RESUMEN
BACKGROUND: Ribosomopathies constitute a class of inherited disorders characterized by defects in ribosome biogenesis and function. Classically, bone marrow (BM) failure is a clinical symptom shared between these syndromes, including Shwachman-Bodian-Diamond syndrome (SBDS). Eukaryotic translation initiation factor 6 (eIF6) is a critical translation factor that rescues the quasilethal effect of the loss of the SBDS protein. OBJECTIVES: To determine whether eIF6 activity is necessary for BM development. METHODS: We used eIF6(+/-) mice and primary BM megakaryocytes to investigate the involvement of eIF6 in the regulation of hematopoiesis. RESULTS: We provide evidence that reduced eIF6 expression negatively impacts on megakaryopoiesis. We show that inhibition of eIF6 leads to a reduction in cell size and mean ploidy level of megakaryocytes and a delay in megakaryocyte maturation by blocking the G1 /S transition. Consistent with this phenotype, only few megakaryocyte-forming proplatelets were found in eIF6(+/-) cells. We also discovered that, in eIF6(+/-) cells, the steady-state abundance of mitochondrial respiratory chain complex I-encoding mRNAs is decreased, resulting in decreased reactive oxygen species (ROS) production. Intriguingly, connectivity map analysis showed that eIF6-mediated changes overlap with specific translational inhibitors. eIF6 is a translation factor acting downstream of insulin/phorbol 12-myristate 13-acetate (PMA) stimulation. PMA treatment significantly restored eIF6(+/-) megakaryocyte maturation, indicating that activation of eIF6 is essential for the rescue of the phenotype. CONCLUSIONS: Taken together, our results show a role for eIF6-driven translation in megakaryocyte development, and unveil the novel connection between translational control and ROS production in this cell subset.
Asunto(s)
Factores de Iniciación de Péptidos/fisiología , Especies Reactivas de Oxígeno/metabolismo , Trombopoyesis/fisiología , Animales , Células de la Médula Ósea/metabolismo , Enfermedades de la Médula Ósea/metabolismo , Tamaño de la Célula , Células Cultivadas , Ensamble y Desensamble de Cromatina/fisiología , Regulación hacia Abajo , Complejo I de Transporte de Electrón/biosíntesis , Complejo I de Transporte de Electrón/genética , Insuficiencia Pancreática Exocrina/metabolismo , Fase G1/fisiología , Lipomatosis/metabolismo , Masculino , Megacariocitos/metabolismo , Megacariocitos/patología , Ratones , Ratones Endogámicos C57BL , Factores de Iniciación de Péptidos/deficiencia , Factores de Iniciación de Péptidos/genética , Fenotipo , Ploidias , Biosíntesis de Proteínas/fisiología , ARN Mensajero/biosíntesis , Subunidades Ribosómicas Grandes de Eucariotas/metabolismo , Síndrome de Shwachman-Diamond , Acetato de Tetradecanoilforbol/farmacologíaRESUMEN
The aim of our research is to use animal models to elucidate the molecular basis for viral clearance and liver disease in the pathogenesis of hepatitis B virus (HBV) and hepatitis C virus (HCV) infections. The results herein discussed provide insight into immunological and virological processes that may lead to the development of new therapeutic strategies to terminate chronic HBV and HCV infections.
Asunto(s)
Hepatitis Viral Humana/etiología , Hepatitis Viral Humana/inmunología , Animales , Modelos Animales de Enfermedad , Hepatitis B/etiología , Hepatitis B/inmunología , Hepatitis C/etiología , Hepatitis C/inmunología , HumanosRESUMEN
We have previously reported that intrahepatic NK T cells activated by alpha-galactosylceramide inhibit hepatitis B virus replication noncytopathically in the liver of transgenic mice. This effect is mediated by antiviral cytokines directly produced by activated NK T cells and/or by other cytokine-producing inflammatory cells that are recruited into the liver. In this study, we demonstrated that IFN-gamma produced by activated NK T cells induced parenchymal and nonparenchymal cells of the liver to produce high levels of CXC chemokine ligands 9 and 10, which mediated the intrahepatic recruitment of lymphomononuclear inflammatory cells. Recruitment of these cells was not necessary for the antiviral activity, indicating that direct activation of the intrahepatic resident NK T cell is sufficient to control viral replication in this model.
