RESUMEN
The antiviral role of the tripartite motif-containing (TRIM) protein family , a member of the E3-ubiquitin ligase family, has recently been actively studied. Hepatitis B virus (HBV) infection is a major contributor to liver diseases; however, the host factors regulated by cytokine-inducible TRIM21 to suppress HBV remain unclear. In this study, we showed the antiviral efficacy of TRIM21 against HBV in hepatoma cell lines, primary human hepatocytes isolated from patient liver tissues, and mouse model. Using TRIM21 knock-out cells, we confirmed that the antiviral effects of interferon-gamma, which suppress HBV replication, are diminished when TRIM21 is deficient. Northern blot analysis confirmed a reduction of HBV RNA levels by TRIM21. Using Luciferase reporter assay, we also discovered that TRIM21 decreases the activity of HBV enhancers, which play a crucial role in covalently closed circular DNA transcription. The participation of the RING domain and PRY-SPRY domain in the anti-HBV effect of TRIM21 was demonstrated through experiments using deletion mutants. We identified a novel interaction between TRIM21 and hepatocyte nuclear factor 4α (HNF4α) through co-immunoprecipitation assay. More specifically, ubiquitination assay revealed that TRIM21 promotes ubiquitin-mediated proteasomal degradation of HNF4α. HNF1α transcription is down-regulated as a result of the degradation of HNF4α, an activator for the HNF1α promoter. Therefore, the reduction of key HBV enhancer activators, HNF4α and HNF1α, by TRIM21 resulted in a decline in HBV transcription, ultimately leading to the inhibition of HBV replication.IMPORTANCEDespite extensive research efforts, a definitive cure for chronic hepatitis B remains elusive, emphasizing the persistent importance of this viral infection as a substantial public health concern. Although the risks associated with hepatitis B virus (HBV) infection are well known, host factors capable of suppressing HBV are largely uncharacterized. This study elucidates that tripartite motif-containing protein 21 (TRIM21) suppresses HBV transcription and consequently inhibits HBV replication by downregulating the hepatocyte nuclear factors, which are host factors associated with the HBV enhancers. Our findings demonstrate a novel anti-HBV mechanism of TRIM21 in interferon-gamma-induced anti-HBV activity. These findings may contribute to new strategies to block HBV.
Asunto(s)
Virus de la Hepatitis B , Factor Nuclear 4 del Hepatocito , Hepatocitos , Interferón gamma , Ribonucleoproteínas , Replicación Viral , Humanos , Virus de la Hepatitis B/fisiología , Animales , Ratones , Interferón gamma/farmacología , Interferón gamma/metabolismo , Hepatocitos/virología , Hepatocitos/metabolismo , Factor Nuclear 4 del Hepatocito/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Hepatitis B/virología , Hepatitis B/metabolismo , Células Hep G2 , Línea Celular TumoralRESUMEN
AIMS: Sirtuin 7 (SIRT7) plays an important role in tumor development, and has been characterized as a potent regulator of cellular stress. However, the effect of SIRT7 on sorafenib acquired resistance remains unclear and a possible anti-tumor mechanism beyond this process in HCC has not been clarified. We examined the therapeutic potential of SIRT7 and determined whether it functions synergistically with sorafenib to overcome chemoresistance. METHODS: Cancer Genome Atlas-liver HCC data and unbiased gene set enrichment analyses were used to identify SIRT7 as a potential effector molecule in sorafenib acquired resistance. Two types of SIRT7 chemical inhibitors were developed to evaluate its therapeutic properties when synergized with sorafenib. Mass spectrometry was performed to discover a direct target of SIRT7, DDX3X, and DDX3X deacetylation levels and protein stability were explored. Moreover, an in vivo xenograft model was used to confirm anti-tumor effect of SIRT7 and DDX3X chemical inhibitors combined with sorafenib. RESULTS: SIRT7 inhibition mediated DDX3X depletion can re-sensitize acquired sorafenib resistance by disrupting NLRP3 inflammasome assembly, finally suppressing hyperactive ERK1/2 signaling in response to NLRP3 inflammasome-mediated IL-1ß inhibition. CONCLUSIONS: SIRT7 is responsible for sorafenib acquired resistance, and its inhibition would be beneficial when combined with sorafenib by suppressing hyperactive pro-cell survival ERK1/2 signaling.
Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Sirtuinas , Humanos , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Sorafenib/farmacología , Sorafenib/uso terapéutico , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Inflamasomas/metabolismo , Inflamasomas/farmacología , Fosforilación , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Sistema de Señalización de MAP Quinasas , Resistencia a Antineoplásicos/genética , Línea Celular Tumoral , Proliferación Celular , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , ARN Helicasas DEAD-box/farmacología , Sirtuinas/genética , Sirtuinas/metabolismo , Sirtuinas/farmacologíaRESUMEN
Hepatic organoids might provide a golden opportunity for realizing precision medicine in various hepatic diseases. Previously described hepatic organoid protocols from pluripotent stem cells rely on complicated multiple differentiation steps consisting of both 2D and 3D differentiation procedures. Therefore, the spontaneous formation of hepatic organoids from 2D monolayer culture is associated with a low-throughput production, which might hinder the standardization of hepatic organoid production and hamper the translation of this technology to the clinical or industrial setting. Here we describe the stepwise and fully 3D production of hepatic organoids from human pluripotent stem cells. We optimized every differentiation step by screening for optimal concentrations and timing of differentiation signals in each differentiation step. Hepatic organoids are stably expandable without losing their hepatic functionality. Moreover, upon treatment of drugs with known hepatotoxicity, we found hepatic organoids are more sensitive to drug-induced hepatotoxicity compared with 2D hepatocytes differentiated from PSCs, making them highly suitable for in vitro toxicity screening of drug candidates. The standardized fully 3D protocol described in the current study for producing functional hepatic organoids might serve as a novel platform for the industrial and clinical translation of hepatic organoid technology.
Asunto(s)
Enfermedad Hepática Inducida por Sustancias y Drogas , Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Humanos , Diferenciación Celular/genética , OrganoidesRESUMEN
Since its discovery in 1965, our understanding of the hepatitis B virus (HBV) replication cycle and host immune responses has increased markedly. In contrast, our knowledge of the molecular biology of hepatitis delta virus (HDV), which is associated with more severe liver disease, is less well understood. Despite the progress made, critical gaps remain in our knowledge of HBV and HDV replication and the mechanisms underlying viral persistence and evasion of host immunity. The International HBV Meeting is the leading annual scientific meeting for presenting the latest advances in HBV and HDV molecular virology, immunology, and epidemiology. In 2023, the annual scientific meeting was held in Kobe, Japan and this review summarises some of the advances presented at the Meeting and lists gaps in our knowledge that may facilitate the development of new therapies.
Asunto(s)
Virus de la Hepatitis B , Hepatitis B , Virus de la Hepatitis Delta , Replicación Viral , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Virus de la Hepatitis B/inmunología , Humanos , Virus de la Hepatitis Delta/genética , Virus de la Hepatitis Delta/fisiología , Hepatitis B/virología , Hepatitis B/inmunología , Biología Molecular , Japón , Hepatitis D/virología , Interacciones Huésped-Patógeno/inmunología , Interacciones Huésped-Patógeno/genéticaRESUMEN
The multifunctional influenza virus protein PB1-F2 plays several roles in deregulation of host innate immune responses and is a known immunopathology enhancer of the 1918 influenza pandemic. Here, we show that the 1918 PB1-F2 protein not only interferes with the mitochondria-dependent pathway of type I interferon (IFN) signaling, but also acquired a novel IFN antagonist function by targeting the DEAD-box helicase DDX3, a key downstream mediator in antiviral interferon signaling, toward proteasome-dependent degradation. Interactome analysis revealed that 1918 PB1-F2, but not PR8 PB1-F2, binds to DDX3 and causes its co-degradation. Consistent with intrinsic protein instability as basis for this gain-of-function, internal structural disorder is associated with the unique cytotoxic sequences of the 1918 PB1-F2 protein. Infusing mice with recombinant DDX3 protein completely rescued them from lethal infection with the 1918 PB1-F2-producing virus. Alongside NS1 protein, 1918 PB1-F2 therefore constitutes a potent IFN antagonist causative for the severe pathogenicity of the 1918 influenza strain. Our identification of molecular determinants of pathogenesis should be useful for the future design of new antiviral strategies against influenza pandemics.
