recent studies underscore the role of epithelial and mesenchymal barrier cells in regulating immune cell function and local homeostatic and inflammatory responses. Here, we highlight immunoregulatory circuits engaging epithelial and mesenchymal cells in the intestine, airways, and skin and discuss how immune communications with hematopoietic cells and the microbiota orchestrate local immune homeostasis and inflammation.
Assuntos
Epitélio/imunologia , Homeostase , Inflamação/imunologia , Mesoderma/imunologia , Animais , Células Epiteliais/imunologia , Humanos , Infecções/imunologia , Intestinos/citologia , Intestinos/imunologia , Intestinos/fisiologia , Mesoderma/citologia , Sistema Respiratório/imunologiaRESUMO
The intestinal mucosal barrier controlling the resident microbiome is dependent on a protective mucus layer generated by goblet cells, impairment of which is a hallmark of the inflammatory bowel disease, ulcerative colitis. Here, we show that IL-18 is critical in driving the pathologic breakdown of barrier integrity in a model of colitis. Deletion of Il18 or its receptor Il18r1 in intestinal epithelial cells (Δ/EC) conferred protection from colitis and mucosal damage in mice. In contrast, deletion of the IL-18 negative regulator Il18bp resulted in severe colitis associated with loss of mature goblet cells. Colitis and goblet cell loss were rescued in Il18bp(-/-);Il18r(Δ/EC) mice, demonstrating that colitis severity is controlled at the level of IL-18 signaling in intestinal epithelial cells. IL-18 inhibited goblet cell maturation by regulating the transcriptional program instructing goblet cell development. These results inform on the mechanism of goblet cell dysfunction that underlies the pathology of ulcerative colitis.
Assuntos
Colite Ulcerativa/patologia , Colite Ulcerativa/fisiopatologia , Interleucina-18/imunologia , Animais , Colite Ulcerativa/induzido quimicamente , Colite Ulcerativa/metabolismo , Sulfato de Dextrana , Células Endoteliais/metabolismo , Células Epiteliais/citologia , Feminino , Células Caliciformes/metabolismo , Células Caliciformes/patologia , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Subunidade alfa de Receptor de Interleucina-18/genética , Subunidade alfa de Receptor de Interleucina-18/metabolismo , Mucosa Intestinal/fisiopatologia , Masculino , Camundongos , Transdução de SinaisRESUMO
Mucus production by goblet cells of the large intestine serves as a crucial antimicrobial protective mechanism at the interface between the eukaryotic and prokaryotic cells of the mammalian intestinal ecosystem. However, the regulatory pathways involved in goblet cell-induced mucus secretion remain largely unknown. Here, we demonstrate that the NLRP6 inflammasome, a recently described regulator of colonic microbiota composition and biogeographical distribution, is a critical orchestrator of goblet cell mucin granule exocytosis. NLRP6 deficiency leads to defective autophagy in goblet cells and abrogated mucus secretion into the large intestinal lumen. Consequently, NLRP6 inflammasome-deficient mice are unable to clear enteric pathogens from the mucosal surface, rendering them highly susceptible to persistent infection. This study identifies an innate immune regulatory pathway governing goblet cell mucus secretion, linking nonhematopoietic inflammasome signaling to autophagy and highlighting the goblet cell as a critical innate immune player in the control of intestinal host-microbial mutualism. PAPERCLIP:
Assuntos
Colo/imunologia , Células Caliciformes/imunologia , Inflamassomos/imunologia , Mucosa Intestinal/imunologia , Receptores de Superfície Celular/imunologia , Animais , Autofagia , Colite/imunologia , Colite/microbiologia , Colo/microbiologia , Células Epiteliais/imunologia , Células Epiteliais/metabolismo , Células Caliciformes/citologia , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Camundongos , Muco/metabolismoRESUMO
Dendritic cells (DCs) have a role in the development and activation of self-reactive pathogenic T cells1,2. Genetic variants that are associated with the function of DCs have been linked to autoimmune disorders3,4, and DCs are therefore attractive therapeutic targets for such diseases. However, developing DC-targeted therapies for autoimmunity requires identification of the mechanisms that regulate DC function. Here, using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies, we identify a regulatory loop of negative feedback that operates in DCs to limit immunopathology. Specifically, we find that lactate, produced by activated DCs and other immune cells, boosts the expression of NDUFA4L2 through a mechanism mediated by hypoxia-inducible factor 1α (HIF-1α). NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs that are involved in the control of pathogenic autoimmune T cells. We also engineer a probiotic that produces lactate and suppresses T cell autoimmunity through the activation of HIF-1α-NDUFA4L2 signalling in DCs. In summary, we identify an immunometabolic pathway that regulates DC function, and develop a synthetic probiotic for its therapeutic activation.