Asunto(s)
Quimiotaxis de Leucocito , Virus de la Hepatitis B/fisiología , Hepatitis B/inmunología , Péptidos y Proteínas de Señalización Intercelular , Células Asesinas Naturales/inmunología , Subgrupos de Linfocitos T/inmunología , Replicación Viral/inmunología , Animales , Antivirales/farmacología , Quimiocina CXCL10 , Quimiocina CXCL9 , Quimiocinas CXC/genética , Quimiocinas CXC/fisiología , Galactosilceramidasa/farmacología , Hepatitis B/genética , Hepatitis B/virología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/crecimiento & desarrollo , Interferón gamma/fisiología , Cinética , Hígado/inmunología , Activación de Linfocitos , Ratones , Ratones Transgénicos , ARN Mensajero/biosíntesis , Receptores CXCR3 , Receptores de Quimiocina/biosíntesis , Receptores de Quimiocina/genéticaRESUMEN
Using transgenic mice that replicate hepatitis B virus (HBV) at high levels in the liver as recipients of HBV-specific cytotoxic T lymphocytes (CTLs), we showed that the chemokines responsive to gamma-2/IFN-gamma inducible protein ([Crg2]IP-10) and monokine induced by interferon-gamma (Mig) are rapidly and strongly induced in the liver after CTL transfer. The transferred CTLs produce neither chemokine; rather, they activate (via the secretion of IFN-gamma) hepatocytes and nonparenchymal cells of the liver to produce (Crg2)IP-10 and Mig. Importantly, blocking these chemokines in vivo reduces the recruitment of host-derived lymphomononuclear cells into the liver and the severity of the liver disease without affecting the IFN-gamma-dependent antiviral potential of the CTLs. The finding that neutralization of these chemokines is associated with maintenance of antiviral effects but diminished tissue damage may be significant for the development of immunotherapeutic approaches for the treatment of chronic HBV infection.
Asunto(s)
Citotoxicidad Inmunológica , Virus de la Hepatitis B/inmunología , Hepatitis B/inmunología , Interferón gamma/inmunología , Monocinas/inmunología , Linfocitos T Citotóxicos/inmunología , Animales , Quimiocina CXCL10 , Hepatitis B/genética , Hepatitis B/patología , Interferón gamma/genética , Hígado/inmunología , Hígado/patología , Hígado/virología , Ratones , Ratones Transgénicos , Monocinas/genéticaRESUMEN
Hepatitis B virus (HBV) RNA is downregulated by inflammatory cytokines induced in the liver by adoptively transferred HBV-specific cytotoxic T lymphocytes (CTLs) and during murine cytomegalovirus (MCMV) infections of the livers of HBV transgenic mice. The disappearance of HBV RNA is tightly associated with the cytokine-induced proteolytic cleavage of a previously defined HBV RNA-binding protein known as La autoantigen. La binds to a predicted stem-loop structure at the 5' end of the posttranscriptional regulatory element of HBV RNA between nucleotides 1243 and 1333. In the present study, we searched for nuclear RNase activities that might be involved in HBV RNA decay. Nuclear extracts derived from control livers and CTL-injected and MCMV-infected livers were analyzed for the ability to cleave HBV RNA. Endonucleolytic activity that cleaved HBV RNA at positions 1269 to 1270 and 1271 to 1272, immediately 5' of the stem-loop bound by the La protein (positions 1272 to 1293), was detected. Furthermore, we provide evidence that the cytokine-dependent downregulation of HBV RNA following MCMV infection is temporally associated with the upregulation of the endonucleolytic activity herein described. Collectively, these results suggest a model in which the steady-state HBV RNA content is controlled by the stabilizing influence of La and the destabilizing influence of nuclear RNase activities.