Asunto(s)
ARN Helicasas DEAD-box/metabolismo , Gripe Humana/virología , Interferones/metabolismo , Orthomyxoviridae/patogenicidad , Proteínas Virales/fisiología , Células A549 , Animales , Perros , Femenino , Células HEK293 , Historia del Siglo XX , Humanos , Gripe Humana/epidemiología , Gripe Humana/historia , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Orthomyxoviridae/metabolismo , Pandemias , Proteolisis , Transducción de Señal , Células U937 , Proteínas Virales/metabolismo , Virulencia/fisiologíaRESUMEN
BACKGROUND AND AIM: Besifovir dipivoxil maleate (BSV) was reported to have comparable antiviral efficacy and superior renal and bone safety to tenofovir disoproxil fumarate (TDF) in chronic hepatitis B (CHB) patients. The present study aims to evaluate changes of liver histology and intrahepatic covalently closed circular DNA (cccDNA) levels by BSV treatment in comparison with TDF therapy. METHODS: This is a subset study of the phase 3 trial comparing BSV with TDF. Among them, only CHB patients willing to participate in a histologic evaluation study were enrolled. Liver histologic examination and intrahepatic cccDNA quantification were performed. RESULTS: A total of 46 CHB patients received liver biopsies (BSV, n = 29; TDF, n = 17). After 48 weeks of treatment, virological response rate was comparable between the groups (P = 0.707). Follow-up liver biopsies showed that necroinflammation was significantly improved in the both groups. However, the histological response rate defined as the proportion of subjects whose modified histologic activity index score decreased by ≥ 2 without deterioration in fibrosis was higher in the BSV group than in the TDF group (77.8% vs 36.4%, P = 0.048). The proportion of subjects with Ishak fibrosis score 3 or more decreased from 77.7% to 55.5% in the BSV and that decreased from 72.7% to 45.4% in the TDF group. The intrahepatic cccDNA significantly decreased from baseline after 48 weeks of BSV or TDF treatment (P < 0.001) without intergroup differences (P = 0.349). CONCLUSIONS: The BSV therapy improves hepatic histology and decreases intrahepatic cccDNA in CHB patients.
Asunto(s)
ADN Circular , Guanina/análogos & derivados , Hepatitis B Crónica , Hígado , Organofosfonatos , Antivirales/uso terapéutico , ADN Circular/efectos de los fármacos , Guanina/uso terapéutico , Hepatitis B Crónica/tratamiento farmacológico , Humanos , Hígado/efectos de los fármacos , Hígado/patología , Organofosfonatos/uso terapéutico , Resultado del TratamientoRESUMEN
The ubiquitin system denotes a potent post-translational modification machinery that is capable of activation or deactivation of target proteins through reversible linkage of a single ubiquitin or ubiquitin chains. Ubiquitination regulates major cellular functions such as protein degradation, trafficking and signaling pathways, innate immune response, antiviral defense, and virus replication. The RNA sensor RIG-I ubiquitination is specifically induced by influenza A virus (IAV) to activate type I IFN production. Influenza virus modulates the activity of major antiviral proteins in the host cell to complete its full life cycle. Its structural and non-structural proteins, matrix proteins and the polymerase complex can regulate host immunity and antiviral response. The polymerase PB1-F2 of mutated 1918 IAV, adapts a novel IFN antagonist function by sending the DDX3 into proteasomal degradation. Ultimately the fate of virus is determined by the outcome of interplay between viral components and host antiviral proteins and ubiquitination has a central role in the encounter of virus and its host cell.
Asunto(s)
Virus de la Influenza A , Gripe Humana , Infecciones por Orthomyxoviridae , Ubiquitinación , Humanos , Inmunidad Innata , Virus de la Influenza A/metabolismo , Gripe Humana/metabolismo , Ubiquitina/metabolismo , Ubiquitinación/fisiología , Replicación Viral/genéticaRESUMEN
Tenofovir disoproxil fumarate (TDF) has been regarded as the most potent drug for treating patients with chronic hepatitis B (CHB). However recently, viral mutations associated with tenofovir have been reported. Here, we found a CHB patient with suboptimal response after more than 4 years of TDF treatment. Clonal analysis of hepatitis B virus (HBV) isolated from sequential sera of this patient identified the seven previously reported TDF-resistant mutations (CYELMVI). Interestingly, a threonine to alanine mutation at the 301 amino acid position of the reverse-transcriptase (RT) domain, (rtT301A), was commonly accompanied with CYELMVI at a high rate (72.7%). Since the rtT301A mutation has not been reported yet, we investigated the role of this naturally occurring mutation on the viral replication and susceptibility to tenofovir in various liver cells (hepatoma cells as well as primary human hepatocytes). A cell-based phenotypic assay revealed that the rtT301A mutation dramatically impaired the replication ability with meaningful reduction in sensitivity to tenofovir in hepatoma cell lines. However, attenuated viral replication by the rtT301A mutation was significantly restored in primary human hepatocytes (PHHs). Our findings suggest that the replication capability and drug sensitivity of HBV is different between hepatoma cell lines and PHHs. Therefore, our study emphasizes that validation studies should be performed not only in the liver cancer cell lines but also in the PHHs to understand the exact viral fitness under antiviral pressure in patients.
