RESUMEN
Hepatocellular carcinoma (HCC) is an aggressive malignancy with its global incidence and mortality rate continuing to rise, although early detection and surveillance are suboptimal. We performed serological profiling of the viral infection history in 899 individuals from an NCI-UMD case-control study using a synthetic human virome, VirScan. We developed a viral exposure signature and validated the results in a longitudinal cohort with 173 at-risk patients who had long-term follow-up for HCC development. Our viral exposure signature significantly associated with HCC status among at-risk individuals in the validation cohort (area under the curve: 0.91 [95% CI 0.87-0.96] at baseline and 0.98 [95% CI 0.97-1] at diagnosis). The signature identified cancer patients prior to a clinical diagnosis and was superior to alpha-fetoprotein. In summary, we established a viral exposure signature that can predict HCC among at-risk patients prior to a clinical diagnosis, which may be useful in HCC surveillance.
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Carcinoma Hepatocelular/patología , Neoplasias Hepáticas/patología , Virosis/patología , Adulto , Anciano , Área Bajo la Curva , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Estudios de Casos y Controles , Estudios de Cohortes , Bases de Datos Genéticas , Femenino , Estudio de Asociación del Genoma Completo , Humanos , Desequilibrio de Ligamiento , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Curva ROC , Factores de Riesgo , Virosis/complicaciones , Adulto Joven , alfa-Fetoproteínas/análisisRESUMEN
Hepatitis B virus (HBV) remains a major public health threat with nearly 300 million people chronically infected worldwide who are at a high risk of developing hepatocellular carcinoma. Current therapies are effective in suppressing HBV replication but rarely lead to cure. Current therapies do not affect the HBV covalently closed circular DNA (cccDNA), which serves as the template for viral transcription and replication and is highly stable in infected cells to ensure viral persistence. In this study, we aim to identify and elucidate the functional role of cccDNA-associated host factors using affinity purification and protein mass spectrometry in HBV-infected cells. Nucleolin was identified as a key cccDNA-binding protein and shown to play an important role in HBV cccDNA transcription, likely via epigenetic regulation. Targeting nucleolin to silence cccDNA transcription in infected hepatocytes may be a promising therapeutic strategy for a functional cure of HBV.
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Hepatitis B , Neoplasias Hepáticas , Humanos , Virus de la Hepatitis B/fisiología , Epigénesis Genética , Replicación Viral/genética , ADN Viral/metabolismo , ADN Circular/genética , ADN Circular/metabolismo , Neoplasias Hepáticas/genética , Hepatitis B/genética , Hepatitis B/metabolismo , NucleolinaRESUMEN
BACKGROUND: A safe and effective vaccine to prevent chronic hepatitis C virus (HCV) infection is a critical component of efforts to eliminate the disease. METHODS: In this phase 1-2 randomized, double-blind, placebo-controlled trial, we evaluated a recombinant chimpanzee adenovirus 3 vector priming vaccination followed by a recombinant modified vaccinia Ankara boost; both vaccines encode HCV nonstructural proteins. Adults who were considered to be at risk for HCV infection on the basis of a history of recent injection drug use were randomly assigned (in a 1:1 ratio) to receive vaccine or placebo on days 0 and 56. Vaccine-related serious adverse events, severe local or systemic adverse events, and laboratory adverse events were the primary safety end points. The primary efficacy end point was chronic HCV infection, defined as persistent viremia for 6 months. RESULTS: A total of 548 participants underwent randomization, with 274 assigned to each group. There was no significant difference in the incidence of chronic HCV infection between the groups. In the per-protocol population, chronic HCV infection developed in 14 participants in each group (hazard ratio [vaccine vs. placebo], 1.53; 95% confidence interval [CI], 0.66 to 3.55; vaccine efficacy, -53%; 95% CI, -255 to 34). In the modified intention-to-treat population, chronic HCV infection developed in 19 participants in the vaccine group and 17 in placebo group (hazard ratio, 1.66; 95% CI, 0.79 to 3.50; vaccine efficacy, -66%; 95% CI, -250 to 21). The geometric mean peak HCV RNA level after infection differed between the vaccine group and the placebo group (152.51×103 IU per milliliter and 1804.93×103 IU per milliliter, respectively). T-cell responses to HCV were detected in 78% of the participants in the vaccine group. The percentages of participants with serious adverse events were similar in the two groups. CONCLUSIONS: In this trial, the HCV vaccine regimen did not cause serious adverse events, produced HCV-specific T-cell responses, and lowered the peak HCV RNA level, but it did not prevent chronic HCV infection. (Funded by the National Institute of Allergy and Infectious Diseases; ClinicalTrials.gov number, NCT01436357.).
