RESUMO
Antiretroviral drugs that target various stages of the Human Immunodeficiency Virus (HIV) life cycle have been effective in curbing the AIDS epidemic. However, drug resistance, off-target effects of antiretroviral therapy (ART), and varying efficacy in prevention underscore the need to develop novel and alternative therapeutics. In this study, we investigated whether targeting the signaling molecule Sphingosine-1-phosphate (S1P) would inhibit HIV-1 infection and generation of the latent reservoir in primary CD4 T cells. We show that FTY720 (Fingolimod), an FDA-approved functional antagonist of S1P receptors, blocks cell-free and cell-to-cell transmission of HIV and consequently reduces detectable latent virus. Mechanistically, FTY720 impacts the HIV-1 life cycle at two levels. Firstly, FTY720 reduces the surface density of CD4, thereby inhibiting viral binding and fusion. Secondly, FTY720 decreases the phosphorylation of the innate HIV restriction factor SAMHD1 which is associated with reduced levels of total and integrated HIV, while reducing the expression of Cyclin D3. In conclusion, targeting the S1P pathway with FTY720 could be a novel strategy to inhibit HIV replication and reduce the seeding of the latent reservoir.
Assuntos
Cloridrato de Fingolimode/farmacologia , Infecções por HIV/tratamento farmacológico , HIV-1/crescimento & desenvolvimento , Proteína 1 com Domínio SAM e Domínio HD/antagonistas & inibidores , Moduladores do Receptor de Esfingosina 1 Fosfato/farmacologia , Linfócitos T/imunologia , Replicação Viral , Infecções por HIV/imunologia , Infecções por HIV/virologia , HIV-1/efeitos dos fármacos , HIV-1/imunologia , Humanos , Lisofosfolipídeos/metabolismo , Fosforilação , Proteína 1 com Domínio SAM e Domínio HD/metabolismo , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Linfócitos T/efeitos dos fármacos , Latência ViralRESUMO
HIV infection has a profound effect on "bystander" cells causing metabolic co-morbidities. This may be mediated by exosomes secreted by HIV-infected cells and containing viral factors. Here we show that exosomes containing HIV-1 protein Nef (exNef) are rapidly taken up by macrophages releasing Nef into the cell interior. This caused down-regulation of ABCA1, reduction of cholesterol efflux and sharp elevation of the abundance of lipid rafts through reduced activation of small GTPase Cdc42 and decreased actin polymerization. Changes in rafts led to re-localization of TLR4 and TREM-1 to rafts, phosphorylation of ERK1/2, activation of NLRP3 inflammasome, and increased secretion of pro-inflammatory cytokines. The effects of exNef on lipid rafts and on inflammation were reversed by overexpression of a constitutively active mutant of Cdc42. Similar effects were observed in macrophages treated with exosomes produced by HIV-infected cells or isolated from plasma of HIV-infected subjects, but not with exosomes from cells and subjects infected with ΔNef-HIV or uninfected subjects. Mice injected with exNef exhibited monocytosis, reduced ABCA1 in macrophages, increased raft abundance in monocytes and augmented inflammation. Thus, Nef-containing exosomes potentiated pro-inflammatory response by inducing changes in cholesterol metabolism and reorganizing lipid rafts. These mechanisms may contribute to HIV-associated metabolic co-morbidities.
Assuntos
Infecções por HIV/metabolismo , Infecções por HIV/virologia , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Efeito Espectador , Colesterol/metabolismo , Exossomos/metabolismo , Exossomos/virologia , Células HEK293 , HIV-1 , Humanos , Inflamação/metabolismo , Inflamação/virologia , Microdomínios da Membrana/metabolismo , Microdomínios da Membrana/virologia , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Células RAW 264.7 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptor 4 Toll-Like/metabolismo , Receptor Gatilho 1 Expresso em Células Mieloides/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
Schistosomiasis is the most important helminthic disease of humanity in terms of morbidity and mortality. Facile manipulation of schistosomes using lentiviruses would enable advances in functional genomics in these and related neglected tropical diseases pathogens including tapeworms, and including their non-dividing cells. Such approaches have hitherto been unavailable. Blood stream forms of the human blood fluke, Schistosoma mansoni, the causative agent of the hepatointestinal schistosomiasis, were infected with the human HIV-1 isolate NL4-3 pseudotyped with vesicular stomatitis virus glycoprotein. The appearance of strong stop and positive strand cDNAs indicated that virions fused to schistosome cells, the nucleocapsid internalized and the RNA genome reverse transcribed. Anchored PCR analysis, sequencing HIV-1-specific anchored Illumina libraries and Whole Genome Sequencing (WGS) of schistosomes confirmed chromosomal integration; >8,000 integrations were mapped, distributed throughout the eight pairs of chromosomes including the sex chromosomes. The rate of integrations in the genome exceeded five per 1,000 kb and HIV-1 integrated into protein-encoding loci and elsewhere with integration bias dissimilar to that of human T cells. We estimated ~ 2,100 integrations per schistosomulum based on WGS, i.e. about two or three events per cell, comparable to integration rates in human cells. Accomplishment in schistosomes of post-entry processes essential for HIV-1replication, including integrase-catalyzed integration, was remarkable given the phylogenetic distance between schistosomes and primates, the natural hosts of the genus Lentivirus. These enigmatic findings revealed that HIV-1 was active within cells of S. mansoni, and provided the first demonstration that HIV-1 can integrate into the genome of an invertebrate.