Asunto(s)
Virus de la Hepatitis B/efectos de los fármacos , Hepatocitos/efectos de los fármacos , Hepatocitos/virología , Tenofovir/farmacología , Antivirales/farmacología , Carcinoma Hepatocelular/genética , Línea Celular Tumoral , Células Cultivadas , Farmacorresistencia Viral/genética , Femenino , Genes Virales , Células Hep G2 , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/genética , Hepatitis B Crónica/virología , Hepatocitos/metabolismo , Humanos , Neoplasias Hepáticas/genética , Persona de Mediana Edad , Mutación Puntual , ADN Polimerasa Dirigida por ARN/genética , Inhibidores de la Transcriptasa Inversa/farmacología , Proteínas Virales/genética , Replicación Viral/efectos de los fármacos , Replicación Viral/genéticaRESUMEN
BACKGROUND AND AIM: Since polymerase and surface genes overlap in hepatitis B virus (HBV), an antiviral-induced mutation in the polymerase gene may alter the surface antigenicity in patients with chronic hepatitis B (CHB), but this possibility has not been clearly confirmed. This study aimed to determine the drug susceptibility and surface antigenicity of the patient-derived mutants. PATIENTS AND METHODS: Full-length HBV genomes isolated from four entecavir-resistant CHB patients were cloned and sequenced. Around 10 clones of full-length HBV obtained from each patient were analysed and registered in the NCBI GenBank. Representative clones were further characterized by in vitro drug susceptibility and surface antigenicity assays. RESULTS: The rtL180M + rtM204V mutations were common among all the clones analysed. Additionally, the ETV resistance mutations rtT184A/L, rtS202G and rtM250V were found among three patients. Most of the ETV-resistant mutants had amino acid alterations within the known epitopes recognized by T- and B-cells in the HBV surface and core antigens. The in vitro drug susceptibility assay showed that all tested clones were resistant to ETV treatment. However, they were all susceptible to ADV and TDF. More importantly, the rtI169T mutation in the RT domain, led to the sF161L mutation in the overlapping S gene, which decreased in surface antigenicity. CONCLUSIONS: The ETV resistance mutations can affect the antigenicity of the HBsAg proteins due to changes in the overlapping sequence of this surface antigen. Thus, the apparent decline or disappearance of HBsAg needs to be interpreted cautiously in patients with previous or current antiviral resistance mutations.
Asunto(s)
Virus de la Hepatitis B , Hepatitis B Crónica , Antígenos de Superficie/uso terapéutico , Antivirales/farmacología , Antivirales/uso terapéutico , Farmacorresistencia Viral/genética , Guanina/análogos & derivados , Guanina/uso terapéutico , Virus de la Hepatitis B/genética , Hepatitis B Crónica/tratamiento farmacológico , Humanos , Lamivudine/uso terapéutico , MutaciónRESUMEN
BACKGROUND AND AIM: Interferon-stimulated gene 20 (ISG20) is an interferon-inducible exonuclease that inhibits the replication of several RNA viruses. In patients with chronic hepatitis B, ISG20 expression is related to the interferon-α treatment response. However, the molecular mechanism of ISG20-mediated anti-hepatitis B virus (HBV) activity is unclear. METHODS: We have investigated the effect of ISG20 on antiviral activity to address that. The life cycle of HBV was analyzed by the ectopic expression of ISG20 in HepG2 and HepG2-NTCP cells. Finally, to provide physiological relevance of our study, the expression of ISG20 from chronic hepatitis B patients was examined. RESULTS: Interferon-stimulated gene 20 was mainly induced by interferon-ß and dramatically inhibited HBV replication. In addition, ISG20 decreased HBV gene expression and transcription. Although ISG20 inhibited HBV replication by reducing viral enhancer activity, the expression of transcription factors that bind the HBV enhancer was not affected. Particularly, ISG20 suppressed HBV enhancer activity by binding to the enhancer II and core promoter (EnhII/Cp) region. CONCLUSION: Our findings suggest that ISG20 exerts the anti-HBV activity by acting as a putative repressor binding to the HBV EnhII/Cp region.
Asunto(s)
Exorribonucleasas/genética , Exorribonucleasas/metabolismo , Expresión Génica , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/genética , Interferón-alfa/uso terapéutico , Interferón beta/uso terapéutico , Activación Viral/genética , Exorribonucleasas/fisiología , Células Hep G2 , Humanos , Interferón-alfa/farmacología , Interferón beta/farmacología , Regiones Promotoras Genéticas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Replicación Viral/genéticaRESUMEN
Hepatitis B virus (HBV) infection is a major factor in the development of various liver diseases such as hepatocellular carcinoma (HCC). Among HBV encoded proteins, HBV X protein (HBx) is known to play a key role in the development of HCC. Hepatocyte nuclear factor 4α (HNF4α) is a nuclear transcription factor which is critical for hepatocyte differentiation. However, the expression level as well as its regulatory mechanism in HBV infection have yet to be clarified. Here, we observed the suppression of HNF4α in cells which stably express HBV whole genome or HBx protein alone, while transient transfection of HBV replicon or HBx plasmid had no effect on the HNF4α level. Importantly, in the stable HBV- or HBx-expressing hepatocytes, the downregulated level of HNF4α was restored by inhibiting the ERK signaling pathway. Our data show that HNF4α was suppressed during long-term HBV infection in cultured HepG2-NTCP cells as well as in a mouse model following hydrodynamic injection of pAAV-HBV or in mice intravenously infected with rAAV-HBV. Importantly, HNF4α downregulation increased cell proliferation, which contributed to the formation and development of tumor in xenograft nude mice. The data presented here provide proof of the effect of HBV infection in manipulating the HNF4α regulatory pathway in HCC development.