Assuntos
Doenças Autoimunes , Sistema Nervoso Central , Células Dendríticas , Subunidade alfa do Fator 1 Induzível por Hipóxia , Ácido Láctico , Humanos , Doenças Autoimunes/imunologia , Doenças Autoimunes/metabolismo , Doenças Autoimunes/prevenção & controle , Autoimunidade , Sistema Nervoso Central/citologia , Sistema Nervoso Central/imunologia , Sistema Nervoso Central/patologia , Células Dendríticas/imunologia , Células Dendríticas/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/química , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Ácido Láctico/metabolismo , Probióticos/uso terapêutico , Espécies Reativas de Oxigênio/metabolismo , Linfócitos T/imunologia , Retroalimentação Fisiológica , Lactase/genética , Lactase/metabolismo , Análise de Célula ÚnicaRESUMO
APOBEC3 proteins catalyze deamination of cytidines in single-stranded DNA (ssDNA), providing innate protection against retroviral replication by inducing deleterious dC > dU hypermutation of replication intermediates. APOBEC3G expression is induced in mitogen-activated lymphocytes; however, no physiologic role related to lymphoid cell proliferation has yet to be determined. Moreover, whether APOBEC3G cytidine deaminase activity transcends to processing cellular genomic DNA is unknown. Here we show that lymphoma cells expressing high APOBEC3G levels display efficient repair of genomic DNA double-strand breaks (DSBs) induced by ionizing radiation and enhanced survival of irradiated cells. APOBEC3G transiently accumulated in the nucleus in response to ionizing radiation and was recruited to DSB repair foci. Consistent with a direct role in DSB repair, inhibition of APOBEC3G expression or deaminase activity resulted in deficient DSB repair, whereas reconstitution of APOBEC3G expression in leukemia cells enhanced DSB repair. APOBEC3G activity involved processing of DNA flanking a DSB in an integrated reporter cassette. Atomic force microscopy indicated that APOBEC3G multimers associate with ssDNA termini, triggering multimer disassembly to multiple catalytic units. These results identify APOBEC3G as a prosurvival factor in lymphoma cells, marking APOBEC3G as a potential target for sensitizing lymphoma to radiation therapy.
Assuntos
Citidina Desaminase/metabolismo , Reparo do DNA/fisiologia , Linfoma/metabolismo , Linfoma/radioterapia , Tolerância a Radiação/fisiologia , Desaminase APOBEC-3G , Domínio Catalítico , Linhagem Celular Tumoral , Sobrevivência Celular , Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/química , Citidina Desaminase/genética , Quebras de DNA de Cadeia Dupla , Reparo do DNA por Junção de Extremidades , DNA de Neoplasias/metabolismo , DNA de Neoplasias/efeitos da radiação , Técnicas de Silenciamento de Genes , Humanos , Linfoma/patologia , Microscopia de Força Atômica , Multimerização ProteicaRESUMO
APOE4 is the strongest genetic risk factor for Alzheimer's disease (AD), with increased odds ratios in female carriers. Targeting amyloid plaques shows modest improvement in male non-APOE4 carriers. Leveraging single-cell transcriptomics across APOE variants in both sexes, multiplex flow cytometry and validation in two independent cohorts of APOE4 female carriers with AD, we identify a new subset of neutrophils interacting with microglia associated with cognitive impairment. This phenotype is defined by increased interleukin (IL)-17 and IL-1 coexpressed gene modules in blood neutrophils and in microglia of cognitively impaired female APOE ε4 carriers, showing increased infiltration to the AD brain. APOE4 female IL-17+ neutrophils upregulated the immunosuppressive cytokines IL-10 and TGFß and immune checkpoints, including LAG3 and PD-1, associated with accelerated immune aging. Deletion of APOE4 in neutrophils reduced this immunosuppressive phenotype and restored the microglial response to neurodegeneration, limiting plaque pathology in AD mice. Mechanistically, IL-17F upregulated in APOE4 neutrophils interacts with microglial IL-17RA to suppress the induction of the neurodegenerative phenotype, and blocking this axis supported cognitive improvement in AD mice. These findings provide a translational basis to target IL-17F in APOE ε4 female carriers with cognitive impairment.
