RESUMEN
In the human genome, the APOBEC3 gene has expanded into a tandem array of genes termed APOBEC3A-H. Several members of this family have potent anti-HIV-1 activity. Here we demonstrate that APOBEC-3B/3C/3F and -3G are expressed in all major cellular components of the CNS. Moreover, we show that both interferon-alpha (IFN-alpha) and IFN-gamma significantly enhance the expression of APOBEC-3G/3F and drastically inhibit HIV-1 replication in primary human brain microvascular endothelial cells (BMVECs), the major component of blood-brain barrier (BBB). As the viral inhibition can be neutralized by APOBEC3G-specific siRNA, APOBEC3G plays a key role to mediate the anti-HIV-1 activity of IFN-alpha and/or IFN-gamma. Our findings suggest that, in addition to the restriction at viral entry level, the restriction from APOBEC3 family could account for the low-level replication of HIV-1 in BMVECs. The manipulation of IFN-APOBEC3 signaling pathway could be a potent therapeutic strategy to prevent HIV invasion to central nervous system (CNS).
Asunto(s)
Barrera Hematoencefálica/metabolismo , Sistema Nervioso Central/virología , Citidina Desaminasa/fisiología , Infecciones por VIH/prevención & control , VIH-1/efectos de los fármacos , Inmunidad/fisiología , Interferones/farmacología , Desaminasa APOBEC-3G , Antivirales/farmacología , Citidina Desaminasa/metabolismo , Infecciones por VIH/inmunología , VIH-1/fisiología , Humanos , Inmunidad/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Replicación Viral/fisiologíaRESUMEN
In the current study, we extended our previous works on natural endogenous reverse transcription (NERT) and further examined its potential as a virucide molecular target in sexual transmission of primate lentiviruses. HIV-1 and SIV virions were pretreated with select nucleoside (NRTIs) and nonnucleoside RT inhibitors (NNRTIs), either alone or in combination with NERT-stimulating substances. The effects of these antiretrovirals on virion inactivation were analyzed in human T cell lines and primary cell cultures. Pretreatment of HIV-1 virions with physiologic NERT-stimulants and 3'-azido-3'-deoxythymidine 5'-triphosphate (AZT-TP) or nevirapine potently inactivated cell-free HIV-1 virions and resulted in strong inhibition of the viral infectivity. Pretreatment of chimeric SHIV-RT virions with NERT-stimulating cocktail and select antiretrovirals also resulted in virion inactivation and inhibition of viral infectivity in T cell lines. Our findings demonstrate the potential clinical utility of approaches based on inhibiting NERT in sexual transmission of HIV-1, through the development of effective anti-HIV-1 microbicides, such as NRTIs and NNRTIs.
Asunto(s)
Antivirales/farmacología , VIH-1/efectos de los fármacos , Nevirapina/farmacología , Inhibidores de la Transcriptasa Inversa/farmacología , Virus de la Inmunodeficiencia de los Simios/efectos de los fármacos , Nucleótidos de Timina/farmacología , Transcripción Genética/efectos de los fármacos , Zidovudina/análogos & derivados , Células Cultivadas , Didesoxinucleótidos , Transmisión de Enfermedad Infecciosa/prevención & control , VIH-1/genética , Humanos , Infecciones por Lentivirus/prevención & control , Infecciones por Lentivirus/transmisión , Virus de la Inmunodeficiencia de los Simios/genética , Linfocitos T , Zidovudina/farmacologíaRESUMEN
The persistence of HIV-1 in virally suppressed infected individuals on highly active antiretroviral therapy (HAART) remains a major therapeutic problem. The use of cytokines has been envisioned as an additional therapeutic strategy to stimulate latent proviruses in these individuals. Immune activation therapy using IL-2 has shown some promise. In the present study, we found that IL-7 was significantly more effective at enhancing HIV-1 proviral reactivation than either IL-2 alone or IL-2 combined with phytohemagglutinin (PHA) in CD8-depleted PBMCs. IL-7 also showed a positive trend for inducing proviral reactivation from resting CD4(+) T lymphocytes from HIV-1-infected patients on suppressive HAART. Moreover, the phylogenetic analyses of viral envelope gp120 genes from induced viruses indicated that distinct proviral quasispecies had been activated by IL-7, as compared with those activated by the PHA/IL-2 treatment. These studies thus demonstrate that different activators of proviral latency may perturb and potentially deplete only selected, specific portions of the proviral archive in virally suppressed individuals. The known immunomodulatory effects of IL-7 could be combined with its ability to stimulate HIV-1 replication from resting CD4(+) T lymphocytes, in addition to other moieties, to potentially deplete HIV-1 reservoirs and lead to the rational design of immune-antiretroviral approaches.