Asunto(s)
Autoantígenos/metabolismo , Virus de la Hepatitis B/genética , Hígado/enzimología , ARN Viral/metabolismo , Ribonucleasas/metabolismo , Ribonucleoproteínas/metabolismo , Animales , Secuencia de Bases , Sitios de Unión , Núcleo Celular/enzimología , Humanos , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , ARN Viral/química , Antígeno SS-BRESUMEN
This review describes the contribution of noncytolytic mechanisms to the control of viral infections with a particular emphasis on the role of cytokines in these processes. It has long been known that most cell types in the body respond to an incoming viral infection by rapidly secreting antiviral cytokines such as interferon alpha/beta (IFN-alpha/beta). After binding to specific receptors on the surface of infected cells, IFN-alpha/beta has the potential to trigger the activation of multiple noncytolytic intracellular antiviral pathways that can target many steps in the viral life cycle, thereby limiting the amplification and spread of the virus and attenuating the infection. Clearance of established viral infections, however, requires additional functions of the immune response. The accepted dogma is that complete clearance of intracellular viruses by the immune response depends on the destruction of infected cells by the effector cells of the innate and adaptive immune system [natural killer (NK) cells and cytotoxic T cells (CTLs)]. This notion, however, has been recently challenged by experimental evidence showing that much of the antiviral potential of these cells reflects their ability to produce antiviral cytokines such as IFN-gamma and tumor necrosis factor (TNF)-alpha at the site of the infection. Indeed, these cytokines can purge viruses from infected cells noncytopathically as long as the cell is able to activate antiviral mechanisms and the virus is sensitive to them. Importantly, the same cytokines also control viral infections indirectly, by modulating the induction, amplification, recruitment, and effector functions of the immune response and by upregulating antigen processing and display of viral epitopes at the surface of infected cells. In keeping with these concepts, it is not surprising that a number of viruses encode proteins that have the potential to inhibit the antiviral activity of cytokines.
Asunto(s)
Virosis/inmunología , Animales , Anticuerpos Antivirales/inmunología , Presentación de Antígeno , Antígenos Virales/inmunología , Citocinas/fisiología , Células Dendríticas/inmunología , Granulocitos/inmunología , Humanos , Inmunidad Innata , Interferones/fisiología , Células Asesinas Naturales/inmunología , Macrófagos/fisiología , Ratones , Ratones Transgénicos , Modelos Inmunológicos , Subgrupos de Linfocitos T/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Factor de Necrosis Tumoral alfa/fisiología , Proteínas Virales/fisiología , Replicación ViralRESUMEN
The role of hepatocyte nuclear factor 1alpha (HNF1 alpha) in the regulation of hepatitis B virus (HBV) transcription and replication in vivo was investigated using a HNF1 alpha-null HBV transgenic mouse model. HBV transcription was not measurably affected by the absence of the HNF1 alpha transcription factor. However, intracellular viral replication intermediates were increased two- to fourfold in mice lacking functional HNF1 alpha protein. The increase in encapsidated cytoplasmic replication intermediates in HNF1 alpha-null HBV transgenic mice was associated with the appearance of nonencapsidated nuclear covalently closed circular (CCC) viral genomic DNA. Viral CCC DNA was not readily detected in HNF1 alpha-expressing HBV transgenic mice. This indicates the synthesis of nuclear HBV CCC DNA, the proposed viral transcriptional template found in natural infection, is regulated either by subtle alterations in the levels of viral transcripts or by changes in the physiological state of the hepatocyte in this in vivo model of HBV replication.
