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[This corrects the article DOI: 10.1371/journal.ppat.1003490.].
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[This corrects the article DOI: 10.1371/journal.ppat.1003490.].
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The intrahepatic immune environment is normally biased towards tolerance. Nonetheless, effective antiviral immune responses can be induced against hepatotropic pathogens. To examine the immunological basis of this paradox we studied the ability of hepatocellularly expressed hepatitis B virus (HBV) to activate immunologically naïve HBV-specific CD8⺠T cell receptor (TCR) transgenic T cells after adoptive transfer to HBV transgenic mice. Intrahepatic priming triggered vigorous in situ T cell proliferation but failed to induce interferon gamma production or cytolytic effector function. In contrast, the same T cells differentiated into cytolytic effector T cells in HBV transgenic mice if Programmed Death 1 (PD-1) expression was genetically ablated, suggesting that intrahepatic antigen presentation per se triggers negative regulatory signals that prevent the functional differentiation of naïve CD8⺠T cells. Surprisingly, coadministration of an agonistic anti-CD40 antibody (αCD40) inhibited PD-1 induction and restored T cell effector function, thereby inhibiting viral gene expression and causing a necroinflammatory liver disease. Importantly, the depletion of myeloid dendritic cells (mDCs) strongly diminished the αCD40 mediated functional differentiation of HBV-specific CD8⺠T cells, suggesting that activation of mDCs was responsible for the functional differentiation of HBV-specific CD8⺠T cells in αCD40 treated animals. These results demonstrate that antigen-specific, PD-1-mediated CD8⺠T cell exhaustion can be rescued by CD40-mediated mDC-activation.
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Inmunidad Adaptativa , Antígenos CD40/agonistas , Linfocitos T CD8-positivos/inmunología , Virus de la Hepatitis B/fisiología , Hepatitis B/inmunología , Interacciones Huésped-Patógeno , Receptor de Muerte Celular Programada 1/metabolismo , Animales , Presentación de Antígeno , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Antígenos Virales/metabolismo , Antígenos CD40/genética , Antígenos CD40/metabolismo , Linfocitos T CD8-positivos/metabolismo , Linfocitos T CD8-positivos/patología , Linfocitos T CD8-positivos/virología , Diferenciación Celular , Proliferación Celular , Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Células Dendríticas/patología , Células Dendríticas/virología , Regulación Viral de la Expresión Génica , Hepatitis B/metabolismo , Hepatitis B/patología , Hepatitis B/virología , Virus de la Hepatitis B/inmunología , Hígado/inmunología , Hígado/metabolismo , Hígado/patología , Hígado/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Receptor de Muerte Celular Programada 1/genética , Receptores de Antígenos de Linfocitos T/genética , Receptores de Antígenos de Linfocitos T/metabolismo , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/metabolismo , Linfocitos T Citotóxicos/patología , Linfocitos T Citotóxicos/virologíaRESUMEN
UNLABELLED: Approximately 50% of patients with chronic hepatitis C (CHC) have ongoing expression of interferon stimulated genes (ISGs) in the liver. It is unclear why this endogenous antiviral response is inefficient in eradicating the infection. Several viral escape strategies have been identified in vitro, including inhibition of interferon (IFN) induction and ISG messenger RNA (mRNA) translation. The in vivo relevance of these mechanisms is unknown, because reliable methods to identify hepatitis C virus (HCV)-infected cells in human liver are lacking. We developed a highly sensitive in situ hybridization (ISH) system capable of HCV RNA and ISG mRNA detection in human liver biopsies and applied it to study the interaction of HCV with the endogenous IFN system. We simultaneously monitored HCV RNA and ISG mRNA using HCV isolate- and ISG mRNA-specific probes in liver biopsy sections from 18 CHC patients. The signals were quantified at the single-cell resolution in a series of random high-power fields. The proportion of infected hepatocytes ranged from 1%-54% and correlated with viral load, but not with HCV genotype or ISG expression. Infected cells occurred in clusters, pointing to cell-to-cell spread as the predominant mode of HCV transmission. ISG mRNAs were readily detected in HCV-infected cells, challenging previously proposed mechanisms of viral interference with the immune system. Conversely, infected cells and neighboring cells showed increased ISG mRNA levels, demonstrating that the stimulus driving ISG expression originates from HCV-infected hepatocytes. CONCLUSION: HCV infection in human hepatocytes during CHC does not efficiently interfere with IFN induction, IFN signaling, or transcription of ISG mRNA.