Assuntos
Genoma Helmíntico , Infecções por HIV , HIV-1 , Schistosoma mansoni/virologia , Esquistossomose mansoni/virologia , Integração Viral , Animais , Animais Geneticamente Modificados , Camundongos , Reação em Cadeia da Polimerase , Transdução GenéticaRESUMO
High density lipoproteins (HDL) are key components of reverse cholesterol transport pathway. HDL removes excessive cholesterol from peripheral cells, including macrophages, providing protection from cholesterol accumulation and conversion into foam cells, which is a key event in pathogenesis of atherosclerosis. The mechanism of cellular cholesterol efflux stimulation by HDL involves interaction with the ABCA1 lipid transporter and ensuing transfer of cholesterol to HDL particles. In this study, we looked for additional proteins contributing to HDL-dependent cholesterol efflux. Using RNAseq, we analyzed mRNAs induced by HDL in human monocyte-derived macrophages and identified three genes, fatty acid desaturase 1 (FADS1), insulin induced gene 1 (INSIG1), and the low-density lipoprotein receptor (LDLR), expression of which was significantly upregulated by HDL. We individually knocked down these genes in THP-1 cells using gene silencing by siRNA, and measured cellular cholesterol efflux to HDL. Knock down of FADS1 did not significantly change cholesterol efflux (pâ¯=â¯0.70), but knockdown of INSIG1 and LDLR resulted in highly significant reduction of the efflux to HDL (67% and 75% of control, respectively, pâ¯<â¯0.001). Importantly, the suppression of cholesterol efflux was independent of known effects of these genes on cellular cholesterol content, as cells were loaded with cholesterol using acetylated LDL. These results indicate that HDL particles stimulate expression of genes that enhance cellular cholesterol transfer to HDL.
Assuntos
HDL-Colesterol/genética , Macrófagos/fisiologia , Transportador 1 de Cassete de Ligação de ATP/genética , Aterosclerose/fisiopatologia , Transporte Biológico , Colesterol , HDL-Colesterol/metabolismo , Dessaturase de Ácido Graxo Delta-5 , Ácidos Graxos Dessaturases/genética , Ácidos Graxos Dessaturases/metabolismo , Células Espumosas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/genética , Inativação Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lipoproteínas HDL/genética , Lipoproteínas HDL/metabolismo , Macrófagos/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , RNA Mensageiro , RNA Interferente Pequeno , Receptores de LDL/genética , Receptores de LDL/metabolismo , Células THP-1 , Regulação para CimaRESUMO
OBJECTIVE: HIV-infected patients are at an increased risk of developing atherosclerosis, in part because of downmodulation and functional impairment of ATP-binding cassette A1 (ABCA1) cholesterol transporter by the HIV-1 protein Nef. The mechanism of this effect involves Nef interacting with an ER chaperone calnexin and disrupting calnexin binding to ABCA1, leading to ABCA1 retention in ER, its degradation and resulting suppression of cholesterol efflux. However, molecular details of Nef-calnexin interaction remained unknown, limiting the translational impact of this finding. APPROACH AND RESULTS: Here, we used molecular modeling and mutagenesis to characterize Nef-calnexin interaction and to identify small molecule compounds that could block it. We demonstrated that the interaction between Nef and calnexin is direct and can be reconstituted using recombinant proteins in vitro with a binding affinity of 89.1 nmol/L measured by surface plasmon resonance. The cytoplasmic tail of calnexin is essential and sufficient for interaction with Nef, and binds Nef with an affinity of 9.4 nmol/L. Replacing lysine residues in positions 4 and 7 of Nef with alanines abrogates Nef-calnexin interaction, prevents ABCA1 downregulation by Nef, and preserves cholesterol efflux from HIV-infected cells. Through virtual screening of the National Cancer Institute library of compounds, we identified a compound, 1[(7-oxo-7H-benz[de]anthracene-3-yl)amino]anthraquinone, which blocked Nef-calnexin interaction, partially restored ABCA1 activity in HIV-infected cells, and reduced foam cell formation in a culture of HIV-infected macrophages. CONCLUSION: This study identifies potential targets that can be exploited to block the pathogenic effect of HIV infection on cholesterol metabolism and prevent atherosclerosis in HIV-infected subjects.