Asunto(s)
ADN Circular/metabolismo , Proteínas de Unión al ADN , Genoma Viral , Virus de la Hepatitis B/fisiología , Hepatitis B/virología , Hígado/virología , Proteínas Nucleares , Factores de Transcripción/fisiología , Animales , ADN Viral/metabolismo , Modelos Animales de Enfermedad , Regulación Viral de la Expresión Génica , Antígenos del Núcleo de la Hepatitis B/metabolismo , Antígenos e de la Hepatitis B/metabolismo , Factor Nuclear 1 del Hepatocito , Factor Nuclear 1-alfa del Hepatocito , Factor Nuclear 1-beta del Hepatocito , Hígado/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , ARN Viral/metabolismo , Factores de Transcripción/genética , Transcripción Genética , Replicación ViralRESUMEN
The sequence of the hepatitis B virus (HBV) major envelope (Env) protein (ayw subtype) was scanned for the presence of H-2(d,b) motifs. Following binding and immunogenicity testing, two new H-2(d)-restricted epitopes (Env.362 and Env.364) were identified. These epitopes induced CTLs capable of recognizing naturally processed HBV-Env, but were apparently generated with lower efficiency than the previously defined dominant Env.28 epitope. Next, HBV-transgenic mice that express all of the HBV proteins and produce fully infectious particles were immunized with a mixture of lipopeptides encompassing the Env.28, Env.362, and Env.364 epitopes. Significant CTL responses were obtained, but they had no effect on viral replication in the liver, nor did they induce an inflammatory liver disease. However, in adoptive transfer experiments, CTL lines generated from the HBV-transgenic mice following immunization were able to inhibit viral replication in vivo without causing hepatitis. This is in contrast to CTL lines derived from nontransgenic mice that displayed both antiviral and cytopathic effects, presumably because they displayed higher avidity for the viral epitopes than the transgenic CTLs. These results suggest that T cell tolerance to HBV can be broken with appropriate immunization but the magnitude and characteristics of the resultant T cell response are significantly different from the response in HBV-naive individuals since their antiviral potential is stronger than their cytotoxic potential. This has obvious implications for immunotherapy of chronic HBV infection.
Asunto(s)
Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , Tolerancia Inmunológica/genética , Linfocitos T Citotóxicos/inmunología , Traslado Adoptivo , Animales , Presentación de Antígeno/genética , Citotoxicidad Inmunológica/genética , Relación Dosis-Respuesta Inmunológica , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Epítopos de Linfocito T/inmunología , Epítopos de Linfocito T/metabolismo , Hepatitis B/genética , Hepatitis B/inmunología , Hepatitis B/patología , Antígenos de Superficie de la Hepatitis B/biosíntesis , Antígenos de Superficie de la Hepatitis B/metabolismo , Epítopos Inmunodominantes/inmunología , Epítopos Inmunodominantes/metabolismo , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Fragmentos de Péptidos/inmunología , Fragmentos de Péptidos/metabolismo , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/trasplante , Linfocitos T Citotóxicos/virología , Células Tumorales Cultivadas , Proteínas del Envoltorio Viral/genética , Proteínas del Envoltorio Viral/inmunología , Proteínas del Envoltorio Viral/metabolismoRESUMEN
We have previously reported that hepatitis B virus (HBV)-specific CD8(+) cytotoxic T lymphocytes and CD4(+) helper T lymphocytes can inhibit HBV replication in the liver of HBV transgenic mice by secreting interferon (IFN)-gamma when they recognize viral antigen. To determine whether an activated innate immune system can also inhibit HBV replication, in this study we activated natural killer T (NKT) cells in the liver of HBV transgenic mice by a single injection of alpha-galactosylceramide (alpha-GalCer), a glycolipid antigen presented to Valpha14(+)NK1.1(+) T cells by the nonclassical major histocompatibility complex class I-like molecule CD1d. Within 24 h of alpha-GalCer injection, IFN-gamma and IFN-alpha/beta were detected in the liver of HBV transgenic mice and HBV replication was abolished. Both of these events were temporally associated with the rapid disappearance of NKT cells from the liver, presumably reflecting activation-induced cell death, and by the recruitment of activated NK cells into the organ. In addition, prior antibody-mediated depletion of CD4(+) and CD8(+) T cells from the mice did not diminish the ability of alpha-GalCer to trigger the disappearance of HBV from the liver, indicating that conventional T cells were not downstream mediators of this effect. Finally, the antiviral effect of alpha-GalCer was inhibited in mice that are genetically deficient for either IFN-gamma or the IFN-alpha/beta receptor, indicating that most of the antiviral activity of alpha-GalCer is mediated by these cytokines. Based on these results, we conclude that alpha-GalCer inhibits HBV replication by directly activating NKT cells and by secondarily activating NK cells to secrete antiviral cytokines in the liver. In view of these findings, we suggest that, if activated, the innate immune response, like the adaptive immune response, has the potential to control viral replication during natural HBV infection. In addition, the data suggest that therapeutic activation of NKT cells may represent a new strategy for the treatment of chronic HBV infection.
