RESUMEN
HIV-associated neurocognitive disorders (HAND) are a spectrum of cognitive impairments that continue to affect approximately half of all HIV-positive individuals despite effective viral suppression through antiretroviral therapy (ART). White matter pathologies have persisted in the ART era, and the degree of white matter damage correlates with the degree of neurocognitive impairment in patients with HAND. The HIV protein Nef has been implicated in HAND pathogenesis, but its effect on white matter damage has not been well characterized. Here, utilizing in vivo, ex vivo, and in vitro methods, we demonstrate that Nef-containing extracellular vesicles (Nef EVs) disrupt myelin sheaths and inflict damage upon oligodendrocytes within the murine central nervous system. Intracranial injection of Nef EVs leads to reduced myelin basic protein (MBP) staining and a decreased number of CC1 + oligodendrocytes in the corpus callosum. Moreover, cerebellar slice cultures treated with Nef EVs exhibit diminished MBP expression and increased presence of unmyelinated axons. Primary mixed brain cultures and enriched oligodendrocyte precursor cell cultures exposed to Nef EVs display a decreased number of O4 + cells, indicative of oligodendrocyte impairment. These findings underscore the potential contribution of Nef EV-mediated damage to oligodendrocytes and myelin maintenance in the pathogenesis of HAND.
Asunto(s)
Vesículas Extracelulares , VIH-1 , Oligodendroglía , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Animales , Ratones , Células Cultivadas , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Sistema Nervioso Central/virología , Vesículas Extracelulares/metabolismo , VIH-1/metabolismo , Ratones Endogámicos C57BL , Vaina de Mielina/metabolismo , Vaina de Mielina/patología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Oligodendroglía/metabolismo , Oligodendroglía/patología , Oligodendroglía/virologíaRESUMEN
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.
Asunto(s)
Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismo , Animales , Efecto Espectador , Colesterol/metabolismo , Exosomas/metabolismo , Exosomas/virología , Células HEK293 , VIH-1 , Humanos , Inflamación/metabolismo , Inflamación/virología , Microdominios de Membrana/metabolismo , Microdominios de Membrana/virología , Ratones , Ratones Endogámicos C57BL , Modelos Biológicos , Células RAW 264.7 , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptor Toll-Like 4/metabolismo , Receptor Activador Expresado en Células Mieloides 1/metabolismo , Proteína de Unión al GTP cdc42/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
BACKGROUND: Endothelial progenitor cells (EPCs) has been shown to be dysfunctional in both type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD) leading to poor regeneration of endothelium and renal perfusion. EPCs have been shown to be a robust cardiovascular disease (CVD) risk indicator. Effect of sodium glucose channel inhibitors (SGLT2i) such as Canagliflozin (CG) on a cellular biomarker such as CD34+ve progenitor cells, which may help predict CVD risk, in patients with T2DM with established CKD has not been explored. METHODS: This is a pilot study where 29 subjects taking metformin and/or Insulin were enrolled in a 16 week, double blind, randomized placebo matched trial, with a low dose 100 mg CG as the intervention group compared to matched placebo. Type 2 diabetes subjects (30-70 years old), with hemoglobin A1c (HbA1c) of 7-10%, were enrolled. CD34+ve cell number, migratory function, gene expression along with vascular parameters such as arterial stiffness, serum biochemistry pertaining to cardio-metabolic health, resting energy expenditure and body composition were measured. Data were collected at week 0, 8 and 16. A mixed model regression analysis was done and p value less than 0.05 was considered statistically significant. RESULTS: A significant expression of CXCR4 receptor with a concomittant increase in migratory function of CD34+ve cells was observed in CG treated group as compared to placebo group. Gene expression analysis of CD34+ve cells showed an increase in expression of antioxidants (superoxide dismutase 2 or SOD2, Catalase and Glutathione Peroxidase or GPX) and notable endothelial markers (PECAM1, VEGF-A, and NOS3). A significant reduction in glucose and HbA1c levels were observed along with improved systolic and diastolic blood pressure in the CG group. A significant increase in adiponectin (p = 0.006) was also noted in treatment group. Urinary exosomal protein leak in urine, examining podocyte health (podocalyxin, Wilm's tumor and nephrin) showed reduction with CG CONCLUSION: Low dose Canagliflozin has a beneficial effect on CD34+ cell function, serum biochemistry and urinary podocyte specific exosomes in type 2 diabetes.