Assuntos
Antraquinonas/farmacologia , Aterosclerose/prevenção & controle , Calnexina/metabolismo , Colesterol/metabolismo , Desenho de Fármacos , Infecções por HIV/tratamento farmacológico , Hipolipemiantes/farmacologia , Simulação de Acoplamento Molecular , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Antraquinonas/química , Aterosclerose/metabolismo , Aterosclerose/virologia , Transporte Biológico , Calnexina/química , Calnexina/genética , Desenho Assistido por Computador , Células Espumosas/efeitos dos fármacos , Células Espumosas/metabolismo , Células HEK293 , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Humanos , Hipolipemiantes/química , Lisina , Mutação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Relação Estrutura-Atividade , Transfecção , Produtos do Gene nef do Vírus da Imunodeficiência Humana/química , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
HIV-infected patients are at increased risk of developing atherosclerosis, in part due to an altered high density lipoprotein profile exacerbated by down-modulation and impairment of ATP-binding cassette transporter A1 (ABCA1) activity by the HIV-1 protein Nef. However, the mechanisms of this Nef effect remain unknown. Here, we show that Nef interacts with an endoplasmic reticulum chaperone calnexin, which regulates folding and maturation of glycosylated proteins. Nef disrupted interaction between calnexin and ABCA1 but increased affinity and enhanced interaction of calnexin with HIV-1 gp160. The Nef mutant that did not bind to calnexin did not affect the calnexin-ABCA1 interaction. Interaction with calnexin was essential for functionality of ABCA1, as knockdown of calnexin blocked the ABCA1 exit from the endoplasmic reticulum, reduced ABCA1 abundance, and inhibited cholesterol efflux; the same effects were observed after Nef overexpression. However, the effects of calnexin knockdown and Nef on cholesterol efflux were not additive; in fact, the combined effect of these two factors together did not differ significantly from the effect of calnexin knockdown alone. Interestingly, gp160 and ABCA1 interacted with calnexin differently; although gp160 binding to calnexin was dependent on glycosylation, glycosylation was of little importance for the interaction between ABCA1 and calnexin. Thus, Nef regulates the activity of calnexin to stimulate its interaction with gp160 at the expense of ABCA1. This study identifies a mechanism for Nef-dependent inactivation of ABCA1 and dysregulation of cholesterol metabolism.
Assuntos
Transportador 1 de Cassete de Ligação de ATP/metabolismo , Calnexina/metabolismo , Retículo Endoplasmático/metabolismo , Proteína gp160 do Envelope de HIV/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Aterosclerose/metabolismo , Colesterol/metabolismo , Glicosilação , Células HEK293 , HIV-1/metabolismo , Células HeLa , Humanos , Ligação Proteica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas em TandemRESUMO
Previous studies demonstrated that liver X receptor (LXR) agonists inhibit human immunodeficiency virus (HIV) replication by upregulating cholesterol transporter ATP-binding cassette A1 (ABCA1), suppressing HIV production, and reducing infectivity of produced virions. In this study, we extended these observations by analyzing the effect of the LXR agonist T0901317 [N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide] on the ongoing HIV infection and investigating the possibility of using LXR agonist for pre-exposure prophylaxis of HIV infection in a humanized mouse model. Pre-exposure of monocyte-derived macrophages to T0901317 reduced susceptibility of these cells to HIV infection in vitro. This protective effect lasted for up to 4 days after treatment termination and correlated with upregulated expression of ABCA1, reduced abundance of lipid rafts, and reduced fusion of the cells with HIV. Pre-exposure of peripheral blood leukocytes to T0901317 provided only a short-term protection against HIV infection. Treatment of HIV-exposed humanized mice with LXR agonist starting 2 weeks postinfection substantially reduced viral load. When eight humanized mice were pretreated with LXR agonist prior to HIV infection, five animals were protected from infection, two had viral load at the limit of detection, and one had viral load significantly reduced relative to mock-treated controls. T0901317 pretreatment also reduced HIV-induced dyslipidemia in infected mice. In conclusion, these results reveal a novel link between LXR stimulation and cell resistance to HIV infection and suggest that LXR agonists may be good candidates for development as anti-HIV agents, in particular for pre-exposure prophylaxis of HIV infection.