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Anticuerpos contra la Hepatitis C/sangre , Hepatitis C Crónica/prevención & control , Inmunogenicidad Vacunal , Vacunas contra Hepatitis Viral/inmunología , Adenovirus de los Simios/genética , Adolescente , Adulto , Animales , Método Doble Ciego , Femenino , Vectores Genéticos , Hepatitis C Crónica/epidemiología , Hepatitis C Crónica/inmunología , Humanos , Incidencia , Masculino , Persona de Mediana Edad , Pan troglodytes , Abuso de Sustancias por Vía Intravenosa , Linfocitos T/inmunología , Vacunas Sintéticas/inmunología , Vacunas contra Hepatitis Viral/efectos adversos , Adulto JovenRESUMEN
Despite the development of highly effective hepatitis C virus (HCV) treatments, an effective prophylactic vaccine is still lacking. HCV infection is mediated by its envelope glycoproteins, E1 and E2, during the entry process, with E2 binding to cell receptors and E1 mediating endosomal fusion. The structure of E1E2 has only been partially resolved by X-ray crystallography of the core domain of E2 protein (E2c) and its complex with various neutralizing antibodies. Structural understanding of the E1E2 heterodimer in its native form can advance the design of candidates for HCV vaccine development. Here, we analyze the structure of the recombinant HCV E1E2 heterodimer with the aid of well-defined monoclonal anti-E1 and E2 antibodies, as well as a small-molecule chlorcyclizine-diazirine-biotin that can target and cross-link the putative E1 fusion domain. Three-dimensional (3D) models were generated after extensive 2D classification analysis with negative-stain single-particle data sets. We modeled the available crystal structures of the E2c and Fabs into 3D volumes of E1E2-Fab complexes based on the shape and dimension of the domain density. The E1E2 heterodimer exists in monomeric form and consists of a main globular body, presumably depicting the E1 and E2 stem/transmembrane domain, and a protruding structure representing the E2c region, based on anti-E2 Fab binding. At low resolution, a model generated from negative-stain analysis revealed the unique binding and orientation of individual or double Fabs onto the E1 and E2 components of the complex. Cryo-electron microscopy (cryo-EM) of the double Fab complexes resulted in a refined structural model of the E1E2 heterodimer, presented here. IMPORTANCE Recombinant HCV E1E2 heterodimer is being developed as a vaccine candidate. Using electron microscopy, we demonstrated unique features of E1E2 in complex with various neutralizing antibodies and small molecule inhibitors that are important to understanding its antigenicity and induction of immune response.
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Hepacivirus , Proteínas del Envoltorio Viral , Humanos , Anticuerpos Neutralizantes/química , Microscopía por Crioelectrón , Electrones , Hepacivirus/fisiología , Hepatitis C , Imagenología Tridimensional , Proteínas del Envoltorio Viral/química , Conformación ProteicaRESUMEN
BACKGROUND AND AIMS: Mutations within the precore (PC) and basal core promoter (BCP) regions of the HBV genome are associated with fulminant hepatitis and HBV reactivation. These mutations may enhance viral replication, but little is known about whether they directly induce damage to the liver. We investigated mechanisms of direct cytopathic effects induced by the infection with PC/BCP mutants in the absence of immune response in vitro and in vivo . APPROACH AND RESULTS: Mice with humanized livers and hepatocytes derived from humanized mice were infected with either wild-type or mutant-type PC/BCP HBV, and the HBV replication and human hepatocyte damage were evaluated. HBV proliferated vigorously in mice with PC/BCP-mutant infection, and the severe loss of human hepatocytes with a slight human ALT elevation subsequently occurred only in PC/BCP mutant mice. In PC/BCP mutant infection, the accumulation of HBsAg in humanized livers colocalized with the endoplasmic reticulum, leading to apoptosis through unfolded protein response in HBV-infected hepatocytes. RNA-sequencing revealed the molecular characteristics of the phenotype of PC/BCP mutant infection in a humanized mouse model. Reduced ALT elevation and higher HBV DNA levels in this model are consistent with characteristics of HBV reactivation, indicating that the hepatocyte damage in this model might mimic HBV reactivation followed by hepatocyte damage under immunosuppressive conditions. CONCLUSION: PC and BCP mutations were associated with enhanced viral replication and cell death induced by ER stress using HBV infection models. These mutations might be associated with liver damage in patients with fulminant hepatitis or HBV reactivation.