Asunto(s)
Terapia Antirretroviral Altamente Activa , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/efectos de los fármacos , Interleucina-7/farmacología , Activación Viral/efectos de los fármacos , Replicación Viral/efectos de los fármacos , Fármacos Anti-VIH/uso terapéutico , Linfocitos T CD4-Positivos/citología , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/inmunología , Células Cultivadas , Regulación Viral de la Expresión Génica/efectos de los fármacos , Proteína gp120 de Envoltorio del VIH/genética , Infecciones por VIH/sangre , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/genética , Duplicado del Terminal Largo de VIH/genética , VIH-1/genética , VIH-1/fisiología , Humanos , Interleucina-2/farmacología , Activación de Linfocitos/efectos de los fármacos , Filogenia , Fitohemaglutininas/farmacología , Provirus/efectos de los fármacos , Provirus/fisiología , ARN Viral/genética , ARN Viral/metabolismo , Especificidad de la EspecieRESUMEN
Cell surface heparan sulfate proteoglycans (HSPGs) mediate internalization of HIV-1 Tat. Herein, we report that human WiDr cells, which express perlecan but no other HSPGs, can internalize 125I-labeled Tat with minimal lysosomal degradation. Pre-treatment of cells with heparitinase almost completely abolished 125I-Tat surface binding, while the use of an HIV-1 long terminal repeat (LTR) promoter-reporter construct demonstrated that transactivation was potently blocked by pretreatment of cells with heparitinase, indicating an essential role for perlecan in the biologic effects of Tat. We conclude that the perlecan mediates Tat uptake and is required for HIV-1 LTR-directed transactivation in this human cell type.
Asunto(s)
Productos del Gen tat/metabolismo , VIH-1/fisiología , Proteoglicanos de Heparán Sulfato/metabolismo , Transporte de Proteínas , Línea Celular , Genes Reporteros , Duplicado del Terminal Largo de VIH , Humanos , Polisacárido Liasas/metabolismo , Regiones Promotoras Genéticas , Activación Transcripcional , Productos del Gen tat del Virus de la Inmunodeficiencia HumanaAsunto(s)
Productos del Gen vif/metabolismo , VIH-1/fisiología , Proteínas/metabolismo , Replicación Viral , Desaminasa APOBEC-3G , Cisteína Endopeptidasas , Citidina Desaminasa , Citosina/metabolismo , ADN Viral/metabolismo , Desaminación , VIH-1/química , Complejos Multienzimáticos , Mutación , Nucleósido Desaminasas , Complejo de la Endopetidasa Proteasomal , Unión Proteica , Proteínas Represoras , Uracilo/metabolismo , Virión/química , Productos del Gen vif del Virus de la Inmunodeficiencia HumanaRESUMEN
Several studies have reported a crucial role for cholesterol-enriched membrane lipid rafts and cell-associated heparan sulfate proteoglycans (HSPGs), a class of molecules that can localize in lipid rafts, in the entry of human immunodeficiency virus type 1 (HIV-1) into permissive cells. For the present study, we examined the role of these cell surface moieties in HIV-1 entry into primary human brain microvascular endothelial cells (BMVECs), which represent an important HIV-1 central nervous system-based cell reservoir and a portal for neuroinvasion. Cellular cholesterol was depleted by exposure to beta-cyclodextrins and 3-hydroxy-3-methylglutaryl (HMG)-coenzyme A reductase inhibitors (statins), the loss of cholesterol was quantitated, and disruption of membrane rafts was verified by immunofluorescence. Nevertheless, these treatments did not affect binding of several strains of HIV-1 virions to BMVECs at 4 degrees C or their infectivities at 37 degrees C. In contrast, we confirmed that cholesterol depletion and raft disruption strongly inhibited HIV-1 binding and infection of Jurkat T cells. Enzymatic digestion of cell-associated HSPGs on human BMVECs dramatically inhibited HIV-1 infection, and our data from quantitative HIV-1 DNA PCR analysis strongly suggest that cell-associated chondroitin sulfate proteoglycans greatly facilitate infective entry of HIV-1 into human BMVECs. These findings, in combination with our earlier work showing that human BMVECs lack CD4, indicate that the molecular mechanisms for HIV-1 entry into BMVECs are fundamentally different from that of viral entry into T cells, in which lipid rafts, CD4, and probably HSPGs play important roles.