RESUMEN
Two major interferon (IFN)-mediated antiviral defense enzymes are double-stranded (ds)RNA-dependent 2',5'-oligoadenylate (2-5A) synthetase (2-5OAS) and p68 kinase (PKR). When activated by dsRNA, 2-5OAS synthesizes 2-5A, which binds to and activates RNase L. Activated RNase L hydrolyzes single-stranded viral RNA, thereby inhibiting viral protein synthesis. HIV-1 inhibits the IFN-mediated intracellular antiviral pathways. We have reported the synthesis and characterization of a nuclease-resistant 2-5A agonist (2-5A(N6B)) that overcomes the HIV-1 induced blockades by restoring the 2-5OAS/RNase L antiviral pathway (Homan JW, et al., J Acquir Immune Defic Syndr 2002;30:9-20). The objective of this study was to test the effect of 2-5A(N6B) on chronically infected CD4(+) T lymphocytes and CD14(+) monocytes derived from HIV-1-seropositive individuals. Wild-type HIV-1 replication was effectively inhibited by 2-5A(N6B) in CD4(+) T lymphocytes and CD14(+) monocytes purified from HIV-1 seropositive individuals (n = 18) compared to untreated cells. We also assessed the cytotoxicity of 2-5A(N6B) and report that 2-5A(N6B) exerts its anti-HIV-1 activity with no evidence of cytotoxicity (IC(90) > 100,000 nM). Furthermore, 2-5A(N6B) did not alter the cellular RNA profile, affect CCR5 or CXCR4 coreceptor expression, or activate caspase-dependent apoptosis. Evidence is also provided to show that 2-5A(N6B), and naturally occurring 2-5A(4), act as ligands to activate human Toll-like receptor 4. These results indicate that the 2-5A agonist 2-5A(N6B) has the potential to enhance host cell innate and acquired immune defense mechanisms against HIV-1 infection.
Asunto(s)
Nucleótidos de Adenina/farmacología , Fármacos Anti-VIH/farmacología , Linfocitos T CD4-Positivos/virología , Infecciones por VIH/metabolismo , VIH-1/efectos de los fármacos , Receptores de Lipopolisacáridos , Oligorribonucleótidos/farmacología , Nucleótidos de Adenina/agonistas , Nucleótidos de Adenina/síntesis química , Adulto , Anciano , Recuento de Linfocito CD4 , Linfocitos T CD4-Positivos/efectos de los fármacos , Células Cultivadas , Relación Dosis-Respuesta a Droga , Femenino , Citometría de Flujo , Infecciones por VIH/tratamiento farmacológico , Seropositividad para VIH , VIH-1/fisiología , Humanos , Factores Inmunológicos/farmacología , Masculino , Persona de Mediana Edad , Monocitos/citología , Monocitos/inmunología , Oligorribonucleótidos/agonistas , Oligorribonucleótidos/síntesis química , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Receptor Toll-Like 4/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
Previous studies from this laboratory evaluated the role of p68 kinase (PKR) in the control of HIV-1 replication via retrovirus-mediated gene transfer. PKR was studied because it is a key component of the interferon (IFN)-associated innate antiviral defense pathway in mammalian cells. In this study, CD34(+) hematopoietic stem cells (HSC) were transduced with an HIV-1-based lentiviral vector encoding the PKR transgene (pHIV-PIB) and cultured under conditions that support in vitro differentiation. With high-titer pseudotyped vector stocks, the histogram suggests 100% transduction of the HSC because the cells were blasticidin resistant. Analysis of transduced cells by hybridization revealed an average proviral vector copy number of 1.8 and 2.1 copies of vector sequence per cell. Increased PKR expression and activity (phosphorylation of eukaryotic initiation factor 2alpha [eIF2alpha]) were demonstrated in PKR-transduced, differentiated HSC. There was minimal reduction in cell viability and no induction of apoptosis after transduction of PKR. HSC transduced with the pHIV-PIB lentiviral vector demonstrated normal differentiation into CD34-derived T cell progeny. Two days after HIV-1 infection, lentivirus-mediated transduction of PKR inhibited HIV-1 replication by 72% in T cell progeny compared with cells transduced with the empty vector control (pHIV-IB). By days 5 and 7 post-HIV-1 infection, the surviving PKR-transduced cells were protected from HIV-1 infection, as evidenced by a decrease in p24 antigen expression of at least two orders of magnitude. Our results demonstrate that PKR can be effectively delivered to HSC by a lentiviral vector and can protect CD34-derived T cell progeny from HIV-1 infection. These results provide support for application of the innate antiviral defense pathway in a gene therapy setting to the treatment of HIV-1 infection.