Asunto(s)
Antígenos CD34/metabolismo , Glucemia/efectos de los fármacos , Canagliflozina/uso terapéutico , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Células Progenitoras Endoteliales/efectos de los fármacos , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Adulto , Anciano , Biomarcadores/sangre , Glucemia/metabolismo , Canagliflozina/efectos adversos , Células Cultivadas , Quimiotaxis/efectos de los fármacos , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/patología , Diabetes Mellitus Tipo 2/fisiopatología , Método Doble Ciego , Quimioterapia Combinada , Células Progenitoras Endoteliales/metabolismo , Células Progenitoras Endoteliales/patología , Exosomas/efectos de los fármacos , Exosomas/metabolismo , Femenino , Hemoglobina Glucada/metabolismo , Humanos , Insulina/uso terapéutico , Masculino , Metformina/uso terapéutico , Persona de Mediana Edad , Fenotipo , Proyectos Piloto , Inhibidores del Cotransportador de Sodio-Glucosa 2/efectos adversos , Factores de Tiempo , Resultado del TratamientoRESUMEN
BACKGROUND: Endothelial Progenitor cells (EPCs) has been shown to be dysfunctional in both type 2 diabetes mellitus (T2DM) and chronic kidney disease (CKD) leading to poor regeneration of endothelium and renal perfusion. EPCs have been shown to be a robust cardiovascular disease (CVD) risk indicator. Cellular mechanisms of DPP4 inhibitors such as linagliptin (LG) on CVD risk, in patients with T2DM with established CKD has not been established. Linagliptin, a DPP4 inhibitor when added to insulin, metformin or both may improve endothelial dysfunction in a diabetic kidney disease (DKD) population. METHODS: 31 subjects taking metformin and/or Insulin were enrolled in this 12 weeks, double blind, randomized placebo matched trial, with 5 mg LG compared to placebo. Type 2 diabetes subjects (30-70 years old), HbA1c of 6.5-10%, CKD Stage 1-3 were included. CD34+ cell number, migratory function, gene expression along with vascular parameters such as arterial stiffness, biochemistry, resting energy expenditure and body composition were measured. Data were collected at week 0, 6 and 12. A mixed model regression analysis was done with p value < 0.05 considered significant. RESULTS: A double positive CD34/CD184 cell count had a statistically significant increase (p < 0.02) as determined by flow cytometry in LG group where CD184 is SDF1a cell surface receptor. Though mRNA differences in CD34+ve was more pronounced CD34- cell mRNA analysis showed increase in antioxidants (superoxide dismutase 2 or SOD2, Catalase and Glutathione Peroxidase or GPX) and prominent endothelial markers (PECAM1, VEGF-A, vWF and NOS3). Arterial stiffness measures such as augmentation Index (AI) (p < 0.04) and pulse wave analysis (PWV) were improved (reduced in stiffness) in LG group. A reduction in LDL: HDL ratio was noted in treatment group (p < 0.04). Urinary exosome protein examining podocyte health (podocalyxin, Wilms tumor and nephrin) showed reduction or improvement. CONCLUSIONS: In DKD subjects, Linagliptin promotes an increase in CXCR4 expression on CD34 + progenitor cells with a concomitant improvement in vascular and renal parameters at 12 weeks. Trial Registration Number NCT02467478 Date of Registration: 06/08/2015.
Asunto(s)
Antígenos CD34/sangre , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Nefropatías Diabéticas/tratamiento farmacológico , Inhibidores de la Dipeptidil-Peptidasa IV/uso terapéutico , Células Progenitoras Endoteliales/efectos de los fármacos , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Linagliptina/uso terapéutico , Metformina/uso terapéutico , Insuficiencia Renal Crónica/tratamiento farmacológico , Adulto , Anciano , Biomarcadores/sangre , Células Cultivadas , Diabetes Mellitus Tipo 2/sangre , Diabetes Mellitus Tipo 2/diagnóstico , Nefropatías Diabéticas/sangre , Nefropatías Diabéticas/diagnóstico , Inhibidores de la Dipeptidil-Peptidasa IV/efectos adversos , District of Columbia , Método Doble Ciego , Quimioterapia Combinada , Células Progenitoras Endoteliales/metabolismo , Células Progenitoras Endoteliales/patología , Femenino , Humanos , Hipoglucemiantes/efectos adversos , Insulina/efectos adversos , Linagliptina/efectos adversos , Masculino , Metformina/efectos adversos , Persona de Mediana Edad , Proyectos Piloto , Receptores CXCR4/sangre , Insuficiencia Renal Crónica/sangre , Insuficiencia Renal Crónica/diagnóstico , Factores de Tiempo , Resultado del TratamientoRESUMEN
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.