Asunto(s)
Antivirales/inmunología , Galactosilceramidas/inmunología , Virus de la Hepatitis B/inmunología , Células Asesinas Naturales/inmunología , Activación de Linfocitos/inmunología , Replicación Viral/inmunología , Animales , Antivirales/administración & dosificación , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD8-positivos/inmunología , Relación Dosis-Respuesta a Droga , Femenino , Galactosilceramidas/administración & dosificación , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Humanos , Células Asesinas Naturales/efectos de los fármacos , Activación de Linfocitos/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Factores de Tiempo , Replicación Viral/efectos de los fármacosRESUMEN
We have previously shown that hepatitis B virus (HBV) replication is abolished in the liver of HBV transgenic mice by inflammatory cytokines induced by HBV-specific cytotoxic T cells and during unrelated viral infections of the liver. We now report that intrahepatic HBV replication is also inhibited in mice infected by the malaria species Plasmodium yoelii 17X NL. P. yoelii infection triggers an intrahepatic inflammatory response characterized by the influx of natural killer cells, macrophages, and T cells. During this process, interferon (IFN)-gamma and IFN-alpha/beta suppress HBV gene expression and replication in the liver. Collectively, the data suggest that malaria infection might influence the course and pathogenesis of HBV infection in coinfected humans.
Asunto(s)
Virus de la Hepatitis B/inmunología , Hepatitis B/inmunología , Interferones/inmunología , Hígado/inmunología , Malaria/inmunología , Plasmodium yoelii/fisiología , Alanina Transaminasa/sangre , Animales , Northern Blotting , Citocinas/biosíntesis , Citocinas/genética , Citocinas/inmunología , Hepatitis B/complicaciones , Hepatitis B/virología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Humanos , Interferones/biosíntesis , Interferones/genética , Hígado/parasitología , Hígado/patología , Hígado/virología , Macrófagos/inmunología , Malaria/complicaciones , Malaria/parasitología , Ratones , Ratones Transgénicos , Linfocitos T/inmunología , Replicación ViralRESUMEN
Although coinfection of hepatitis B virus (HBV) and Schistosoma mansoni is a frequent event in humans, little is known about the interactions between these two pathogens. S. mansoni infection induces T helper cell type 2 (Th2)-type cytokines in the liver of humans and mice. The intrahepatic induction of nitric oxide (NO) and Th1-type cytokines, such as interferon (IFN)-gamma and IFN-alpha/beta, inhibits HBV replication noncytopathically in the liver of transgenic mice. To examine whether S. mansoni infection and the accompanying induction of Th2-type cytokines could interfere with HBV replication in the liver, HBV transgenic mice were infected with S. mansoni. By 5 wk after infection, HBV replication disappeared concomitant with the intrahepatic induction of NO and Th1-type cytokines, and in the absence of Th2-type cytokines. By 6-8 wk after infection, HBV replication remained undetectable and this was associated with further induction of NO and Th1-type cytokines together with the appearance of Th2-type cytokines. The S. mansoni-dependent antiviral effect was partially blocked by genetically deleting IFN-gamma, although it was unaffected by deletion of IFN-alpha/beta. These results indicate that IFN-gamma (probably via NO) mediates most of this antiviral activity and that Th2-type cytokines do not counteract the antiviral effect of IFN-gamma. Similar events may suppress HBV replication during human S. mansoni infection.
Asunto(s)
Virus de la Hepatitis B/fisiología , Esquistosomiasis mansoni/virología , Replicación Viral , Animales , Interferón gamma/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Óxido Nítrico/fisiología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo IIRESUMEN
We have previously identified two antiviral cytokines (interferon [IFN]-gamma and IFN-alpha/beta) that downregulate hepatitis B virus (HBV) replication in the liver of transgenic mice. The cytokine-inducible downstream events that inhibit HBV replication have not been identified. One possible factor is nitric oxide (NO), a pleiotropic free radical with antiviral activity that is produced in the liver by the inducible NO synthase (iNOS). To examine the role of NO in our model, we crossed transgenic mice that replicate HBV with mice that lack a functional iNOS. Importantly, iNOS-deficient mice were almost completely resistant to the noncytopathic inhibitory effect of HBV-specific cytotoxic T lymphocytes on viral replication, an effect that we have shown previously to depend on the intrahepatic induction of IFN-gamma. Conversely, iNOS-deficient mice were not resistant to the antiviral effect of IFN-alpha/beta induced by either polyinosinic-polycytidylic acid complex or by lymphocytic choriomeningitis virus (LCMV) infection. These results indicate that NO mediates the antiviral activity of IFN-gamma, whereas the antiviral activity of IFN-alpha/beta is NO independent. We also compared the relative sensitivity of LCMV to control by NO in these animals. Interestingly, LCMV replicated to higher levels in the liver of iNOS-deficient mice than control mice, indicating that NO controls LCMV replication in the liver, as well as HBV.