Asunto(s)
Antígenos CD34 , Infecciones por VIH/genética , VIH-1/genética , Células Madre Hematopoyéticas/fisiología , Transducción Genética , Replicación Viral/genética , eIF-2 Quinasa/genética , Antígenos CD34/metabolismo , Linfocitos T CD4-Positivos/fisiología , Diferenciación Celular/genética , Diferenciación Celular/fisiología , Células Cultivadas , Vectores Genéticos , Infecciones por VIH/metabolismo , Células Madre Hematopoyéticas/virología , Humanos , Subgrupos de Linfocitos T/fisiología , Replicación Viral/fisiologíaRESUMEN
A 2',5'-oligoadenylate (2-5A)-dependent 37-kDa form of RNase L has been reported in extracts of peripheral blood mononuclear cells (PBMC) from individuals with chronic fatigue syndrome (CFS). In the current study, analytic gel permeation FPLC, azido photoaffinity labeling, two-dimensional (2-D) gel electrophoresis, and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) have been used to examine the biochemical relationship between the 80-kDa RNase L in healthy control PBMC and the 37-kDa RNase L in PBMC from individuals with CFS. Like the 80-kDa RNase L, the 37-kDa RNase L is present as a catalytically inactive heterodimer complex with the RNase L inhibitor (RLI). Formation of a 37-kDa RNase L-RLI complex indicates that the 37-kDa RNase L is structurally similar to the 80-kDa RNase L at the N-terminus, which contains the 2-5A binding domain. The enzymatically active monomer form of 37-kDa RNase L resolved by 2-D gel electrophoresis has a pI of 6.1. RT-PCR and Southern blot analyses demonstrated that the 37-kDa RNase L is not formed by alternative splicing. In-gel tryptic digestion of the 37-kDa RNase L that was excised from 2-D gels and subsequent MALDI-MS analysis identified three peptide masses that are identical to three predicted peptide masses in the 80-kDa RNase L. The electrophoretic mobility of 2-5A azido photolabeled/immunoprecipitated 37-kDa RNase L was the same under reducing and nonreducing conditions. The results presented show that the 37-kDa form of RNase L in PBMC shares structural and functional features with the native 80-kDa RNase L, in particular in the 2-5A binding and catalytic domains.
Asunto(s)
Adenosina/análogos & derivados , Endorribonucleasas/química , Endorribonucleasas/fisiología , Síndrome de Fatiga Crónica/enzimología , Adenosina/química , Marcadores de Afinidad/química , Azidas/química , Extractos Celulares/análisis , Células Cultivadas , Cromatografía en Gel , Disulfuros/metabolismo , Electroforesis en Gel Bidimensional , Endorribonucleasas/genética , Humanos , Células K562 , Leucocitos Mononucleares/enzimología , Peso Molecular , ARN Mensajero/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónAsunto(s)
Transportadoras de Casetes de Unión a ATP , Chaperoninas , Síndrome de Fatiga Crónica/metabolismo , Gastroenteritis/metabolismo , Interferones/fisiología , Enfermedad Aguda , Endorribonucleasas/metabolismo , Síndrome de Fatiga Crónica/enzimología , Gastroenteritis/enzimología , Humanos , Ligasas/metabolismo , Proteínas/metabolismoRESUMEN
Opioids potentiate HIV-1 infection in vitro at least partly by suppressing immunoresponsive processes in human lymphocytes and monocytes. For example, it appears that morphine inhibits the interferon (IFN)-alpha, -beta, and -gamma-mediated natural antiviral defense pathways in human peripheral blood mononuclear cells (PBMC). In this study, we show that restoration of a key component of the antiviral pathway reverses morphine-potentiated HIV-1 infection of human PBMC. The data show that HIV-1 replication is potentiated and RNase L activity is inhibited after morphine administration. Because HIV-1 inhibits the antiviral pathway at the level of 2',5'-oligoadenylate (2-5A) synthetase and p68 kinase, antiviral enzymes that require double-stranded RNA, we overcame this blockade by the addition of the nuclease-resistant, nontoxic 2-5A agonist, 2-5A(N6B), to PBMC in culture. Addition of 2-5A(N6B), but not zidovudine or saquinavir, to morphine-treated PBMC completely reversed the morphine-induced potentiation of HIV-1 infection. Further, 2-5A(N6B) significantly enhanced expression of both IFN-alpha and IFN-gamma. Also, increased expression of IFN-gamma was associated with a significant increase in expression of RANTES and monocyte chemotactic protein (MCP)-1, chemokines that may inhibit HIV-1 infection by blocking viral attachment to CCR2 and CCR5 co-receptors. Our results suggest that reactivation of the antiviral pathway by 2-5A agonists may be useful to inhibit opioid-potentiated HIV-1 replication.