Asunto(s)
Antraquinonas/farmacología , Aterosclerosis/prevención & control , Calnexina/metabolismo , Colesterol/metabolismo , Diseño de Fármacos , Infecciones por VIH/tratamiento farmacológico , Hipolipemiantes/farmacología , Simulación del Acoplamiento Molecular , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismo , Antraquinonas/química , Aterosclerosis/metabolismo , Aterosclerosis/virología , Transporte Biológico , Calnexina/química , Calnexina/genética , Diseño Asistido por Computadora , Células Espumosas/efectos de los fármacos , Células Espumosas/metabolismo , Células HEK293 , Infecciones por VIH/metabolismo , Infecciones por VIH/virología , Humanos , Hipolipemiantes/química , Lisina , Mutación , Unión Proteica , Dominios y Motivos de Interacción de Proteínas , Relación Estructura-Actividad , Transfección , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/química , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
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.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Calnexina/metabolismo , Retículo Endoplásmico/metabolismo , Proteínas gp160 de Envoltorio del VIH/metabolismo , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Aterosclerosis/metabolismo , Colesterol/metabolismo , Glicosilación , Células HEK293 , VIH-1/metabolismo , Células HeLa , Humanos , Unión Proteica , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masas en TándemRESUMEN
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.
Asunto(s)
Vesículas Extracelulares , Infecciones por VIH , VIH-1 , Productos del Gen nef del Virus de la Inmunodeficiencia Humana , Vesículas Extracelulares/metabolismo , Humanos , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , VIH-1/metabolismo , Infecciones por VIH/metabolismo , Transportador 1 de Casete de Unión a ATP/metabolismoRESUMEN
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.
Asunto(s)
Vesículas Extracelulares , Infecciones por VIH , Seropositividad para VIH , VIH-1 , Humanos , Ratones , Animales , VIH-1/genética , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genética , Vesículas Extracelulares/metabolismo , Macrófagos/metabolismo , Inflamación/metabolismoRESUMEN
BACKGROUND: Human immunodeficiency virus type 1 (HIV-1) and herpes simplex virus type 2 (HSV-2) are responsible for 2 intersecting epidemics in which the disease caused by 1 virus facilitates the transmission of and pathogenesis by the other. Therefore, suppression of one virus infection will affect the other. Acyclovir, a common antiherpetic drug, was shown to directly suppress both viruses in coinfected tissues. However, both antiviral activities of acyclovir are dependent on phosphorylation by the nucleoside kinase activity of coinfecting human herpesviruses. METHODS: We developed acyclovir ProTides, monophosphorylated acyclovir with the phosphate group masked by lipophilic groups to allow efficient cellular uptake, and investigated their antiviral potential in cell lines and in human tissues ex vivo. RESULTS: Acyclovir ProTides suppressed both HIV-1 and HSV-2 at median effective concentrations in the submicromolar range in ex vivo lymphoid and cervicovaginal human tissues and at 3-12 micromol/L in CD4(+) T cells. Acyclovir ProTides retained activity against acyclovir-resistant HSV-2. CONCLUSIONS: Acyclovir ProTides represent a new class of antivirals that suppress both HIV-1 and HSV-2 by directly and independently blocking the key replicative enzymes of both viruses. Further optimization of such compounds may lead to double-targeted antivirals that can prevent viral transmission and treat the 2 synergistic diseases caused by HIV-1 and HSV-2. To our knowledge, the acyclovir ProTides described here represent the first example of acyclic nucleoside monophosphate prodrugs being active against HIV-1.