Asunto(s)
Virus de la Hepatitis B/inmunología , Hígado/virología , Óxido Nítrico/inmunología , Replicación Viral/inmunología , Animales , Femenino , Antígenos de Superficie de la Hepatitis B/inmunología , Virus de la Hepatitis B/fisiología , Inductores de Interferón/inmunología , Interferón-alfa/inmunología , Interferón beta/inmunología , Interferón gamma/inmunología , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Óxido Nítrico Sintasa/genética , Óxido Nítrico Sintasa/inmunología , Óxido Nítrico Sintasa de Tipo II , Poli I-C/inmunología , Linfocitos T Citotóxicos/inmunologíaRESUMEN
We have previously shown that hepatitis B virus (HBV) replication is inhibited noncytopathically in the livers of transgenic mice following injection of HBV-specific cytotoxic T lymphocytes (CTLs) or infection with unrelated hepatotropic viruses, including lymphocytic choriomeningitis virus (LCMV) and adenovirus. These effects are mediated by gamma interferon (IFNgamma), tumor necrosis factor alpha (TNFalpha), and IFNalpha/beta. In the present study, we crossed HBV transgenic mice with mice genetically deficient for IFNgamma (IFNgammaKO), the TNFalpha receptor (TNFalphaRKO), or the IFNalpha/beta receptor (IFNalpha/betaRKO) in order to determine the relative contribution of each cytokine to the antiviral effects observed in each of these systems. Interestingly, we showed that HBV replicates in unmanipulated IFNgammaKO and IFNalpha/betaRKO mice at levels higher than those observed in control mice, implying that baseline levels of these cytokines control HBV replication in the absence of inflammation. We also showed that IFNgamma mediates most of the antiviral effect of the CTLs while IFNalpha/beta is primarily responsible for the early inhibitory effect of LCMV and adenovirus on HBV replication. In addition, we showed that the hepatic induction of IFNalpha/beta observed after injection of poly(I. C) is sufficient to inhibit HBV replication and that a similar antiviral effect is achieved by systemic administration of very high doses of IFNalpha. We also compared the relative sensitivity of LCMV and adenovirus to control by IFNgamma, TNFalpha, or IFNalpha/beta in these animals. Importantly, IFNalpha/betaRKO mice, and to a lesser extent IFNgammaKO mice, showed higher hepatic levels of LCMV RNA and adenovirus DNA and RNA than control mice, underscoring the importance of both interferons in controlling these other viral infections as well.