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Regulación Viral de la Expresión Génica , Hepatitis C/virología , Interferones/fisiología , Hígado/virología , Hepatitis C/genética , Hepatitis C/metabolismo , Hepatocitos/virología , Humanos , Hibridación in Situ , Hígado/metabolismo , ARN Viral/genética , Carga Viral/genéticaRESUMEN
Hepatitis C virus (HCV) genome replication is thought to occur in a membranous cellular compartment derived from the endoplasmic reticulum (ER). The molecular mechanisms by which these membrane-associated replication complexes are formed during HCV infection are only starting to be unraveled, and both viral and cellular factors contribute to their formation. In this study, we describe the discovery of nonopioid sigma-1 receptor (S1R) as a cellular factor that mediates the early steps of viral RNA replication. S1R is a cholesterol-binding protein that resides in lipid-rich areas of the ER and in mitochondrion-associated ER membranes (MAMs). Several functions have been ascribed to this ER-resident chaperone, many of which are related to Ca(2+) signaling at the MAMs and lipid storage and trafficking. Downregulation of S1R expression by RNA interference (RNAi) in Huh-7 cells leads to a proportional decrease in susceptibility to HCV infection, as shown by reduced HCV RNA accumulation and intra- and extracellular infectivity in single-cycle infection experiments. Similar RNAi studies in persistently infected cells indicate that S1R expression is not rate limiting for persistent HCV RNA replication, as marked reduction in S1R in these cells does not lead to any decrease in HCV RNA or viral protein expression. However, subgenomic replicon transfection experiments indicate that S1R expression is rate limiting for HCV RNA replication without impairing primary translation. Overall, our data indicate that the initial steps of HCV infection are regulated by S1R, a key component of MAMs, suggesting that these structures could serve as platforms for initial RNA replication during HCV infection.
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Regulación Viral de la Expresión Génica , Hepacivirus/genética , Hepatitis C/metabolismo , Hepatitis C/virología , ARN Viral/genética , Receptores sigma/metabolismo , Replicación Viral , Línea Celular , Retículo Endoplásmico/genética , Retículo Endoplásmico/metabolismo , Hepacivirus/fisiología , Hepatitis C/genética , Humanos , ARN Viral/metabolismo , Receptores sigma/genética , Receptor Sigma-1RESUMEN
To better define the mechanism(s) likely responsible for viral clearance during hepatitis B virus (HBV) infection, viral clearance was studied in a panel of immunodeficient mouse strains that were hydrodynamically transfected with a plasmid containing a replication-competent copy of the HBV genome. Neither B cells nor perforin were required to clear the viral DNA transcriptional template from the liver. In contrast, the template persisted for at least 60 days at high levels in NOD/Scid mice and at lower levels in the absence of CD4(+) and CD8(+) T cells, NK cells, Fas, IFN-gamma (IFN-gamma), IFN-alpha/beta receptor (IFN-alpha/betaR1), and TNF receptor 1 (TNFR1), indicating that each of these effectors was required to eliminate the transcriptional template from the liver. Interestingly, viral replication was ultimately terminated in all lineages except the NOD/Scid mice, suggesting the existence of redundant pathways that inhibit HBV replication. Finally, induction of a CD8(+) T cell response in these animals depended on the presence of CD4(+) T cells. These results are consistent with a model in which CD4(+) T cells serve as master regulators of the adaptive immune response to HBV; CD8(+) T cells are the key cellular effectors mediating HBV clearance from the liver, apparently by a Fas-dependent, perforin-independent process in which NK cells, IFN-gamma, TNFR1, and IFN-alpha/betaR play supporting roles. These results provide insight into the complexity of the systems involved in HBV clearance, and they suggest unique directions for analysis of the mechanism(s) responsible for HBV persistence.