Assuntos
Fármacos Anti-HIV/farmacologia , Infecções por HIV/tratamento farmacológico , HIV/efeitos dos fármacos , Hidrocarbonetos Fluorados/farmacologia , Receptores Nucleares Órfãos/agonistas , Receptores Nucleares Órfãos/metabolismo , Sulfonamidas/farmacologia , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Células HEK293 , Infecções por HIV/metabolismo , Infecções por HIV/virologia , Humanos , Leucócitos/efeitos dos fármacos , Leucócitos/virologia , Receptores X do Fígado , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Camundongos , Camundongos Endogâmicos NOD , Regulação para Cima/efeitos dos fármacos , Carga Viral/métodosRESUMO
Extracellular vesicles (EVs) serve as pivotal mediators of intercellular communication in both health and disease, delivering biologically active molecules from vesicle-producing cells to recipient cells. In the context of HIV infection, EVs have been shown to carry the viral protein Nef, a key pathogenic factor associated with HIV-related co-morbidities. Despite this recognition, the specific localisation of Nef within the vesicles has remained elusive. This study addresses this critical knowledge gap by investigating Nef-containing EVs. Less than 1% of the total released Nef was associated with EVs; most Nef existed as free protein released by damaged cells. Nevertheless, activity of EV-associated Nef in downregulating the major cholesterol transporter ABCA1, a critical aspect linked to the pathogenic effects of Nef, was comparable to that of free Nef present in the supernatant. Through a series of biochemical and microscopic assays, we demonstrate that the majority of EV-associated Nef molecules are localised on the external surface of the vesicles. This distinctive distribution prompts the consideration of Nef-containing EVs as potential targets for immunotherapeutic interventions aimed at preventing or treating HIV-associated co-morbidities. In conclusion, our results shed light on the localisation and functional activity of Nef within EVs, providing valuable insights for the development of targeted immunotherapies to mitigate the impact of HIV-associated co-morbidities.
Assuntos
Vesículas Extracelulares , Infecções por HIV , HIV-1 , Produtos do Gene nef do Vírus da Imunodeficiência Humana , Vesículas Extracelulares/metabolismo , Humanos , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , HIV-1/metabolismo , Infecções por HIV/metabolismo , Transportador 1 de Cassete de Ligação de ATP/metabolismoRESUMO
HIV infection, through the actions of viral accessory protein Nef, impairs activity of cholesterol transporter ABCA1, inhibiting cholesterol efflux from macrophages and elevating the risk of atherosclerosis. Nef also induces lipid raft formation. In this study, we demonstrate that these activities are tightly linked and affect macrophage function and HIV replication. Nef stimulated lipid raft formation in macrophage cell line RAW 264.7, and lipid rafts were also mobilized in HIV-1-infected human monocyte-derived macrophages. Nef-mediated transfer of cholesterol to lipid rafts competed with the ABCA1-dependent pathway of cholesterol efflux, and pharmacological inhibition of ABCA1 functionality or suppression of ABCA1 expression by RNAi increased Nef-dependent delivery of cholesterol to lipid rafts. Nef reduced cell-surface accessibility of ABCA1 and induced ABCA1 catabolism via the lysosomal pathway. Despite increasing the abundance of lipid rafts, expression of Nef impaired phagocytic functions of macrophages. The infectivity of the virus produced in natural target cells of HIV-1 negatively correlated with the level of ABCA1. These findings demonstrate that Nef-dependent inhibition of ABCA1 is an essential component of the viral replication strategy and underscore the role of ABCA1 as an innate anti-HIV factor.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Colesterol/metabolismo , HIV-1/patogenicidade , Macrófagos/metabolismo , Microdomínios da Membrana/metabolismo , Produtos do Gene nef do Vírus da Imunodeficiência Humana/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Animais , Transporte Biológico , Cloreto de Cálcio/farmacologia , Cloroquina/farmacologia , Infecções por HIV/virologia , HIV-1/fisiologia , Células HeLa , Humanos , Hidrocarbonetos Fluorados/farmacologia , Lisossomos/metabolismo , Macrófagos/efeitos dos fármacos , Macrófagos/virologia , Camundongos , Estabilidade Proteica , Proteólise , Interferência de RNA , Sulfonamidas/farmacologia , Transfecção , Replicação Viral , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genéticaRESUMO
HIV-infected subjects are at high risk of developing atherosclerosis, in part due to virus-induced impairment of HDL metabolism. Here, using as a model of HIV infection the NOD.Cg-Prkdc(scid)IL2rg(tm1Wjl)/SzJ (NSG) mice humanized by human stem cell transplantation, we demonstrate that LXR agonist TO901317 potently reduces viral replication and prevents HIV-induced reduction of plasma HDL. These results establish that humanized mice can be used to investigate the mechanisms of HIV-induced impairment of HDL formation, a major feature of dyslipidemia associated with HIV-1 infection, and show potential benefits of developing LXR agonists for treatment of HIV-associated cardio-vascular disease.