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Virus de la Hepatitis B , Necrosis Hepática Masiva , Humanos , Animales , Ratones , Mutación , Fenotipo , Muerte Celular , ADN Viral/genética , Genotipo , Antígenos e de la Hepatitis B/genéticaRESUMEN
Global elimination of hepatitis C virus (HCV) will be difficult to attain without an effective HCV vaccine. Controlled human infection (CHI) studies with HCV were not considered until recently, when highly effective treatment became available. However, now that successful treatment of a deliberate HCV infection is feasible, it is imperative to evaluate the ethics of establishing a program of HCV CHI research. Here, we evaluate the ethics of studies to develop an HCV CHI model in light of 10 ethical considerations: sufficient social value, reasonable risk-benefit profile, suitable site selection, fair participant selection, robust informed consent, proportionate compensation or payment, context-specific stakeholder engagement, fair and open collaboration, independent review and oversight, and integrated ethics research. We conclude that it can be ethically acceptable to develop an HCV CHI model. Indeed, when done appropriately, developing a model should be a priority on the path toward global elimination of HCV.
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Hepacivirus , Hepatitis C , Humanos , Hepatitis C/epidemiología , Hepatitis C/prevención & control , Hepatitis C/tratamiento farmacológico , Consentimiento Informado , Antivirales/uso terapéuticoRESUMEN
For any controlled human infection model (CHIM), a safe, standardized, and biologically relevant challenge inoculum is necessary. For hepatitis C virus (HCV) CHIM, we propose that human-derived high-titer inocula of several viral genotypes with extensive virologic, serologic, and molecular characterizations should be the most appropriate approach. These inocula should first be tested in human volunteers in a step-wise manner to ensure safety, reproducibility, and curability prior to using them for testing the efficacy of candidate vaccines.
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Hepacivirus , Hepatitis C , Humanos , Hepacivirus/genética , Reproducibilidad de los ResultadosRESUMEN
INTRODUCTION: Bile duct involvement is a key finding of primary biliary cholangitis (PBC). The aim of this study was to evaluate baseline ductopenia and disease progression. METHODS: Retrospective longitudinal histological follow-up of treatment-naive patients with PBC. RESULTS: Eighty-three patients were included, with ductopenia correlated to fibrosis stage at baseline. The cumulative incidence of severe ductopenia remained stable after 5 years, whereas fibrosis continually increased over time. Baseline AST-to-Platelet Ratio Index and elevated alkaline phosphatase >2 times the normal with abnormal bilirubin were associated with ductopenia progression. DISCUSSION: Bile duct injury does not seem to follow the same course as fibrosis in PBC.
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Colangitis , Cirrosis Hepática Biliar , Humanos , Cirrosis Hepática Biliar/complicaciones , Cirrosis Hepática Biliar/diagnóstico , Cirrosis Hepática Biliar/epidemiología , Estudios Retrospectivos , Conductos Biliares/diagnóstico por imagen , Conductos Biliares/patología , Fibrosis , Incidencia , Colangitis/diagnósticoRESUMEN
BACKGROUND AND AIMS: NAFLD is a growing public health burden. However, the pathogenesis of NAFLD has not yet been fully elucidated, and the importance of genetic factors has only recently been appreciated. Genomic studies have revealed a strong association between NAFLD progression and the I148M variant in patatin-like phospholipase domain-containing protein 3 (PNPLA3). Nonetheless, very little is known about the mechanisms by which this gene and its variants can influence disease development. To investigate these mechanisms, we have developed an in vitro model that takes advantage of the unique properties of human-induced pluripotent stem cells (hiPSCs) and the CRISPR/CAS9 gene editing technology. APPROACH AND RESULTS: We used isogenic hiPSC lines with either a knockout (PNPLA3KO ) of the PNPLA3 gene or with the I148M variant (PNPLA3I148M ) to model PNPLA3-associated NAFLD. The resulting hiPSCs were differentiated into hepatocytes, treated with either unsaturated or saturated free fatty acids to induce NAFLD-like phenotypes, and characterized by various functional, transcriptomic, and lipidomic assays. PNPLA3KO hepatocytes showed higher lipid accumulation as well as an altered pattern of response to lipid-induced stress. Interestingly, loss of PNPLA3 also caused a reduction in xenobiotic metabolism and predisposed PNPLA3KO cells to be more susceptible to ethanol-induced and methotrexate-induced toxicity. The PNPLA3I148M cells exhibited an intermediate phenotype between the wild-type and PNPLA3KO cells. CONCLUSIONS: Together, these results indicate that the I148M variant induces a loss of function predisposing to steatosis and increased susceptibility to hepatotoxins.