Asunto(s)
Carcinoma Hepatocelular/virología , Hepatitis B/metabolismo , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Neoplasias Hepáticas/virología , Transactivadores/metabolismo , Proteínas Reguladoras y Accesorias Virales/metabolismo , Animales , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Línea Celular Tumoral , Proliferación Celular , Modelos Animales de Enfermedad , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Células Hep G2 , Hepatitis B/genética , Hepatitis B/virología , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Ratones , Ratones Desnudos , Trasplante de NeoplasiasRESUMEN
BACKGROUND & AIMS: Tenofovir disoproxil fumarate (TDF) is one the most potent nucleot(s)ide analogues for treating chronic hepatitis B virus (HBV) infection. Phenotypic resistance caused by genotypic resistance to TDF has not been reported. This study aimed to characterize HBV mutations that confer tenofovir resistance. METHODS: Two patients with viral breakthrough during treatment with TDF-containing regimens were prospectively enrolled. The gene encoding HBV reverse transcriptase was sequenced. Eleven HBV clones harboring a series of mutations in the reverse transcriptase gene were constructed by site-directed mutagenesis. Drug susceptibility of each clone was determined by Southern blot analysis and real-time PCR. The relative frequency of mutants was evaluated by ultra-deep sequencing and clonal analysis. RESULTS: Five mutations (rtS106C [C], rtH126Y [Y], rtD134E [E], rtM204I/V, and rtL269I [I]) were commonly found in viral isolates from 2 patients. The novel mutations C, Y, and E were associated with drug resistance. In assays for drug susceptibility, the IC50 value for wild-type HBV was 3.8⯱â¯0.6⯵M, whereas the IC50 values for CYE and CYEI mutants were 14.1⯱â¯1.8 and 58.1⯱â¯0.9⯵M, respectively. The IC90 value for wild-type HBV was 30⯱â¯0.5⯵M, whereas the IC90 values for CYE and CYEI mutants were 185⯱â¯0.5 and 790⯱â¯0.2⯵M, respectively. Both tenofovir-resistant mutants and wild-type HBV had similar susceptibility to the capsid assembly modulator NVR 3-778 (IC50â¯<0.4⯵M vs. IC50â¯=â¯0.4⯵M, respectively). CONCLUSIONS: Our study reveals that the quadruple (CYEI) mutation increases the amount of tenofovir required to inhibit HBV by 15.3-fold in IC50 and 26.3-fold in IC90. These results demonstrate that tenofovir-resistant HBV mutants can emerge, although the genetic barrier is high. LAY SUMMARY: Tenofovir is the most potent nucleotide analogue for the treatment of chronic hepatitis B virus infection and there has been no hepatitis B virus mutation that confers >10-fold resistance to tenofovir up to 8â¯years. Herein, we identified, for the first time, a quadruple mutation that conferred 15.3-fold (IC50) and 26.3-fold (IC90) resistance to tenofovir in 2 patients who experienced viral breakthrough during tenofovir treatment.
Asunto(s)
Antivirales/uso terapéutico , Virus de la Hepatitis B/genética , Hepatitis B Crónica/tratamiento farmacológico , Mutación , ADN Polimerasa Dirigida por ARN/genética , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Tenofovir/uso terapéutico , Anciano , Línea Celular Tumoral , Farmacorresistencia Viral/genética , Humanos , MasculinoRESUMEN
BACKGROUND & AIMS: Besifovir dipivoxil maleate (BSV) has activity against hepatitis B virus (HBV). We performed a phase 3 study to compare the antiviral efficacy and safety of BSV vs tenofovir disoproxil fumarate (TDF) in patients with chronic HBV infection in Korea. METHODS: We conducted a double-blind, non-inferiority trial of 197 patients with chronic HBV infection at 22 sites in South Korea, from November 2013 through February 2016. Patients were randomly assigned to groups given BSV (150 mg, n = 99) or TDF (300 mg, n = 98) for 48 weeks. We evaluated virologic responses to therapy (HBV DNA <69 IU/mL or 400 copies/ml), bone mineral density (BMD), and renal outcomes for safety analysis. The main efficacy endpoint was the proportion of patients with a virologic response at week 48. After 48 weeks, TDF was switched to BSV (150 mg) for an additional 48 weeks. RESULTS: After 48 weeks of treatment, 80.9% of patients given BSV and 84.9% of patients given TDF met the efficacy endpoint, indicating the non-inferiority of BSV to TDF. At week 96, 87.2% of patients in the BSV-BSV and 85.7% of patients in the TDF-BSV had a virologic response. At week 48, changes in hip and spine BMD differed significantly between the BSV and TDF groups, whereas the estimated glomerular filtration rate in the TDF group was significantly lower than that in the BSV group. However, at 96 weeks, there were no significant differences in BMD and estimated glomerular filtration rate between the BSV-BSV and TDF-BSV groups. CONCLUSIONS: BSV has antiviral efficacy comparable to that of TDF after 48 weeks of treatment, with durable effects for 96 weeks. BSV has a better safety profile than TDF, in terms of bone and renal outcomes. ClinicalTrials.gov no: NCT01937806.