Asunto(s)
Citocinas/uso terapéutico , Virus de la Hepatitis B/patogenicidad , Hepatitis B/inmunología , Adenoviridae/inmunología , Adenoviridae/patogenicidad , Animales , Citocinas/farmacología , Hepatitis B/tratamiento farmacológico , Hepatitis B/virología , Virus de la Hepatitis B/inmunología , Inductores de Interferón/farmacología , Interferón gamma/deficiencia , Interferón gamma/genética , Transfusión de Linfocitos , Virus de la Coriomeningitis Linfocítica/inmunología , Virus de la Coriomeningitis Linfocítica/patogenicidad , Ratones , Ratones Noqueados , Ratones Transgénicos , Poli I-C/farmacología , Receptores del Factor de Necrosis Tumoral/deficiencia , Receptores del Factor de Necrosis Tumoral/genética , Linfocitos T Citotóxicos , Factor de Necrosis Tumoral alfa/deficiencia , Factor de Necrosis Tumoral alfa/genética , Replicación Viral/efectos de los fármacosRESUMEN
We have previously shown that hepatitis B virus (HBV) replication is abolished in the liver of HBV transgenic mice by stimuli that induce alpha/beta interferon (IFN-alpha/beta) in the liver. The present study was done to identify the step(s) in HBV replication that is affected by this cytokine in transgenic mice treated with the IFN-alpha/beta inducer polyinosinic-polycytidylic acid [poly(I-C)]. Here we show that the pool of cytoplasmic HBV pregenomic RNA (pgRNA)-containing capsids is reduced 10-fold within 9 h after poly(I-C) administration, while there is no change in the abundance of HBV mRNA or in the translational status of cytoplasmic HBV transcripts. In addition, we show that the pool of HBV DNA-containing capsids is not reduced to the same degree until at least 15 h posttreatment, and we show that virus export is not accelerated and the half-life of virions in the serum is unchanged. These results indicate that IFN-alpha/beta triggers intracellular events that either inhibit the assembly of pgRNA-containing capsids or accelerate their degradation, and that maturation and secretion of virus is responsible for clearance of HBV capsids and their cargo of replicative intermediates from the cytoplasm of the hepatocyte.
Asunto(s)
Cápside/inmunología , Virus de la Hepatitis B/inmunología , Interferón-alfa/biosíntesis , Interferón beta/biosíntesis , ARN Viral/metabolismo , Animales , Transporte Biológico , Cápside/metabolismo , Citoplasma/virología , Replicación del ADN , ADN de Cadena Simple , ADN Viral/metabolismo , Expresión Génica , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Inductores de Interferón/farmacología , Interferón-alfa/inmunología , Interferón beta/inmunología , Hígado/virología , Ratones , Ratones Transgénicos , Poli I-C/farmacología , Biosíntesis de Proteínas , ARN/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
The role of the peroxisome proliferator-activated receptor alpha (PPARalpha) in regulating hepatitis B virus (HBV) transcription and replication in vivo was investigated in an HBV transgenic mouse model. Treatment of HBV transgenic mice with the peroxisome proliferators Wy-14,643 and clofibric acid resulted in a less than twofold increase in HBV transcription rates and steady-state levels of HBV RNAs in the livers of these mice. In male mice, this increase in transcription was associated with a 2- to 3-fold increase in replication intermediates, whereas in female mice it was associated with a 7- to 14-fold increase in replication intermediates. The observed increases in transcription and replication were dependent on PPARalpha. HBV transgenic mice lacking this nuclear hormone receptor showed similar levels of HBV transcripts and replication intermediates as untreated HBV transgenic mice expressing PPARalpha but failed to demonstrate alterations in either RNA or DNA synthesis in response to peroxisome proliferators. Therefore, it appears that very modest alterations in transcription can, under certain circumstances, result in relatively large increases in HBV replication in HBV transgenic mice.
Asunto(s)
Virus de la Hepatitis B/fisiología , Proliferadores de Peroxisomas/farmacología , Receptores Citoplasmáticos y Nucleares/metabolismo , Factores de Transcripción/metabolismo , Replicación Viral/efectos de los fármacos , Animales , Ácido Clofíbrico/farmacología , Femenino , Antígenos e de la Hepatitis B/biosíntesis , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Pirimidinas/farmacología , Receptores Citoplasmáticos y Nucleares/genética , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
The function of the X protein in the life cycle of mammalian hepadnaviruses is unclear. Based on tissue culture experiments it has been suggested that this protein represents a transcriptional transactivator which might be essential for the expression of the viral core gene. Here we have examined whether the activity of the human hepatitis B virus (HBV) core gene in vivo depends on X coexpression. To this end we compared core gene expression between four lineages of transgenic mice carrying the HBV core gene in cis arrangement with the X gene (cex lineage) and six lineages containing a modified construct in which the start codon of the X gene had been deleted (ce lineage). Whereas all cex lineages consistently exhibited a high-level hepatic core gene expression, the liver-specific core gene expression pattern of the ce lineages was heterogenous with four lineages virtually not expressing the core gene. This defect was due to a strongly reduced transcription since no core mRNA could be detected by Northern blotting. To test whether core gene expression could be restored by providing an intact X gene in trans, we crossbred mice of two lines which expressed no core mRNA or core protein with transgenic mice expressing the X-gene product under the transcriptional regulation of the liver-specific major-urinary-protein promoter/enhancer (MUP-X mice). The introduction of the MUP-X transgene induced core mRNA expression and core protein biosynthesis in the livers of the double-transgenic mice. This demonstrates that the X-gene product has the capacity to transactivate HBV core gene expression in vivo.