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Hepatocitos/patología , Lipasa/genética , Proteínas de la Membrana/genética , Enfermedad del Hígado Graso no Alcohólico/genética , Sistemas CRISPR-Cas/genética , Diferenciación Celular , Línea Celular , Etanol/toxicidad , Técnicas de Inactivación de Genes , Predisposición Genética a la Enfermedad , Hepatocitos/efectos de los fármacos , Humanos , Células Madre Pluripotentes Inducidas , Metabolismo de los Lípidos/genética , Mutación con Pérdida de Función , Metotrexato/toxicidad , Enfermedad del Hígado Graso no Alcohólico/patología , Polimorfismo de Nucleótido Simple , Pruebas de Toxicidad AgudaRESUMEN
BACKGROUND AND AIMS: An efficient cell-culture system for hepatitis B virus (HBV) is indispensable for research on viral characteristics and antiviral reagents. Currently, for the HBV infection assay in cell culture, viruses derived from HBV genome-integrated cell lines of HepG2.2.15 or HepAD-38 are commonly used. However, these viruses are not suitable for the evaluation of polymorphism-dependent viral characteristics or resistant mutations against antiviral reagents. HBV obtained by the transient transfection of the ordinary HBV molecular clone has limited infection efficiencies in cell culture. APPROACH AND RESULTS: We found that an 11-amino-acid deletion (d11) in the preS1 region enhances the infectivity of cell-culture-generated HBV (HBVcc) to sodium taurocholate cotransporting polypeptide-transduced HepG2 (HepG2/NTCP) cells. Infection of HBVcc derived from a d11-introduced genotype C strain (GTC-d11) was ~10-fold more efficient than infection of wild-type GTC (GTC-wt), and the number of infected cells was comparable between GTC-d11- and HepG2.2.15-derived viruses when inoculated with the same genome equivalents. A time-dependent increase in pregenomic RNA and efficient synthesis of covalently closed circular DNA were detected after infection with the GTC-d11 virus. The involvement of d11 in the HBV large surface protein in the enhanced infectivity was confirmed by an HBV reporter virus and hepatitis D virus infection system. The binding step of the GTC-d11 virus onto the cell surface was responsible for this efficient infection. CONCLUSIONS: This system provides a powerful tool for studying the infection and propagation of HBV in cell culture and also for developing the antiviral strategy against HBV infection.
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Técnicas de Cultivo de Célula/métodos , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/patogenicidad , Hepatitis B/virología , Precursores de Proteínas/metabolismo , Antivirales/farmacología , Antivirales/uso terapéutico , Evaluación Preclínica de Medicamentos/métodos , Células Hep G2 , Hepatitis B/tratamiento farmacológico , Hepatitis B/patología , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Humanos , Precursores de Proteínas/genéticaRESUMEN
BACKGROUND & AIMS: HBV consists of 9 major genotypes (A to I), 1 minor strain (designated J) and multiple subtypes, which may be associated with different clinical characteristics. As only cell lines expressing genotype D3 have been established, herein, we aimed to establish stable cell lines producing high-titer cell culture-generated HBV (HBVcc) of different genotypes and to explore their infectivity, virological features and responses to treatment. METHODS: Stable cell lines producing high titers of HBV genotype A2, B2, C1, E, F1b and H were generated by transfecting plasmids containing a replication-competent 1.3x length HBV genome and an antibiotic marker into HepG2 cells that can support HBV replication. Clones with the highest levels of HBV DNA and/or HBeAg were selected and expanded for large-scale purification of HBVcc. HBVcc of different genotypes were tested in cells and a humanized chimeric mouse model. RESULTS: HBVcc genotypes were infectious in mouse-passaged primary human hepatocytes (PXB cells) and responded differently to human interferon (IFN)-α with variable kinetics of reduction in HBV DNA, HBeAg and HBsAg. HBVcc of all genotypes were infectious in humanized chimeric mice but with variable kinetics of viremia and viral antigen production. Treatment of infected mice with human IFN-α resulted in modest and variable reductions of viremia and viral antigenemia. HBVcc passaged in humanized chimeric mice (HBVmp) infected PXB cells much more efficiently than that of the original HBVcc viral stock. CONCLUSIONS: Herein, we generated stable cell lines producing HBV of various genotypes that are infectious in vitro and in vivo. We observe genotype-associated variations in viral antigen production, infection kinetics and responses to human IFN-α treatment in these models. LAY SUMMARY: Stable cell lines producing high-titer cell culture-generated hepatitis B virus (HBV) of various genotypes were established. HBV genotypes showed stable infectivity in both in vitro and in vivo models, which are valuable tools for antiviral development.