Asunto(s)
Aciclovir/análogos & derivados , Antivirales/farmacología , VIH-1/efectos de los fármacos , Herpesvirus Humano 2/efectos de los fármacos , Profármacos/farmacología , Aciclovir/farmacología , Línea Celular , Cuello del Útero/virología , Femenino , Infecciones por VIH/tratamiento farmacológico , Herpes Genital/tratamiento farmacológico , Histocitoquímica , Humanos , Tonsila Palatina/virología , Linfocitos T/virología , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND: At present, the relatively sudden appearance and explosive spread of HIV throughout Africa and around the world beginning in the 1950s has never been adequately explained. Theorizing that this phenomenon may be somehow related to the eradication of smallpox followed by the cessation of vaccinia immunization, we undertook a comparison of HIV-1 susceptibility in the peripheral blood mononuclear cells from subjects immunized with the vaccinia virus to those from vaccinia naive donors. RESULTS: Vaccinia immunization in the preceding 3-6 months resulted in an up to 5-fold reduction in CCR5-tropic but not in CXCR4-tropic HIV-1 replication in the cells from vaccinated subjects. The addition of autologous serum to the cell cultures resulted in enhanced R5 HIV-1 replication in the cells from unvaccinated, but not vaccinated subjects. There were no significant differences in the concentrations of MIP-1alpha, MIP-1beta and RANTES between the cell cultures derived from vaccinated and unvaccinated subjects when measured in culture medium on days 2 and 5 following R5 HIV-1 challenge. DISCUSSION: Since primary HIV-1 infections are caused almost exclusively by the CCR5-tropic HIV-1 strains, our results suggest that prior immunization with vaccinia virus might provide an individual with some degree of protection to subsequent HIV infection and/or progression. The duration of such protection remains to be determined. A differential elaboration of MIP-1alpha, MIP-1beta and RANTES between vaccinated and unvaccinated subjects, following infection, does not appear to be a mechanism in the noted protection.
Asunto(s)
Infecciones por VIH/inmunología , VIH-1/fisiología , Receptores CCR5/inmunología , Virus Vaccinia/inmunología , Vacunas Virales/inmunología , Adulto , Quimiocina CCL3/biosíntesis , Quimiocina CCL3/inmunología , Quimiocina CCL4/biosíntesis , Quimiocina CCL4/inmunología , Quimiocina CCL5/biosíntesis , Quimiocina CCL5/inmunología , Ensayo de Inmunoadsorción Enzimática , Femenino , Infecciones por VIH/metabolismo , Humanos , Masculino , Receptores CCR5/metabolismo , Receptores CXCR4/inmunología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Replicación Viral/inmunología , Adulto JovenRESUMEN
All primate lentiviruses (HIV-1, HIV-2, SIV) encode Nef proteins, which are important for viral replication and pathogenicity in vivo. It is not known how Nef regulates these processes. It has been suggested that Nef protects infected cells from apoptosis and recognition by cytotoxic T lymphocytes. Other studies suggest that Nef influences the activation state of the infected cell, thereby enhancing the ability of that cell to support viral replication. Here we show that macrophages that express Nef or are stimulated through the CD40 receptor release a paracrine factor that renders T lymphocytes permissive to HIV-1 infection. This activity requires the upregulation of B-cell receptors involved in the alternative pathway of T-lymphocyte stimulation. T lymphocytes stimulated through this pathway become susceptible to viral infection without progressing through the cell cycle. We identify two proteins, soluble CD23 and soluble ICAM, that are induced from macrophages by Nef and CD40L, and which mediate their effects on lymphocyte permissivity. Our results reveal a mechanism by which Nef expands the cellular reservoir of HIV-1 by permitting the infection of resting T lymphocytes.
Asunto(s)
Ligando de CD40/metabolismo , Productos del Gen nef/fisiología , VIH-1/fisiología , Macrófagos/virología , Linfocitos T/virología , Linfocitos B/inmunología , Antígenos CD2/metabolismo , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/virología , Antígenos CD40/metabolismo , Comunicación Celular , Células Cultivadas , Humanos , Molécula 1 de Adhesión Intercelular/metabolismo , Antígenos Comunes de Leucocito/metabolismo , Activación de Linfocitos , Macrófagos/metabolismo , Receptores de IgE/metabolismo , Transducción de Señal , Linfocitos T/inmunología , Latencia del Virus , Replicación Viral , Productos del Gen nef del Virus de la Inmunodeficiencia HumanaRESUMEN
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.