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Virus de la Hepatitis B/genética , Hepatitis B/inmunología , Animales , Linfocitos T CD4-Positivos , Linfocitos T CD8-positivos/inmunología , Carcinoma Hepatocelular/epidemiología , ADN Viral/genética , Ensayo de Inmunoadsorción Enzimática , Regulación Viral de la Expresión Génica , Genoma Viral , Hepatitis B/complicaciones , Hepatitis B/epidemiología , Hepatitis B/genética , Antígenos del Núcleo de la Hepatitis B/genética , Antígenos de Superficie de la Hepatitis B/sangre , Virus de la Hepatitis B/inmunología , Humanos , Neoplasias Hepáticas/epidemiología , Ratones , Ratones Endogámicos NOD , Ratones Noqueados , Ratones SCID , Transcripción Genética , Replicación ViralRESUMEN
The recent development of a cell culture model of hepatitis C virus (HCV) infection based on the JFH-1 molecular clone has enabled discovery of new antiviral agents. Using a cell-based colorimetric screening assay to interrogate a 1,200-compound chemical library for anti-HCV activity, we identified a family of 1,2-diamines derived from trans-stilbene oxide that prevent HCV infection at nontoxic, low micromolar concentrations in cell culture. Structure-activity relationship analysis of ~ 300 derivatives synthesized using click chemistry yielded compounds with greatly enhanced low nanomolar potency and a > 1,000:1 therapeutic ratio. Using surrogate models of HCV infection, we showed that the compounds selectively block the initiation of replication of incoming HCV RNA but have no impact on viral entry, primary translation, or ongoing HCV RNA replication, nor do they suppress persistent HCV infection. Selection of an escape variant revealed that NS5A is directly or indirectly targeted by this compound. In summary, we have identified a family of HCV inhibitors that target a critical step in the establishment of HCV infection in which NS5A translated de novo from an incoming genomic HCV RNA template is required to initiate the replication of this important human pathogen.
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Antivirales/farmacología , Diaminas/farmacología , Hepacivirus/efectos de los fármacos , Estilbenos/farmacología , Replicación Viral/efectos de los fármacos , Antivirales/química , Antivirales/aislamiento & purificación , Antivirales/toxicidad , Línea Celular , Supervivencia Celular/efectos de los fármacos , Diaminas/química , Diaminas/aislamiento & purificación , Diaminas/toxicidad , Evaluación Preclínica de Medicamentos/métodos , Farmacorresistencia Viral , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Humanos , Pruebas de Sensibilidad Microbiana , ARN Viral/metabolismo , Estilbenos/química , Estilbenos/aislamiento & purificación , Estilbenos/toxicidad , Relación Estructura-Actividad , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
Development of hepatitis C virus (HCV) infection cell culture systems has permitted the identification of cellular factors that regulate the HCV life cycle. Some of these cellular factors affect steps in the viral life cycle that are tightly associated with intracellular membranes derived from the endoplasmic reticulum (ER). Here, we describe the discovery of erlin-1 protein as a cellular factor that regulates HCV infection. Erlin-1 is a cholesterol-binding protein located in detergent-resistant membranes within the ER. It is implicated in cholesterol homeostasis and the ER-associated degradation pathway. Silencing of erlin-1 protein expression by siRNA led to decreased infection efficiency characterized by reduction in intracellular RNA accumulation, HCV protein expression and virus production. Mechanistic studies revealed that erlin-1 protein is required early in the infection, downstream of cell entry and primary translation, specifically to initiate RNA replication, and later in the infection to support infectious virus production. This study identifies erlin-1 protein as an important cellular factor regulating HCV infection.