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Genotipo , Hepatitis B/complicaciones , Animales , Técnicas de Cultivo de Célula/métodos , Técnicas de Cultivo de Célula/estadística & datos numéricos , Modelos Animales de Enfermedad , Hepatitis B/genética , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/patogenicidad , RatonesRESUMEN
In as few as 3 months, coronavirus disease 2019 (COVID-19) has spread and ravaged the world at an unprecedented speed in modern history, rivaling the 1918 flu pandemic. Severe acute respiratory syndrome coronavirus-2, the culprit virus, is highly contagious and stable in the environment and transmits predominantly among humans via the respiratory route. Accumulating evidence suggest that this virus, like many of its related viruses, may also be an enteric virus that can spread via the fecal-oral route. Such a hypothesis would also contribute to the rapidity and proliferation of this pandemic. Here we briefly summarize what is known about this family of viruses and literature basis of the hypothesis that severe acute respiratory syndrome coronavirus-2 is capable of infecting the gastrointestinal tract and shedding in the environment for potential human-to-human transmission.
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Betacoronavirus/patogenicidad , Infecciones por Coronavirus/transmisión , Heces/virología , Tracto Gastrointestinal/virología , Neumonía Viral/transmisión , Esparcimiento de Virus , Animales , Betacoronavirus/aislamiento & purificación , COVID-19 , Prueba de COVID-19 , Técnicas de Laboratorio Clínico , Infecciones por Coronavirus/diagnóstico , Infecciones por Coronavirus/epidemiología , Infecciones por Coronavirus/virología , Humanos , Pandemias , Neumonía Viral/diagnóstico , Neumonía Viral/epidemiología , Neumonía Viral/virología , SARS-CoV-2RESUMEN
Hepatitis B virus (HBV) infection affects approximately 300 million people worldwide. Although antiviral therapies have improved the long-term outcomes, patients often require life-long treatment and there is no cure for HBV infection. New technologies can help us learn more about the pathogenesis of HBV infection and develop therapeutic agents to reduce its burden. We review recent advances in development of direct-acting antiviral and host-targeting agents, some of which have entered clinical trials. We also discuss strategies for unbiased high-throughput screens to identify compounds that inhibit HBV and for repurposing existing drugs.
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Antivirales/farmacología , Desarrollo de Medicamentos , Hepatitis B/terapia , Cápside/efectos de los fármacos , Humanos , Factores Inmunológicos/farmacología , Receptores Inmunológicos/efectos de los fármacos , Receptores Virales/efectos de los fármacos , Replicación Viral/efectos de los fármacosRESUMEN
Nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver disease. A single-nucleotide polymorphism (SNP), rs6834314, was associated with serum liver enzymes in the general population, presumably reflecting liver fat or injury. We studied rs6834314 and its nearest gene, 17-beta hydroxysteroid dehydrogenase 13 (HSD17B13), to identify associations with histological features of NAFLD and to characterize the functional role of HSD17B13 in NAFLD pathogenesis. The minor allele of rs6834314 was significantly associated with increased steatosis but decreased inflammation, ballooning, Mallory-Denk bodies, and liver enzyme levels in 768 adult Caucasians with biopsy-proven NAFLD and with cirrhosis in the general population. We found two plausible causative variants in the HSD17B13 gene. rs72613567, a splice-site SNP in high linkage with rs6834314 (r2 = 0.94) generates splice variants and shows a similar pattern of association with NAFLD histology. Its minor allele generates simultaneous expression of exon 6-skipping and G-nucleotide insertion variants. Another SNP, rs62305723 (encoding a P260S mutation), is significantly associated with decreased ballooning and inflammation. Hepatic expression of HSD17B13 is 5.9-fold higher (P = 0.003) in patients with NAFLD. HSD17B13 is targeted to lipid droplets, requiring the conserved amino acid 22-28 sequence and amino acid 71-106 region. The protein has retinol dehydrogenase (RDH) activity, with enzymatic activity dependent on lipid droplet targeting and cofactor binding site. The exon 6 deletion, G insertion, and naturally occurring P260S mutation all confer loss of enzymatic activity. Conclusion: We demonstrate the association of variants in HSD17B13 with specific features of NAFLD histology and identify the enzyme as a lipid droplet-associated RDH; our data suggest that HSD17B13 plays a role in NAFLD through its enzymatic activity.