Assuntos
Anticolesterolemiantes/farmacologia , Infecções por HIV/sangue , HIV-1/efeitos dos fármacos , Hidrocarbonetos Fluorados/farmacologia , Lipoproteínas HDL/sangue , Receptores Nucleares Órfãos/agonistas , Sulfonamidas/farmacologia , Replicação Viral/efeitos dos fármacos , Animais , Modelos Animais de Doenças , Infecções por HIV/virologia , HIV-1/fisiologia , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Receptores X do Fígado , Camundongos , Transplante de Células-TroncoRESUMO
A possible explanation for chronic inflammation in HIV-infected individuals treated with anti-retroviral therapy is hyperreactivity of myeloid cells due to a phenomenon called "trained immunity." Here, we demonstrate that human monocyte-derived macrophages originating from monocytes initially treated with extracellular vesicles containing HIV-1 protein Nef (exNef), but differentiating in the absence of exNef, release increased levels of pro-inflammatory cytokines after lipopolysaccharide stimulation. This effect is associated with chromatin changes at the genes involved in inflammation and cholesterol metabolism pathways and upregulation of the lipid rafts and is blocked by methyl-ß-cyclodextrin, statin, and an inhibitor of the lipid raft-associated receptor IGF1R. Bone-marrow-derived macrophages from exNef-injected mice, as well as from mice transplanted with bone marrow from exNef-injected animals, produce elevated levels of tumor necrosis factor α (TNF-α) upon stimulation. These phenomena are consistent with exNef-induced trained immunity that may contribute to persistent inflammation and associated co-morbidities in HIV-infected individuals with undetectable HIV load.
Assuntos
Vesículas Extracelulares , Infecções por HIV , Soropositividade para HIV , HIV-1 , Humanos , Camundongos , Animais , HIV-1/genética , Produtos do Gene nef do Vírus da Imunodeficiência Humana/genética , Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Inflamação/metabolismoRESUMO
Cholesterol plays an important role in the HIV life cycle, and infectivity of cholesterol-depleted HIV virions is significantly impaired. Recently, we demonstrated that HIV-1, via its protein Nef, inhibits the activity of the major cellular cholesterol transporter ATP binding cassette transporter A1 (ABCA1), suggesting that the virus may use this mechanism to get access to cellular cholesterol. In this study, we investigated the effect on HIV infection of a synthetic liver X receptor (LXR) ligand, N-(2,2,2-trifluoro-ethyl)-N-[4-(2,2,2-trifluoro-1-hydroxy-1-trifluoromethyl-ethyl)-phenyl]-benzenesulfonamide (TO-901317), which is a potent stimulator of ABCA1 expression. We demonstrate that TO-901317 restores cholesterol efflux from HIV-infected T lymphocytes and macrophages. TO-901317 potently suppressed HIV-1 replication in both cell types and inhibited HIV-1 replication in ex vivo cultured lymphoid tissue and in RAG-hu mice infected in vivo. This anti-HIV activity was dependent on ABCA1, because the effect of the drug was significantly reduced in ABCA1-defective T cells from a patient with Tangier disease, and RNA interference-mediated inhibition of ABCA1 expression eliminated the effect of TO-901317 on HIV-1 replication. TO-901317-mediated inhibition of HIV replication was due to reduced virus production and reduced infectivity of produced virions. The infectivity defect was in part due to reduced fusion activity of the virions, which was directly linked to reduced viral cholesterol. These results describe a novel approach to inhibiting HIV infection by stimulating ABCA1 expression.