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Hepacivirus/fisiología , Hepatitis C/metabolismo , Hepatitis C/virología , Interacciones Huésped-Patógeno , Proteínas del Tejido Nervioso/metabolismo , Línea Celular Tumoral , Silenciador del Gen , Hepatitis C/genética , Humanos , Metabolismo de los Lípidos , Proteínas del Tejido Nervioso/genética , ARN Interferente Pequeño/genética , Carga Viral , Internalización del Virus , Replicación ViralRESUMEN
BACKGROUND: Hepatitis C Virus (HCV) is a significant public health burden and small animal models are needed to study the pathology and immunobiology of the virus. In effort to develop experimental HCV mouse models, we screened a panel of HCV replicons to identify clones capable of replicating in mouse hepatocytes. RESULTS: We report the establishment of stable HCV replication in mouse hepatocyte and fibroblast cell lines using replicons derived from the JFH-1 genotype 2a consensus sequence. Viral RNA replication efficiency in mouse cells was comparable to that observed in human Huh-7 replicon cells, with negative-strand HCV RNA and the viral NS5A protein being readily detected by Northern and Western Blot analysis, respectively. Although HCV replication was established in the absence of adaptive mutations that might otherwise compromise the in vitro infectivity of the JFH-1 clone, no infectious virus was detected when the culture medium from full length HCV RNA replicating mouse cells was titrated on Huh-7 cells, suggesting that the mouse cells were unable to support production of infectious progeny viral particles. Consistent with an additional block in viral entry, infectious JFH-1 particles produced in Huh-7 cells were not able to establish detectable HCV RNA replication in naïve mouse cells. CONCLUSION: Thus, this report expands the repertoire of HCV replication systems and possibly represents a step toward developing mouse models of HCV replication, but it also highlights that other species restrictions might continue to make the development of a purely murine HCV infectious model challenging.
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Fibroblastos/virología , Hepacivirus/genética , Hepatitis C/virología , Hepatocitos/virología , Replicón , Replicación Viral , Animales , Línea Celular , Línea Celular Tumoral , Hepacivirus/fisiología , Humanos , Ratones , Modelos Animales , Mutación , Especificidad de la Especie , TransfecciónRESUMEN
Viral nucleic acids often trigger an innate immune response in infected cells. Many viruses, including hepatitis C virus (HCV), have evolved mechanisms to evade intracellular recognition. Nevertheless, HCV-permissive cells can trigger a viral RNA-, TLR7-, and cell-contact-dependent compensatory interferon response in nonpermissive plasmacytoid dendritic cells (pDCs). Here we report that these events are mediated by transfer of HCV-RNA-containing exosomes from infected cells to pDCs. The exosomal viral RNA transfer is dependent on the endosomal sorting complex required for transport (ESCRT) machinery and on Annexin A2, an RNA-binding protein involved in membrane vesicle trafficking, and is suppressed by exosome release inhibitors. Further, purified concentrated HCV-RNA-containing exosomes are sufficient to activate pDCs. Thus, vesicular sequestration and exosomal export of viral RNA may serve both as a viral strategy to evade pathogen sensing within infected cells and as a host strategy to induce an unopposed innate response in replication-nonpermissive bystander cells.
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Células Dendríticas/inmunología , Células Dendríticas/metabolismo , Exosomas/metabolismo , Hepacivirus/inmunología , Inmunidad Innata , ARN Viral/inmunología , ARN Viral/metabolismo , Células Cultivadas , Células Dendríticas/virología , Exosomas/virología , Hepatocitos/virología , HumanosRESUMEN
Hepatitis C virus (HCV) infection is a major worldwide health problem. Our previous results showed that HCV evolved to gain the enhanced infectivity and altered buoyant density distribution during persistent infections in vitro. Here we showed that a point mutation I414T in HCV E2 was mainly responsible for these phenotypic changes. While the I414T mutation had no significant effect on HCV RNA replication and viral entry, it enhanced the production of infectious viral particles and decreased the dependency of viral entry on the levels of HCV receptors. Furthermore, we showed that the I414T mutation reduced the association of viral particles with low-density lipoprotein or very low-density lipoproteins during the virus secretion process, and the infection of the delipidated virus was more sensitive to the blockade by an anti-E2 neutralizing antibody and recombinant CD81 proteins. Our results provided more insights into understanding the roles of lipoprotein associations in HCV life cycle.