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17-Hidroxiesteroide Deshidrogenasas/genética , Enfermedad del Hígado Graso no Alcohólico/genética , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Adulto , Secuencia de Aminoácidos , Estudios de Cohortes , Femenino , Células HEK293 , Células Hep G2 , Humanos , Hígado/metabolismo , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Polimorfismo de Nucleótido Simple , Retinoides/metabolismoRESUMEN
BACKGROUND: Patients with different hepatitis C virus (HCV) genotype infections are associated with varying metabolic disorders. Although alteration of lipid metabolism has been confirmed as a virus-induced metabolic derangement in chronic hepatitis C patients, the impact of various HCV genotypes on hepatic cholesterol metabolism remains elusive. In this study, we thus investigated the HCV genotype-specific lipogenic and cholesterol metabolism profiles in an in vitro cell culture system. METHODS: We first conducted HCV cell culture system (HCVcc) assays by infecting Huh7.5.1 cells with multiple infection-competent HCV strains, including the genotype 2a JFH1 and JFH1-based intergenotypic recombinants 1b and 3a. We then examined the expression levels of various lipid and cholesterol-related genes. RESULTS: The data showed that infection with individual HCV genotypes exerted unique gene expression regulatory effects on lipoproteins and cholesterol metabolism genes. Of note, all HCV strains suppressed cholesterol biosynthesis in hepatocytes through downregulating the expression of HMG-CoA reductase (HMGCR) and farnesyl-diphosphate farnesyltransferase 1 (FDFT1) - two essential enzymes for cholesterol biosynthesis. These HCV-mediated inhibitory effects could be reversed by treatment with sofosbuvir, a pangenotypic NS5B inhibitor. In addition, overexpression of HCV genotype 1b, 2a or 3a core protein significantly suppressed HMGCR mRNA transcription and translation, thus diminished cellular cholesterol biosynthesis. Nonetheless, the core protein had no effect on FDFT1 expression. CONCLUSION: Although HCV infection regulates host lipid metabolism in a genotype-specific manner, its inhibition on hepatocellular cholesterogenic gene expression and total cholesterol biosynthesis is a common effect among HCV genotype 1b, 2a and 3a.