Assuntos
Transportadores de Cassetes de Ligação de ATP/biossíntese , HIV-1/fisiologia , Receptores Nucleares Órfãos/agonistas , Replicação Viral , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Animais Recém-Nascidos , Transporte Biológico , Colesterol/metabolismo , Humanos , Hidrocarbonetos Fluorados/farmacologia , Receptores X do Fígado , Camundongos , Receptores Nucleares Órfãos/metabolismo , Interferência de RNA , Sulfonamidas/farmacologiaRESUMO
Apolipoprotein A-I binding protein (AIBP) is a protein involved in regulation of lipid rafts and cholesterol efflux. AIBP has been suggested to function as a protective factor under several sets of pathological conditions associated with increased abundance of lipid rafts, such as atherosclerosis and acute lung injury. Here, we show that exogenously added AIBP reduced the abundance of lipid rafts and inhibited HIV replication in vitro as well as in HIV-infected humanized mice, whereas knockdown of endogenous AIBP increased HIV replication. Endogenous AIBP was much more abundant in activated T cells than in monocyte-derived macrophages (MDMs), and exogenous AIBP was much less effective in T cells than in MDMs. AIBP inhibited virus-cell fusion, specifically targeting cells with lipid rafts mobilized by cell activation or Nef-containing exosomes. MDM-HIV fusion was sensitive to AIBP only in the presence of Nef provided by the virus or exosomes. Peripheral blood mononuclear cells from donors with the HLA-B*35 genotype, associated with rapid progression of HIV disease, bound less AIBP than cells from donors with other HLA genotypes and were not protected by AIBP from rapid HIV-1 replication. These results provide the first evidence for the role of Nef exosomes in regulating HIV-cell fusion by modifying lipid rafts and suggest that AIBP is an innate factor that restricts HIV replication by targeting lipid rafts.IMPORTANCE Apolipoprotein A-I binding protein (AIBP) is a recently identified innate anti-inflammatory factor. Here, we show that AIBP inhibited HIV replication by targeting lipid rafts and reducing virus-cell fusion. Importantly, AIBP selectively reduced levels of rafts on cells stimulated by an inflammatory stimulus or treated with extracellular vesicles containing HIV-1 protein Nef without affecting rafts on nonactivated cells. Accordingly, fusion of monocyte-derived macrophages with HIV was sensitive to AIBP only in the presence of Nef. Silencing of endogenous AIBP significantly upregulated HIV-1 replication. Interestingly, HIV-1 replication in cells from donors with the HLA-B*35 genotype, associated with rapid progression of HIV disease, was not inhibited by AIBP. These results suggest that AIBP is an innate anti-HIV factor that targets virus-cell fusion.
RESUMO
Despite reductions in mortality from the use of highly active antiretroviral therapy (HAART), the presence of latent or transcriptionally silent proviruses prevents HIV cure/eradication. We have previously reported that DNA-dependent protein kinase (DNA-PK) facilitates HIV transcription by interacting with the RNA polymerase II (RNAP II) complex recruited at HIV LTR. In this study, using different cell lines and peripheral blood mononuclear cells (PBMCs) of HIV-infected patients, we found that DNA-PK stimulates HIV transcription at several stages, including initiation, pause-release and elongation. We are reporting for the first time that DNA-PK increases phosphorylation of RNAP II C-terminal domain (CTD) at serine 5 (Ser5) and serine 2 (Ser2) by directly catalyzing phosphorylation and by augmenting the recruitment of the positive transcription elongation factor (P-TEFb) at HIV LTR. Our findings suggest that DNA-PK expedites the establishment of euchromatin structure at HIV LTR. DNA-PK inhibition/knockdown leads to the severe impairment of HIV replication and reactivation of latent HIV provirus. DNA-PK promotes the recruitment of Tripartite motif-containing 28 (TRIM28) at LTR and assists the release of paused RNAP II through TRIM28 phosphorylation. These results provide the mechanisms through which DNA-PK controls the HIV gene expression and, likely, can be extended to cellular gene expression, including during cell malignancy, where the role of DNA-PK has been well-established.
RESUMO
How HIV-1 affects the monocyte proteome is incompletely understood. We posit that one functional consequence of virus-exposure to the monocyte is the facilitation of protein transformation from the cytosol to the plasma membrane (PM). To test this, cell surface labeling with CyDye fluorophores followed by 2 dimensional differential in-gel electrophoresis (2D DIGE) and liquid chromatography tandem mass spectrometry (LC-MS/MS) was performed. Fifty three percent of HIV-1 induced proteins were PM associated. These were linked, in large measure, to cellular activation and oxidative stress. They included, but not limited to, biliverdin reductase, leukotriene hydrolase A(4), heat shock protein 70, and cystatin B. HIV-1 induced PM protein translocation was associated with cathepsin B- and caspase 9, 3-dependent apoptosis. In contrast, PMA-treated monocytes bypassed caspase 3, 9 pathways and lead to cathepsin B-dependent necrosis. These results demonstrate that HIV-1 uniquely affects monocyte activation and oxidative stress. These do not affect viral infection dynamics but are linked to stress-induced cell death.