Asunto(s)
Regulación Viral de la Expresión Génica , Antígenos del Núcleo de la Hepatitis B/genética , Virus de la Hepatitis B/genética , Transactivadores/metabolismo , Activación Transcripcional , Animales , Humanos , Hígado/patología , Hígado/virología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Ratones Transgénicos , ARN Mensajero/metabolismo , Transactivadores/genética , Transgenes , Proteínas Reguladoras y Accesorias ViralesRESUMEN
The control of viral infections was previously thought to rely exclusive ly on the antigen-specific destruction of infected cells by the antigen-specific destruction of infected cells by the immune system; however, recent studies have shown that several viral infections can be primarily controlled by noncytopathic, cytokine- dependent 'curative' mechanisms (i.e. viral purging). The relative sensitivity of viruses to such curative mechanisms depends not only on the virus but also on the capacity of the specific infected cell to produce the appropriate intracellular antiviral factors.
Asunto(s)
Citocinas/fisiología , Virosis/inmunología , Virus/inmunología , Animales , Humanos , Virosis/virologíaRESUMEN
We recently identified three nuclear proteins (p45, p39, and p26) that bind to a 91-nucleotide (nt) RNA element between nt 1243 and 1333 in hepatitis B virus (HBV) RNA, and we showed that these proteins and HBV RNA are regulated coordinately by gamma interferon and tumor necrosis factor alpha. Purification and sequence analysis of tryptic peptides obtained from p39 revealed sequence homology to the mouse La protein. Immunoprecipitation experiments showed that p45, p39, and p26 were recognized by anti-La-specific antiserum, indicating that p45 is the full-length La protein and that p39 and p26 are likely to be proteolytic La cleavage products. Furthermore, in competition experiments we found that all three La proteins bind, in a phosphorylation-dependent manner, to the same predicted stem-loop structure located between nt 1275 and 1291 of HBV, with Kds of approximately 1.0 nM. Collectively, these results support the notion that the La protein may contribute to HBV RNA stability, constitutively and in response to inflammatory cytokines.
Asunto(s)
Autoantígenos/metabolismo , Virus de la Hepatitis B/genética , ARN Viral/metabolismo , Ribonucleoproteínas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Núcleo Celular/metabolismo , Humanos , Masculino , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Fosforilación , ARN Viral/química , Proteínas de Unión al ARN/metabolismo , Antígeno SS-BRESUMEN
The outcome of viral infections is dependent on the amount of tissue destruction caused either by direct lysis of infected cells and/or by immunopathology resulting from the immune response to the virus. We investigated whether induction of tolerance to only one viral protein could reduce immunopathology caused by nonlytic lymphocytic choriomeningitis virus (LCMV) in perforin-deficient hosts. Earlier studies had shown that LCMV infection results in aplastic anemia and death in most of these mice and that this is associated with bone marrow infiltration by antiviral cytotoxic T lymphocytes (CTL) that secrete inflammatory cytokines. We report here that perforin-deficient mice exhibit severe immunopathology in multiple organs that is characterized by infiltration of anti-LCMV CTL that secrete large amounts of gamma interferon (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha). Importantly, this immunopathology is significantly reduced and long-term survival of LCMV infection is increased in perforin-deficient mice expressing LCMV nucleoprotein (NP) in the thymus (and therefore deleting most of their LCMV-NP CTL) compared to the situation in thymus nonexpressors. This is due to the selective reduction of NP-specific CTL responses and their inflammatory-cytokine (IFN-gamma and TNF-alpha) secretion and to a lack of pathogenetically relevant compensatory responses to other viral proteins. Thus, "selective reduction" of the antiviral immune response to only one viral protein can significantly reduce inflammatory immunopathology and might be a therapeutic possibility for certain nonlytic infections.