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Colesterol/biosíntesis , Hepacivirus/genética , Hepatitis C Crónica/metabolismo , Hepatocitos/metabolismo , Metabolismo de los Lípidos , Línea Celular , Farnesil Difosfato Farnesil Transferasa/genética , Regulación de la Expresión Génica , Genotipo , Hepatitis C Crónica/virología , Hepatocitos/virología , Humanos , Hidroximetilglutaril-CoA Reductasas/genéticaAsunto(s)
Hepatitis C , Experimentación Humana , Vacunas contra Hepatitis Viral , Protocolos de Ensayos Clínicos como Asunto , Regulación Gubernamental , Hepacivirus , Hepatitis C/tratamiento farmacológico , Experimentación Humana/ética , Experimentación Humana/legislación & jurisprudencia , HumanosRESUMEN
BACKGROUND & AIMS: Hepatitis C virus (HCV) co-opts the very-low-density lipoprotein pathway for morphogenesis, maturation, and secretion, and circulates as lipoviroparticles (LVPs). We investigated the functions and underlying mechanisms of the lipid-associated TM6SF2 protein in modulating LVP formation and the HCV life cycle. METHODS: We knocked down or overexpressed TM6SF2 in hepatic cells and examined HCV infection, measuring viral RNA and protein levels and infectious LVP titers. The density of secreted LVPs was evaluated by iodixanol gradient assay. We measured levels and patterns of TM6SF2 in liver biopsies from 73 patients with chronic hepatitis C, livers of HCV-infected humanized Alb-uPA/SCID/beige mice, and HCV-infected Huh7.5.1 cells. RESULTS: TM6SF2 knockdown in hepatocytes reduced viral RNA and infectious viral particle secretion without affecting HCV genome replication, translation, or assembly. Overexpression of TM6SF2 reduced intracellular levels of HCV RNA and infectious LVPs, and conversely increased their levels in the culture supernatants. In HCV-infected cells, TM6SF2 overexpression resulted in production of more infectious LVPs in the lower-density fractions of supernatant. HCV infection increased TM6SF2 expression in cultured cells, humanized livers of mice, and liver tissues of HCV patients. TM6SF2 messenger RNA levels correlated positively with HCV RNA levels in liver biopsies from patients. SREBF2 appears to mediate the ability of HCV to increase the expression of TM6SF2 in hepatic cells. CONCLUSIONS: In studies of cells, mice and human liver tissues, we found TM6SF2 is required for maturation, lipidation, and secretion of infectious LVPs. HCV, in turn, up-regulates expression of TM6SF2 to facilitate productive infection.
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Hepacivirus/metabolismo , Hepatitis C Crónica/virología , Hepatocitos/virología , Lipoproteínas VLDL/metabolismo , Proteínas de la Membrana/fisiología , Animales , Células Cultivadas , Humanos , Hígado/virología , Ratones , Ratones SCID , ARN Viral/metabolismoRESUMEN
Hepatitis B virus (HBV) infection is a major health problem worldwide, and chronically infected individuals are at high risk of developing cirrhosis and hepatocellular carcinoma (HCC). The molecular mechanisms whereby HBV causes HCC are largely unknown. Using a biologically relevant system of HBV infection of primary human hepatocytes (PHHs), we studied how HBV perturbs gene expression and signaling pathways of infected hepatocytes and whether these effects are relevant to productive HBV infection and HBV-associated HCC. Using a human growth factor antibody array, we first showed that HBV infection induced a distinct profile of growth factor production by PHHs, marked particularly by significantly lower levels of the transforming growth factor ß (TGF-ß) family of proteins in the supernatant. Transcriptome profiling next revealed multiple changes in cell proliferation and cell cycle control pathways in response to HBV infection. A human cell cycle PCR array validated deregulation of more than 20 genes associated with the cell cycle in HBV-infected PHHs. Cell cycle analysis demonstrated that HBV-infected PHHs are enriched in the G2/M phase compared to the predominantly G0/G1 phase of cultured PHHs. HBV proviral host factors, such as PPARA, RXRA, and CEBPB, were upregulated upon HBV infection and particularly enriched in cells in the G2/M phase. Together, these results support the notion that HBV deregulates cell cycle control to render a cellular environment that is favorable for productive HBV infection. By perturbing cell cycle regulation of infected cells, HBV may coincidently induce a premalignant phenotype that predisposes infected hepatocytes to subsequent malignant transformation.IMPORTANCE Hepatitis B virus (HBV) infection is a major health problem with high risk of developing hepatocellular carcinoma (HCC). By using a biologically relevant system of HBV infection of primary human hepatocytes (PHHs), we studied how HBV perturbs gene expression and whether these effects are relevant to HBV-associated HCC. HBV induced a distinct profile of growth factor production, marked particularly by significantly lower levels of the transforming growth factor ß (TGF-ß) family of proteins. Transcriptome profiling revealed multiple changes in cell proliferation and cell cycle control pathways. Cell cycle analysis demonstrated that HBV-infected PHHs are enriched in the G2/M phase. HBV proviral host factors were upregulated upon infection and particularly enriched in cells in the G2/M phase. Together, these results support the notion that HBV deregulates cell cycle control to render a cellular environment that is favorable for productive infection. This may coincidently induce a premalignant phenotype that predisposes infected hepatocytes to subsequent malignant transformation.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Ciclo Celular , Transformación Celular Viral/fisiología , Virus de la Hepatitis B/fisiología , Hepatitis B/metabolismo , Neoplasias Hepáticas/metabolismo , Replicación Viral/fisiología , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virología , Células Hep G2 , Hepatitis B/genética , Hepatitis B/patología , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , PPAR alfa/genética , PPAR alfa/metabolismoRESUMEN