Assuntos
Membrana Celular/metabolismo , Infecções por HIV/metabolismo , HIV-1/metabolismo , Monócitos/metabolismo , Proteoma/análise , Carcinógenos/farmacologia , Catepsina B/efeitos dos fármacos , Catepsina B/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/virologia , Sobrevivência Celular/fisiologia , Células Cultivadas , Epóxido Hidrolases/efeitos dos fármacos , Epóxido Hidrolases/metabolismo , Infecções por HIV/virologia , Proteínas de Choque Térmico HSP70/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/metabolismo , Humanos , Monócitos/efeitos dos fármacos , Monócitos/virologia , Oxirredução , Estresse Oxidativo/fisiologia , Acetato de Tetradecanoilforbol/farmacologia , Vitamina E/farmacologia , Vitaminas/farmacologiaRESUMO
Several steps of HIV-1 replication critically depend on cholesterol. HIV infection is associated with profound changes in lipid and lipoprotein metabolism and an increased risk of coronary artery disease. Whereas numerous studies have investigated the role of anti-HIV drugs in lipodystrophy and dyslipidemia, the effects of HIV infection on cellular cholesterol metabolism remain uncharacterized. Here, we demonstrate that HIV-1 impairs ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux from human macrophages, a condition previously shown to be highly atherogenic. In HIV-1-infected cells, this effect was mediated by Nef. Transfection of murine macrophages with Nef impaired cholesterol efflux from these cells. At least two mechanisms were found to be responsible for this phenomenon: first, HIV infection and transfection with Nef induced post-transcriptional down-regulation of ABCA1; and second, Nef caused redistribution of ABCA1 to the plasma membrane and inhibited internalization of apolipoprotein A-I. Binding of Nef to ABCA1 was required for down-regulation and redistribution of ABCA1. HIV-infected and Nef-transfected macrophages accumulated substantial amounts of lipids, thus resembling foam cells. The contribution of HIV-infected macrophages to the pathogenesis of atherosclerosis was supported by the presence of HIV-positive foam cells in atherosclerotic plaques of HIV-infected patients. Stimulation of cholesterol efflux from macrophages significantly reduced infectivity of the virions produced by these cells, and this effect correlated with a decreased amount of virion-associated cholesterol, suggesting that impairment of cholesterol efflux is essential to ensure proper cholesterol content in nascent HIV particles. These results reveal a previously unrecognized dysregulation of intracellular lipid metabolism in HIV-infected macrophages and identify Nef and ABCA1 as the key players responsible for this effect. Our findings have implications for pathogenesis of both HIV disease and atherosclerosis, because they reveal the role of cholesterol efflux impairment in HIV infectivity and suggest a possible mechanism by which HIV infection of macrophages may contribute to increased risk of atherosclerosis in HIV-infected patients.
Assuntos
Colesterol/metabolismo , HIV/patogenicidade , Macrófagos/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/genética , Transportadores de Cassetes de Ligação de ATP/metabolismo , Animais , Transporte Biológico , Linhagem Celular , Regulação para Baixo , Células Espumosas/metabolismo , Produtos do Gene nef/metabolismo , HIV/metabolismo , Infecções por HIV/patologia , Células HeLa , Humanos , Macrófagos/patologia , Camundongos , Dados de Sequência Molecular , Produtos do Gene nef do Vírus da Imunodeficiência HumanaRESUMO
HIV infection is known to be associated with cardiometabolic abnormalities; here we investigated the progression and causes of these abnormalities. Three groups of participants were recruited: HIV-negative subjects and two groups of treatment-naïve HIV-positive subjects, one group initiating antiretroviral treatment, the other remaining untreated. Intima-media thickness (cIMT) increased in HIV-positive untreated group compared to HIV-negative group, but treatment mitigated the difference. We found no increase in diabetes-related metabolic markers or in the level of inflammation in any of the groups. Total cholesterol, low density lipoprotein cholesterol and apoB levels were lower in HIV-positive groups, while triglyceride and Lp(a) levels did not differ between the groups. We found a statistically significant negative association between viral load and plasma levels of total cholesterol, LDL cholesterol, HDL cholesterol, apoA-I and apoB. HIV-positive patients had hypoalphalipoproteinemia at baseline, and we found a redistribution of sub-populations of high density lipoprotein (HDL) particles with increased proportion of smaller HDL in HIV-positive untreated patients, which may result from increased levels of plasma cholesteryl ester transfer protein in this group. HDL functionality declined in the HIV-negative and HIV-positive untreated groups, but not in HIV-positive treated group. We also found differences between HIV-positive and negative groups in plasma abundance of several microRNAs involved in lipid metabolism. Our data support a hypothesis that cardiometabolic abnormalities in HIV infection are caused by HIV and that antiretroviral treatment itself does not influence key cardiometabolic parameters, but mitigates those affected by HIV.