RESUMEN
Nucleic acid measurements are used to follow HIV-1 viral load in clinical applications while p24 ELISA is commonly used to monitor HIV-1 replication in research settings. Current ELISA assays are expensive and offer a narrow dynamic measurement range. This report describes a simple, sensitive and inexpensive bead-based assay offering a wide dynamic measurement range. This cytometric bead assay allows the detection of p24 concentrations over 4 orders of magnitude from less than 0.4pg to up to 20,000pgml(-1) in a volume of 50microl and can be combined with other measurements.
Asunto(s)
Citometría de Flujo/métodos , Proteína p24 del Núcleo del VIH/análisis , VIH-1/química , Técnica del Anticuerpo Fluorescente Directa/métodos , Humanos , Microesferas , Sensibilidad y EspecificidadRESUMEN
Time-resolved confocal microscopy and fluorescence correlation spectroscopy were used to examine the movements of fluorescently labeled HIV-virions (approximately 100 nm) added to samples of human cervical mucus. Particle-tracking analysis indicates that the motion of most virions is decreased 200-fold compared to that in aqueous solution and is not driven by typical diffusion. Rather, the time-dependence of their ensemble-averaged mean-square displacements is proportional to tau(alpha) + v(2)tau(2), describing a combination of anomalous diffusion (alpha approximately 0.3) and flow-like behavior, with tau being the lag time. We attribute the flow-like behavior to slowly relaxing mucus matrix that follows mechanical perturbations such as stretching and twisting of the sample. Further analysis of the tracks and displacements of individual virions indicates differences in the local movements among the virions, including constrained motion and infrequent jumps, perhaps due to abrupt changes in matrix structure. Changes in the microenvironments due to slow structural changes may facilitate movement of the virions, allowing them to reach the epithelial layer.
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Moco del Cuello Uterino/virología , VIH/fisiología , Virión/fisiología , Fenómenos Biomecánicos , Difusión , Infecciones por VIH/virología , Humanos , Cinética , Movimiento (Física) , ReologíaRESUMEN
HIV-infected individuals are at high risk of developing atherosclerosis and cardiovascular disease, in part, due to HIV-induced impairment of cholesterol metabolism. In vitro studies demonstrated that HIV-1 protein Nef inhibits activity of ABCA1, the main cellular cholesterol transporter, leading to cholesterol accumulation in macrophages and conversion of these cells into foam cells, characteristic for atherosclerosis. However, the mechanisms of Nef-mediated effects on cholesterol metabolism in vivo are not well characterized. In this study, we generated Nef-transgenic mice and evaluated the accumulation of neutral lipids in liver and aorta of these animals. Nef expression was low in all transgenic mice, with some mice carrying the Nef transgene, but not expressing the Nef RNA. Using Oil Red O staining, we demonstrated increased levels of neutral lipids in liver and aorta of mice expressing Nef relative to transgenic animals, with no detectable Nef expression or control wild-type mice. These results provide direct evidence that Nef promotes cholesterol deposition in tissues.
Asunto(s)
Aorta/patología , Lípidos/análisis , Hígado/patología , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/biosíntesis , Animales , Histocitoquímica , Ratones Endogámicos C57BL , Ratones Transgénicos , Coloración y Etiquetado , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
BACKGROUND: Members of the United States Armed Forces receive a series of vaccinations during their course of service. To investigate the influence of multiple vaccinations on innate immunity, we measured concentrations of a panel of immunomodulatory and pro-inflammatory cytokines in serum samples from a group of such individuals. RESULTS: Significantly increased levels of macrophage inflammatory protein 1alpha (MIP-1alpha), MIP-1beta and interleukin 8 (IL-8) were detected. Since these cytokines are known to have anti-human immunodeficiency virus (HIV) activity, we tested the effect of serum from these individuals on HIV-1 infectivity and susceptibility of their peripheral blood mononuclear cells (PBMCs) to HIV-1 infection in vitro. Sera from vaccinated military personnel inhibited, and their PBMCs were partially resistant to, infection by HIV-1 strains tropic to CCR5 (R5), but not to CXCR4 (X4), chemokine receptor. CONCLUSION: These findings demonstrate that increased anti-HIV chemokines can be detected in vaccine recipients up to 68 weeks following immunization.