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Hepacivirus/patogenicidad , Lipoproteínas LDL/inmunología , Mutación Puntual , Proteínas del Envoltorio Viral/genética , Anticuerpos Neutralizantes/inmunología , Antígenos CD/inmunología , Línea Celular , Hepacivirus/fisiología , Hepatitis C/inmunología , Hepatitis C/virología , Humanos , ARN Viral/metabolismo , Tetraspanina 28 , Virión/patogenicidad , Virión/fisiología , Internalización del VirusRESUMEN
Virus infection triggers IFN immune defenses in infected cells in part through viral nucleic acid interactions, but the pathways by which dsDNA and DNA viruses trigger innate defenses are only partially understood. Here we present evidence that both retinoic acid-induced gene I (RIG-I) and mitochondrial antiviral signaling protein (MAVS) are required for dsDNA-induced IFN-beta promoter activation in a human hepatoma cell line (Huh-7), and that activation is efficiently blocked by the hepatitis C virus NS3/4A protease, which is known to block dsRNA signaling by cleaving MAVS. These findings suggest that dsDNA and dsRNA share a common pathway to trigger the innate antiviral defense response in human cells, although dsDNA appears to trigger that pathway upstream of the dsRNA-interacting protein RIG-I.
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ADN/metabolismo , ARN Bicatenario/metabolismo , Transducción de Señal , Simplexvirus/inmunología , Antivirales , Línea Celular Tumoral , Citosol/metabolismo , Proteína 58 DEAD Box , ARN Helicasas DEAD-box/metabolismo , ADN/genética , ADN/inmunología , Regulación de la Expresión Génica , Humanos , Inmunidad Innata/inmunología , Interferón beta/genética , Interferón beta/metabolismo , Regiones Promotoras Genéticas , ARN Bicatenario/genética , ARN Bicatenario/inmunología , Receptores Inmunológicos , Proteínas no Estructurales Virales/genética , Proteínas no Estructurales Virales/metabolismoRESUMEN
The virological and cellular consequences of persistent hepatitis C virus (HCV) infection have been elusive due to the absence of the requisite experimental systems. Here, we report the establishment and the characteristics of persistent in vitro infection of human hepatoma-derived cells by a recently described HCV genotype 2a infectious molecular clone. Persistent in vitro infection was characterized by the selection of viral variants that displayed accelerated expansion kinetics, higher peak titers, and increased buoyant densities. Sequencing analysis revealed the selection of a single adaptive mutation in the HCV E2 envelope protein that was largely responsible for the variant phenotype. In parallel, as the virus became more aggressive, cells that were resistant to infection emerged, displaying escape mechanisms operative at the level of viral entry, HCV RNA replication, or both. Collectively, these results reveal the existence of coevolutionary events during persistent HCV infection that favor survival of both virus and host.
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Adaptación Biológica , Hepacivirus/fisiología , Hepatocitos/virología , Línea Celular Tumoral , Análisis Mutacional de ADN , Hepacivirus/genética , Humanos , Mutación Missense , ARN Viral/química , ARN Viral/genética , Análisis de Secuencia de ADN , Proteínas del Envoltorio Viral , Internalización del Virus , Replicación ViralRESUMEN
The chimpanzee (Pan troglodytes) is an important model for studying the immune response to several human pathogens, but the study of correlates of immunity has been hindered by the fact that little is known about the epitope-binding specificity of chimpanzee (Patr) class I MHC. In the present study we have characterized the peptide binding specificity of several common Patr class I molecules. Using single amino acid substitution analogs and large peptide libraries, quantitative peptide binding motifs have been derived for Patr A*0101, A*0701, A*0901, B*0101, and B*2401. Each molecule was found to bind peptides using position 2 and the C terminus as main anchor contacts. On the other hand, each Patr molecule is associated with a unique binding specificity, and the range of specificities is similar to that seen amongst HLA alleles. A high degree of cross-reactivity was noted between Patr A*0701 and Patr A*0901, suggesting the existence of a Patr-specific supertype. Consistent with previous studies suggesting that some cross-reactivity may exist between HLA and Patr alleles, Patr A*0901 was found to have an appreciable degree of cross-reactivity with molecules of the HLA A24-supertype. Finally, utilizing motif scans and peptide binding and intracellular cytokine staining assays, 77 hepatitis B virus (HBV)-derived epitopes were identified in five chimpanzees that were recently convalescent from acute HBV infection. Because the Patr alleles studied herein were found to be very common in two different chimpanzee populations, the present data should facilitate the use of chimpanzees for immunological studies.