Host cells harbor various intrinsic mechanisms to restrict viral infections as a first line of antiviral defense. Viruses have evolved various countermeasures against these antiviral mechanisms. Here we show that N-Myc downstream-regulated gene 1 (NDRG1) limits productive hepatitis C virus (HCV) infection by inhibiting viral assembly. Interestingly, HCV infection downregulates NDRG1 protein and mRNA expression. The loss of NDRG1 increases the size and number of lipid droplets, which are the sites of HCV assembly. HCV suppresses NDRG1 expression by upregulating MYC, which directly inhibits the transcription of NDRG1 The upregulation of MYC also leads to the reduced expression of the NDRG1-specific kinase serum/glucocorticoid-regulated kinase 1 (SGK1), resulting in a markedly diminished phosphorylation of NDRG1. The knockdown of MYC during HCV infection rescues NDRG1 expression and phosphorylation, suggesting that MYC regulates NDRG1 at both the transcriptional and posttranslational levels. Overall, our results suggest that NDRG1 restricts HCV assembly by limiting lipid droplet formation. HCV counteracts this intrinsic antiviral mechanism by downregulating NDRG1 via a MYC-dependent mechanism.IMPORTANCE Hepatitis C virus (HCV) is an enveloped single-stranded RNA virus that targets hepatocytes in the liver. HCV is a leading cause of chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma, and estimates suggest a global prevalence of 2.35%. Up to 80% of acutely infected individuals will develop chronic infection, and as many as 5% eventually progress to liver cancer. An understanding of the mechanisms behind virus-host interactions and viral carcinogenesis is still lacking. The significance of our research is that it identifies a previously unknown relationship between HCV and a known tumor-associated gene. Furthermore, our data point to a new role for this gene in the liver and in lipid metabolism. Thus, HCV infection serves as a great biological model to advance our knowledge of liver functions and the development of liver cancer.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Hepacivirus/fisiología , Hepatitis C/metabolismo , Hepatitis C/virología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Gotas Lipídicas/metabolismo , Ensamble de Virus , Replicación Viral , Proteínas de Ciclo Celular/genética , Línea Celular , Regulación de la Expresión Génica , Hepacivirus/aislamiento & purificación , Hepatitis C/genética , Hepatocitos/metabolismo , Hepatocitos/virología , Interacciones Huésped-Patógeno , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Metabolismo de los Lípidos , Modelos Biológicos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Interferente Pequeño/genéticaRESUMEN
Hepatitis C virus (HCV) infection induces interferon (IFN)-stimulated genes (ISGs) and downstream innate immune responses. This study investigated whether baseline and on-treatment differences in these responses predict response versus virological breakthrough during therapy with direct-acting antivirals (DAAs). Thirteen HCV genotype 1b-infected patients who had previously failed a course of pegylated IFN/ribavirin were retreated with asunaprevir/daclatasvir for 24 weeks. After pretreatment biopsy, patients were randomized to undergo a second biopsy at week 2 or 4 on therapy. Microarray and NanoString analyses were performed on paired liver biopsies and analyzed using linear mixed models. As biomarkers for peripheral IFN responses, peripheral blood natural killer cells were assessed for phosphorylated signal transducer and activator of transcription 1 (pSTAT1) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and degranulation. Nine of 13 (69%) patients achieved sustained virological response at 12 weeks off therapy (SVR12), and 4 experienced virological breakthroughs between weeks 4 and 12. Patients who achieved SVR12 displayed higher ISG expression levels in baseline liver biopsies and a higher frequency of pSTAT1 and TRAIL-expressing, degranulating natural killer cells in baseline blood samples than those who experienced virological breakthrough. Comparing gene expression levels from baseline and on-therapy biopsies, 408 genes (±1.2-fold, P < 0.01) were differentially expressed. Genes down-regulated on treatment were predominantly ISGs. Down-regulation of ISGs was rapid and correlated with HCV RNA suppression. Conclusion: An enhanced IFN signature is observed at baseline in liver and blood of patients who achieve SVR12 compared to those who experience a virological breakthrough; the findings suggest that innate immunity may contribute to clearance of HCV during DAA therapy by preventing the emergence of resistance-associated substitutions that lead to viral breakthrough during DAA therapy.