Asunto(s)
Quimiocinas/sangre , Personal Militar , Vacunación , Adulto , Quimiocina CCL3 , Quimiocina CCL4 , Susceptibilidad a Enfermedades , VIH-1/fisiología , Humanos , Interleucina-8/sangre , Leucocitos Mononucleares/virología , Proteínas Inflamatorias de Macrófagos/sangre , Masculino , Receptores CCR5/fisiología , Receptores CXCR4/fisiología , Estados UnidosRESUMEN
Cholesterol is an essential component of the cellular membranes and, by extension, of the HIV envelope membrane, which is derived from the host cell plasma membrane. Depletion of the cellular cholesterol has an inhibitory effect on HIV assembly, reduces infectivity of the produced virions, and makes the cell less susceptible to HIV infection. It is not surprising that the virus has evolved to gain access to cellular proteins regulating cholesterol metabolism. One of the key mechanisms used by HIV to maintain high levels of cholesterol in infected cells is Nef-mediated inhibition of cholesterol efflux and the cholesterol transporter responsible for this process, ABCA1. In this chapter, we describe methods to investigate these effects of HIV-1 infection.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/metabolismo , Colesterol/metabolismo , Infecciones por VIH/metabolismo , VIH-1/fisiología , Microscopía Confocal/métodos , Productos del Gen nef del Virus de la Inmunodeficiencia Humana/metabolismo , Transporte Biológico , Línea Celular , Humanos , Procesamiento de Imagen Asistido por Computador/métodosRESUMEN
The post-entry events of HIV-1 infection occur within reverse transcription complexes derived from the viral cores entering the target cell. HIV-1 cores contain host proteins incorporated from virus-producing cells. In this report, we show that MCM5, a subunit of the hexameric minichromosome maintenance (MCM) DNA helicase complex, associates with Gag polyprotein and is incorporated into HIV-1 virions. The progeny virions depleted of MCM5 demonstrated reduced reverse transcription in newly infected cells, but integration and subsequent replication steps were not affected. Interestingly, increased packaging of MCM5 into the virions also led to reduced reverse transcription, but here viral replication was impaired. Our data suggest that incorporation of physiological amounts of MCM5 promotes aberrant reverse transcription, leading to partial incapacitation of cDNA, whereas increased MCM5 abundance leads to reduced reverse transcription and infection. Therefore, MCM5 has the properties of an inhibitory factor that interferes with production of an integration-competent cDNA product.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , VIH-1/fisiología , Virión , Replicación Viral , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD4-Positivos/virología , Línea Celular , Células Cultivadas , Infecciones por VIH/virología , Humanos , Macrófagos/metabolismo , Macrófagos/virología , Complejos Multiproteicos/metabolismo , Unión Proteica , Transporte de Proteínas , Productos del Gen gag del Virus de la Inmunodeficiencia Humana/metabolismoRESUMEN
Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across the cervicovaginal mucosa in women is influenced by many factors including the microbiota and the presence of underlying inflammation. It is important that potential HIV preventative agents do not alter the mucosal environment in a way that enhances HIV acquisition. We examined the impact of a "live" microbicide on the vaginal mucosal environment in a rhesus macaque repeated vaginal simian-HIV (SHIVSF162P3) challenge model. The microbicide contained a human vaginal Lactobacillus jensenii expressing the HIV-1 entry inhibitor, modified Cyanovirin-N (mCV-N), and henceforth called LB-mCV-N. Macaques were colonized vaginally each week with LB-mCV-N and sampled six days after colonization for culturable bacteria, pH and cervical-vaginal cytokines during the duration of the six-week study. We show that macaques that retained the engineered LB-mCV-N strain in their vaginal microbiota, during SHIV challenge, had lower pH, when colonization levels were higher, and had no evidence of inflammatory cytokines. Indeed, Interleukin-13, a mediator of inflammation, was detected less often in LB-mCV-N colonized macaques than in controls and we found higher levels of Interleukin 1 receptor antagonist (IL-1RA) in LB-mCV-N colonized macaques during the SHIV challenge period. We noted an inverse correlation between levels of mucosal IL-1RA and peak plasma viral load, thus higher IL-1RA correlated with lower viral load in LB-mCV-N treated macaques. These data support the use of LB-mCV-N as a safe "live" microbicide and suggest that lactobacilli themselves may positively impact the mucosal environment.