Asunto(s)
Antivirales/uso terapéutico , ADN Viral/sangre , Guanina/análogos & derivados , Hepatitis B Crónica/tratamiento farmacológico , Organofosfonatos/uso terapéutico , Tenofovir/uso terapéutico , Absorciometría de Fotón , Adulto , Alanina Transaminasa/sangre , Densidad Ósea , Enfermedades Óseas Metabólicas/inducido químicamente , Método Doble Ciego , Farmacorresistencia Viral , Femenino , Tasa de Filtración Glomerular , Guanina/uso terapéutico , Antígenos de Superficie de la Hepatitis B/sangre , Antígenos e de la Hepatitis B/sangre , Hepatitis B Crónica/sangre , Cadera/diagnóstico por imagen , Humanos , Vértebras Lumbares/diagnóstico por imagen , Masculino , Maleatos , Persona de Mediana Edad , Osteoporosis/inducido químicamente , Insuficiencia Renal/inducido químicamente , Respuesta Virológica Sostenida , Resultado del TratamientoRESUMEN
Hepatitis B virus (HBV) infection is a leading cause of liver diseases; however, the host factors which facilitate the replication and persistence of HBV are largely unidentified. Cellular FLICE inhibitory protein (c-FLIP) is a typical antiapoptotic protein. In many cases of liver diseases, the expression level of c-FLIP is altered, which affects the fate of hepatocytes. We previously found that c-FLIP and its cleaved form interact with HBV X protein (HBx), which is essential for HBV replication, and regulate diverse cellular signals. In this study, we investigated the role of endogenous c-FLIP in HBV replication and its underlying mechanisms. The knockdown of endogenous c-FLIP revealed that this protein regulates HBV replication through two different mechanisms. (i) c-FLIP interacts with HBx and protects it from ubiquitin-dependent degradation. The N-terminal DED1 domain of c-FLIP is required for HBx stabilization. (ii) c-FLIP regulates the expression or stability of hepatocyte nuclear factors (HNFs), which have critical roles in HBV transcription and maintenance of hepatocytes. c-FLIP regulates the stability of HNFs through physical interactions. We verified our findings in three HBV infection systems: HepG2-NTCP cells, differentiated HepaRG cells, and primary human hepatocytes. In conclusion, our results identify c-FLIP as an essential factor in HBV replication. c-FLIP regulates viral replication through its multiple effects on viral and host proteins that have critical roles in HBV replication.IMPORTANCE Although the chronic hepatitis B virus (HBV) infection still poses a major health concern, the host factors which are required for the replication of HBV are largely uncharacterized. Our studies identify cellular FLICE inhibitory protein (c-FLIP) as an essential factor in HBV replication. We found the dual roles of c-FLIP in regulation of HBV replication: c-FLIP interacts with HBx and enhances its stability and regulates the expression or stability of hepatocyte nuclear factors which are essential for transcription of HBV genome. Our findings may provide a new target for intervention in persistent HBV infection.
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Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Virus de la Hepatitis B/fisiología , Interacciones Huésped-Patógeno , Transactivadores/metabolismo , Replicación Viral , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/genética , Técnicas de Silenciamiento del Gen , Hepatocitos/virología , Humanos , Proteínas Reguladoras y Accesorias ViralesRESUMEN
Intrinsic disorders are a common feature of hub proteins in eukaryotic interactomes controlling the signaling pathways. The intrinsically disordered proteins (IDPs) are prone to misfolding, and maintaining their functional stability remains a major challenge in validating their therapeutic potentials. Considering that IDPs are highly enriched in RNA-binding proteins (RBPs), here we reasoned and confirmed that IDPs could be stabilized by fusion to RBPs. Dickkopf2 (DKK2), Wnt antagonist and a prototype IDP, was fused with lysyl-tRNA synthetase (LysRS), with or without the fragment crystallizable (Fc) domain of an immunoglobulin and expressed predominantly as a soluble form from a bacterial host. The functional competence was confirmed by in vitro Wnt signaling reporter and tube formation in human umbilical vein endothelial cells (HUVECs) and in vivo Matrigel plug assay. The removal of LysRS by site-specific protease cleavage prompted the insoluble aggregation, confirming that the linkage to RBP chaperones the functional competence of IDPs. While addressing to DKK2 as a key modulator for cancer and ischemic vascular diseases, our results suggest the use of RBPs as stabilizers of disordered proteinaceous materials for acquiring and maintaining the structural stability and functional competence, which would impact the druggability of a variety of IDPs from human proteome.