RESUMO
Gut inflammation involves contributions from immune and non-immune cells, whose interactions are shaped by the spatial organization of the healthy gut and its remodeling during inflammation. The crosstalk between fibroblasts and immune cells is an important axis in this process, but our understanding has been challenged by incomplete cell-type definition and biogeography. To address this challenge, we used MERFISH to profile the expression of 940 genes in 1.35 million cells imaged across the onset and recovery from a mouse colitis model. We identified diverse cell populations; charted their spatial organization; and revealed their polarization or recruitment in inflammation. We found a staged progression of inflammation-associated tissue neighborhoods defined, in part, by multiple inflammation-associated fibroblasts, with unique expression profiles, spatial localization, cell-cell interactions, and healthy fibroblast origins. Similar signatures in ulcerative colitis suggest conserved human processes. Broadly, we provide a framework for understanding inflammation-induced remodeling in the gut and other tissues.
RESUMO
Dendritic cells (DCs) control the generation of self-reactive pathogenic T cells. Thus, DCs are considered attractive therapeutic targets for autoimmune diseases. Using single-cell and bulk transcriptional and metabolic analyses in combination with cell-specific gene perturbation studies we identified a negative feedback regulatory pathway that operates in DCs to limit immunopathology. Specifically, we found that lactate, produced by activated DCs and other immune cells, boosts NDUFA4L2 expression through a mechanism mediated by HIF-1α. NDUFA4L2 limits the production of mitochondrial reactive oxygen species that activate XBP1-driven transcriptional modules in DCs involved in the control of pathogenic autoimmune T cells. Moreover, we engineered a probiotic that produces lactate and suppresses T-cell autoimmunity in the central nervous system via the activation of HIF-1α/NDUFA4L2 signaling in DCs. In summary, we identified an immunometabolic pathway that regulates DC function, and developed a synthetic probiotic for its therapeutic activation.
RESUMO
Deamination of cytidine residues in viral DNA is a major mechanism by which APOBEC3G (A3G) inhibits vif-deficient human immunodeficiency virus type 1 (HIV-1) replication. dC-to-dU transition following RNase-H activity leads to viral cDNA degradation, production of non-functional proteins, formation of undesired stop codons and decreased viral protein synthesis. Here, we demonstrate that A3G provides an additional layer of defense against HIV-1 infection dependent on inhibition of proviral transcription. HIV-1 transcription elongation is regulated by the trans-activation response (TAR) element, a short stem-loop RNA structure required for elongation factors binding. Vif-deficient HIV-1-infected cells accumulate short viral transcripts and produce lower amounts of full-length HIV-1 transcripts due to A3G deamination of the TAR apical loop cytidine, highlighting the requirement for TAR loop integrity in HIV-1 transcription. We further show that free single-stranded DNA (ssDNA) termini are not essential for A3G activity and a gap of CCC motif blocked with juxtaposed DNA or RNA on either or 3'+5' ends is sufficient for A3G deamination. These results identify A3G as an efficient mutator and that deamination of (-)SSDNA results in an early block of HIV-1 transcription.
Assuntos
Citidina Desaminase/metabolismo , DNA de Cadeia Simples/genética , DNA Viral/genética , Transcriptase Reversa do HIV/metabolismo , HIV-1/fisiologia , Elementos de Resposta/genética , Ativação Viral/fisiologia , Desaminase APOBEC-3G , Pareamento de Bases , Sequência de Bases , Northern Blotting , Ensaio de Desvio de Mobilidade Eletroforética , Transcriptase Reversa do HIV/antagonistas & inibidores , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Viral/genética , Transcrição Gênica , Replicação Viral , Produtos do Gene vif do Vírus da Imunodeficiência Humana/genéticaRESUMO
High frequency of cytidine to thymidine conversions was identified in the genome of several types of cancer cells. In breast cancer cells, these mutations are clustered in long DNA regions associated with single-strand DNA (ssDNA), double-strand DNA breaks (DSB), and genomic rearrangements. The observed mutational pattern resembles the deamination signature of cytidine to uridine carried out by members of the APOBEC3 family of cellular deaminases. Consistently, APOBEC3B (A3B) was recently identified as the mutational source in breast cancer cells. A3G is another member of the cytidine deaminases family predominantly expressed in lymphoma cells, where it is involved in mutational DSB repair following ionizing radiation treatments. This activity provides us with a new paradigm for cancer cell survival and tumor promotion and a mechanistic link between ssDNA, DSBs, and clustered mutations. Cancer Res; 73(12); 3494-8. ©2013 AACR.