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Antígenos de Histocompatibilidad Clase I/inmunología , Epítopos Inmunodominantes/inmunología , Modelos Animales , Pan troglodytes/inmunología , Péptidos/inmunología , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Reacciones Cruzadas , Antígenos HLA-A/genética , Antígenos HLA-A/inmunología , Antígenos de la Hepatitis B/inmunología , Antígenos de Histocompatibilidad Clase I/genética , Humanos , Epítopos Inmunodominantes/genética , Datos de Secuencia Molecular , Pan troglodytes/genética , Biblioteca de Péptidos , Péptidos/genéticaRESUMEN
Persistent hepatitis B virus (HBV) infection is characterized by a weak and narrowly focused CD8+ T-cell response to HBV that is thought to reflect the induction of central and/or peripheral tolerance to HBV proteins in neonatal and adult onset infections, respectively. Immunotherapeutic strategies that overcome tolerance and boost these suboptimal responses may lead to viral clearance in chronically infected individuals. The present study was performed to compare the relative immunogenicities and tolerogenicities of HBV structural (envelope [ENV]) and nonstructural (polymerase [POL]) proteins at the CD8+ cytotoxic T lymphocyte (CTL) level in transgenic mice that replicate HBV in the liver and secrete infectious virus into the blood, thus representing an excellent model of persistent HBV infection. Interestingly, the mice were tolerant to the ENV but not to the POL proteins at the CTL level. Furthermore, the POL-specific CTLs had no impact on HBV replication or liver function in vivo, even though they were readily induced and reached the liver after DNA immunization, reflecting their relatively low avidity and the low level at which the POL protein is expressed by the hepatocyte. Collectively, these results suggest that the factors that make POL less tolerogenic also make POL-specific CTLs relatively inefficient effector cells when they reach the target organ. Immunotherapeutic strategies to control HBV infection by inducing virus-specific CTL responses in chronically infected subjects should be evaluated in light of this observation.
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Productos del Gen env/inmunología , Productos del Gen pol/inmunología , Vacunas contra Hepatitis B/inmunología , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/prevención & control , Tolerancia Inmunológica , Animales , Linfocitos T CD8-positivos/inmunología , Productos del Gen env/genética , Productos del Gen pol/genética , Vacunas contra Hepatitis B/administración & dosificación , Virus de la Hepatitis B/genética , Hepatitis B Crónica/inmunología , Inmunización , Hígado/inmunología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Virus Vaccinia/genéticaRESUMEN
Hepatitis B virus (HBV) is a prototype for liver-specific pathogens in which the failure of the immune system to mount an effective response leads to chronic infection. Our understanding of the immune response to HBV is incomplete, largely due to the narrow host restriction of this pathogen and the limitations of existing experimental models. We have developed a murine model for studying human HBV replication, immunogenicity, and control. After transfection of hepatocytes in vivo with a replication-competent, over-length, linear HBV genome, viral antigens and replicative intermediates were synthesized and virus was secreted into the blood. Viral antigens disappeared from the blood as early as 7 days after transfection, coincident with the appearance of antiviral antibodies. HBV transcripts and replicative intermediates disappeared from the liver by day 15, after the appearance of antiviral CD8 + T cells. In contrast, the virus persisted for at least 81 days after transfection of NOD/Scid mice, which lack functional T cells, B cells, and natural killer (NK) cells. Thus, the outcome of hydrodynamic transfection of HBV depends on the host immune response, as it is during a natural infection. The methods we describe will allow the examination of viral dynamics in a tightly controlled in vivo system, the application of mutagenesis methods to the study of the HBV life cycle in vivo, and the dissection of the immune response to HBV using genetically modified mice whose immunoregulatory and immune effector functions have been deleted or overexpressed. In addition, this methodology represents a prototype for the study of other known and to-be-discovered liver-specific pathogens.