Asunto(s)
Proteínas Intrínsecamente Desordenadas/química , Proteínas Intrínsecamente Desordenadas/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Humanos , Péptidos y Proteínas de Señalización Intercelular/química , Péptidos y Proteínas de Señalización Intercelular/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Lisina-ARNt Ligasa/química , Lisina-ARNt Ligasa/genética , Lisina-ARNt Ligasa/metabolismo , Motivos de Unión al ARN , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Vía de Señalización Wnt/genética , Vía de Señalización Wnt/fisiologíaRESUMEN
OBJECTIVE: Interferons (IFNs) mediate direct antiviral activity. They play a crucial role in the early host immune response against viral infections. However, IFN therapy for HBV infection is less effective than for other viral infections. DESIGN: We explored the cellular targets of HBV in response to IFNs using proteome-wide screening. RESULTS: Using LC-MS/MS, we identified proteins downregulated and upregulated by IFN treatment in HBV X protein (HBx)-stable and control cells. We found several IFN-stimulated genes downregulated by HBx, including TRIM22, which is known as an antiretroviral protein. We demonstrated that HBx suppresses the transcription of TRIM22 through a single CpG methylation in its 5'-UTR, which further reduces the IFN regulatory factor-1 binding affinity, thereby suppressing the IFN-stimulated induction of TRIM22. CONCLUSIONS: We verified our findings using a mouse model, primary human hepatocytes and human liver tissues. Our data elucidate a mechanism by which HBV evades the host innate immune system.
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Regiones no Traducidas 5'/genética , Islas de CpG/genética , Virus de la Hepatitis B/inmunología , Interferones/inmunología , Antígenos de Histocompatibilidad Menor/genética , Proteínas Represoras/genética , Proteínas de Motivos Tripartitos/genética , Animales , Regulación hacia Abajo/genética , Regulación hacia Abajo/inmunología , Epigénesis Genética , Regulación de la Expresión Génica/inmunología , Hepatocitos/metabolismo , Humanos , Evasión Inmune , Hígado/metabolismo , Metilación , Ratones , Antígenos de Histocompatibilidad Menor/biosíntesis , Proteoma , Proteínas Represoras/biosíntesis , Proteínas de Motivos Tripartitos/biosíntesisRESUMEN
BACKGROUND & AIMS: Cytokines are key molecules implicated in the defense against virus infection. Tumor necrosis factor-alpha (TNF-α) is well known to block the replication of hepatitis B virus (HBV). However, the molecular mechanism and the downstream effector molecules remain largely unknown. METHODS: In this study, we investigated the antiviral effect and mechanism of p22-FLIP (FLICE-inhibitory protein) by ectopic expression in vitro and in vivo. In addition, to provide the biological relevance of our study, we examined that the p22-FLIP is involved in TNF-α-mediated suppression of HBV in primary human hepatocytes. RESULTS: We found that p22-FLIP, a newly discovered c-FLIP cleavage product, inhibited HBV replication at the transcriptional level in both hepatoma cells and primary human hepatocytes, and that c-FLIP conversion to p22-FLIP was stimulated by the TNF-α/NF-κB pathway. p22-FLIP inhibited HBV replication through the upregulation of HNF3ß but downregulation of HNF4α, thus inhibiting both HBV enhancer elements. Finally, p22-FLIP potently inhibited HBV DNA replication in a mouse model of HBV replication. CONCLUSIONS: Taken together, these findings suggest that the anti-apoptotic p22-FLIP serves a novel function of inhibiting HBV transcription, and mediates the antiviral effect of TNF-α against HBV replication.