Assuntos
Antirretrovirais/administração & dosagem , Aterosclerose/sangue , Infecções por HIV/sangue , HIV-1 , Hipoalfalipoproteinemias/sangue , Lipídeos/sangue , Adulto , Aterosclerose/prevenção & controle , Feminino , Infecções por HIV/tratamento farmacológico , Humanos , Hipoalfalipoproteinemias/prevenção & controle , Masculino , MicroRNAs/sangue , Pessoa de Meia-Idade , Estudos ProspectivosRESUMO
It has been demonstrated that the p53 pathway plays an important role in HIV-1 infection. Previous work from our lab has established a model demonstrating how p53 could become inactivated in HIV-1 infected cells through binding to Tat. Subsequently, p53 was inactivated and lost its ability to transactivate its downstream target gene p21/waf1. P21/waf1 is a well-known cdk inhibitor (CKI) that can lead to cell cycle arrest upon DNA damage. Most recently, the p21/waf1 function was further investigated as a molecular barrier for HIV-1 infection of stem cells. Therefore, we reason that the restoration of the p53 and p21/waf1 pathways could be a possible theraputical arsenal for combating HIV-1 infection. In this current study, we show that a small chemical molecule, 9-aminoacridine (9AA) at low concentrations, could efficiently reactivate p53 pathway and thereby restoring the p21/waf1 function. Further, we show that the 9AA could significantly inhibit virus replication in activated PBMCs, likely through a mechanism of inhibiting the viral replication machinery. A mechanism study reveals that the phosphorylated p53ser15 may be dissociated from binding to HIV-1 Tat protein, thereby activating the p21/waf1 gene. Finally, we also show that the 9AA-activated p21/waf1 is recruited to HIV-1 preintegration complex, through a mechanism yet to be elucidated.
Assuntos
Aminacrina/farmacologia , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , HIV-1/efeitos dos fármacos , Antivirais/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , HIV-1/fisiologia , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/virologia , Fosforilação , Transcrição Reversa/efeitos dos fármacos , Transdução de Sinais , Linfócitos T/efeitos dos fármacos , Linfócitos T/virologia , Proteína Supressora de Tumor p53/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismoRESUMO
BACKGROUND: A hallmark of atherosclerosis is its complex pathogenesis, which is dependent on altered cholesterol metabolism and inflammation. Both arms of pathogenesis involve myeloid cells. Monocytes migrating into the arterial walls interact with modified low-density lipoprotein (LDL) particles, accumulate cholesterol and convert into foam cells, which promote plaque formation and also contribute to inflammation by producing proinflammatory cytokines. A number of studies characterized transcriptomics of macrophages following interaction with modified LDL, and revealed alteration of the expression of genes responsible for inflammatory response and cholesterol metabolism. However, it is still unclear how these two processes are related to each other to contribute to atherosclerotic lesion formation. METHODS: We attempted to identify the main mater regulator genes in macrophages treated with atherogenic modified LDL using a bioinformatics approach. RESULTS: We found that most of the identified genes were involved in inflammation, and none of them was implicated in cholesterol metabolism. Among the key identified genes were interleukin (IL)-7, IL-7 receptor, IL- 15 and CXCL8. CONCLUSION: Our results indicate that activation of the inflammatory pathway is the primary response of the immune cells to modified LDL, while the lipid metabolism genes may be a secondary response triggered by inflammatory signalling.
Assuntos
Perfilação da Expressão Gênica , Inflamação/genética , Inflamação/metabolismo , Lipoproteínas LDL/genética , Lipoproteínas LDL/metabolismo , Macrófagos/metabolismo , Macrófagos/patologia , Voluntários Saudáveis , Humanos , Lipoproteínas LDL/químicaRESUMO
In this mini-review, the role of macrophage phenotypes in atherogenesis is considered. Recent studies on distribution of M1 and M2 macrophages in different types of atherosclerotic lesions indicate that macrophages exhibit a high degree of plasticity of phenotype in response to various conditions in microenvironment. The effect of the accumulation of cholesterol, a key event in atherogenesis, on the macrophage phenotype is also discussed. The article presents the results of transcriptome analysis of cholesterol-loaded macrophages revealing genes involved in immune response whose expression rate has changed the most. It turned out that the interaction of macrophages with modified LDL leads to higher expression levels of pro-inflammatory marker TNF-α and antiinflammatory marker CCL18. Phenotypic profile of macrophage activation could be a good target for testing of novel anti-atherogenic immunocorrectors. A number of anti-atherogenic drugs were tested as potential immunocorrectors using primary macrophage-based model.