Assuntos
Citosina Desaminase/metabolismo , Quebras de DNA de Cadeia Dupla , Reparo do DNA , Neoplasias/genética , Desaminases APOBEC , Desaminase APOBEC-3G , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/terapia , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citidina Desaminase/antagonistas & inibidores , Citidina Desaminase/metabolismo , Citosina Desaminase/antagonistas & inibidores , DNA de Cadeia Simples , Inibidores Enzimáticos/uso terapêutico , Humanos , Linfoma/genética , Linfoma/metabolismo , Linfoma/terapia , Antígenos de Histocompatibilidade Menor , Modelos Genéticos , Mutação , Neoplasias/metabolismo , Neoplasias/terapiaRESUMO
The innate immune system has evolved in multicellular organisms to detect and respond to situations that compromise tissue homeostasis. It comprises a set of tissue-resident and circulating leukocytes primarily designed to sense pathogens and tissue damage through hardwired receptors and eliminate noxious sources by mediating inflammatory processes. While indispensable to immunity, the inflammatory mediators produced in situ by activated innate cells during injury or infection are also associated with increased cancer risk and tumorigenesis. Here, we outline basic principles of innate immune cell functions in inflammation and discuss how these functions converge upon cancer development.
Assuntos
Imunidade Inata/imunologia , Inflamação/imunologia , Neoplasias/imunologia , Células Dendríticas/imunologia , Humanos , Macrófagos/imunologia , Monócitos/imunologia , Neutrófilos/imunologiaRESUMO
Inflammation is a fundamental innate immune response to perturbed tissue homeostasis. Chronic inflammatory processes affect all stages of tumour development as well as therapy. In this Review, we outline the principal cellular and molecular pathways that coordinate the tumour-promoting and tumour-antagonizing effects of inflammation and we discuss the crosstalk between cancer development and inflammatory processes. In addition, we discuss the recently suggested role of commensal microorganisms in inflammation-induced cancer and we propose that understanding this microbial influence will be crucial for targeted therapy in modern cancer treatment.
Assuntos
Regulação Neoplásica da Expressão Gênica , Inflamação/complicações , Inflamação/imunologia , Neoplasias/etiologia , Neoplasias/imunologia , Neoplasias/patologia , Animais , Carcinogênese , Proliferação de Células , Transformação Celular Neoplásica/imunologia , Genoma , Homeostase , Humanos , Imunidade Inata , Inflamação/microbiologia , Camundongos , Microbiota , Metástase Neoplásica , Neoplasias/microbiologia , Transdução de SinaisAssuntos
Bacteriófago M13/genética , Bacteriófago M13/isolamento & purificação , DNA de Cadeia Simples/química , DNA Viral/química , Técnicas de Amplificação de Ácido Nucleico/métodos , Sequência de Bases , Catálise , DNA Catalítico/metabolismo , DNA de Cadeia Simples/análise , DNA de Cadeia Simples/genética , DNA Viral/análise , DNA Viral/genética , Conformação de Ácido NucleicoRESUMO
In the absence of human immunodeficiency virus type 1 (HIV-1) Vif protein, the host antiviral deaminase apolipoprotein B mRNA-editing enzyme-catalytic polypeptide-like 3G (A3G) restricts the production of infectious HIV-1 by deamination of dC residues in the negative single-stranded DNA produced by reverse transcription. The Vif protein averts the lethal threat of deamination by precluding the packaging of A3G into assembling virions by mediating proteasomal degradation of A3G. In spite of this robust Vif activity, residual A3G molecules that escape degradation and incorporate into newly assembled virions are potentially deleterious to the virus. We hypothesized that virion-associated Vif inhibits A3G enzymatic activity and therefore prevents lethal mutagenesis of the newly synthesized viral DNA. Here, we show that (i) Vif-proficient HIV-1 particles released from H9 cells contain A3G with lower specific activity compared with Δvif-virus-associated A3G, (ii) encapsidated HIV-1 Vif inhibits the deamination activity of recombinant A3G, and (iii) purified HIV-1 Vif protein and the Vif-derived peptide Vif25-39 inhibit A3G activity in vitro at nanomolar concentrations in an uncompetitive manner. Our results manifest the potentiality of Vif to control the deamination threat in virions or in the pre-integration complexes following entry to target cells. Hence, virion-associated Vif could serve as a last line of defense, protecting the virus against A3G antiviral activity.