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Proteína Reguladora de Apoptosis Similar a CASP8 y FADD/metabolismo , Virus de la Hepatitis B , Factor de Necrosis Tumoral alfa , Replicación Viral/efectos de los fármacos , Animales , Antivirales/farmacología , Línea Celular , ADN Viral/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/fisiología , Factores Nucleares del Hepatocito/metabolismo , Hepatocitos/metabolismo , Humanos , Ratones , Modelos Animales , Transducción de Señal/efectos de los fármacos , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Hepatocystin/80K-H is known as a causative gene for autosomal dominant polycystic liver disease. However, the role of hepatocystin in hepatitis B virus-related liver disease remains unknown. Here, we investigated the role of hepatocystin on the cytokine-mediated antiviral response against hepatitis B virus infection. We investigated the antiviral effect and mechanism of hepatocystin by ectopic expression and RNAi knockdown in cell culture and mouse livers. Hepatocystin suppressed the replication of hepatitis B virus both in vitro and in vivo. This inhibitory effect was HBx-independent and mediated by the transcriptional regulation of viral genome via the activation of exogenous signal-regulated kinase 1/2 and the reduced expression of hepatocyte nuclear factor 4α, a transcription factor essential for hepatitis B virus replication. The amino-terminal region of hepatocystin was essential for regulation of this antiviral signaling pathway. We also found that hepatocystin acts as a critical component in interferon-mediated mitogen-activated protein kinase signaling pathway, and the interferon-induced antiviral activity against hepatitis B virus is associated with the expression levels of hepatocystin. We demonstrated that hepatocystin plays a critical role in modulating the susceptibility of hepatitis B virus to interferon, suggesting that the modulation of hepatocystin expression is important for cytokine-mediated viral clearance during hepatitis B virus infection.
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Antivirales/uso terapéutico , Carcinoma Hepatocelular/prevención & control , Regulación de la Expresión Génica , Glucosidasas/metabolismo , Hepatitis B/prevención & control , Factor Nuclear 4 del Hepatocito/metabolismo , Interferón gamma/uso terapéutico , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Animales , Northern Blotting , Southern Blotting , Western Blotting , Proteínas de Unión al Calcio , Carcinoma Hepatocelular/inmunología , Carcinoma Hepatocelular/virología , Células Cultivadas , Sinergismo Farmacológico , Glucosidasas/genética , Hepatitis B/inmunología , Hepatitis B/virología , Virus de la Hepatitis B/patogenicidad , Humanos , Técnicas para Inmunoenzimas , Péptidos y Proteínas de Señalización Intracelular/genética , Neoplasias Hepáticas/inmunología , Neoplasias Hepáticas/prevención & control , Neoplasias Hepáticas/virología , Masculino , Ratones , Ratones Endogámicos BALB C , Transducción de Señal , Replicación ViralRESUMEN
UNLABELLED: The emergence of drug-resistant hepatitis B virus (HBV) is a major problem for antiviral treatment in chronic hepatitis B infection. In this study, we analyzed the evolution of drug-resistant mutations and characterized the effects of the rtA181T and rtI233V mutations on viral replication and drug resistance. We performed a clonal analysis of the HBV polymerase gene from serum samples during viral breakthrough treated with antiviral agents. A series of mutant clones containing rtA181T and/or rtI233V mutations were constructed and determined the effect of these mutations on the replication ability and drug resistance. An in vitro study revealed that the effect of the rtA181T mutation on viral replication and drug resistance is dependent on the mutations in the overlapping surface gene. Compared to the rtA181T surface missense mutation (rtA181T/sW172S), the introduction of rtA181T surface nonsense mutation (rtA181T/sW172*) resulted in decreased viral replication and increased drug resistance. Complementation assay revealed that the truncated PreS1 is responsible for reduced replication of rtA181T/sW172* mutant. Moreover, the rtA181T/sW172* mutant exhibited a defect in viral particle secretion. The rtI233V mutation that emerged during adefovir therapy reduced viral replication and conferred resistance to adefovir. Our data suggest that the impact of the rtA181T mutation on replication and drug resistance differs based on the mutation status of the corresponding surface gene. The rtI233V mutation also affects replication ability and drug resistance. This observation suggests the need for genotypic analysis of overlapping surface genes to manage antiviral drug resistance if clinical isolates harbor the rtA181T mutation. IMPORTANCE: The emergence of drug-resistant HBV that are no longer susceptible to nucleos(t)ide analogues is a major problem for antiviral treatment in chronic hepatitis B infection. Among drug-resistant mutations, the single rtA181T mutation is known to confer cross-resistance to antiviral drugs. This mutation causes intermediate or reduced susceptibility to tenofovir. Moreover, the clinical occurrence of the rtA181T mutation during antiviral therapy is also high. Our study revealed that the effect of the rtA181T mutation on viral replication and drug resistance is dependent on the mutations in the overlapping surface gene. This observation suggests the need for genotypic analysis of overlapping surface genes to manage antiviral drug resistance if clinical isolates harbor the rtA181T mutation. We believe that our study will not only extend the understanding of the drug resistance mechanism, but it will also ultimately provide new treatment options for patients with multidrug resistant HBV.
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Antivirales/farmacología , Farmacorresistencia Viral , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/enzimología , Hepatitis B/virología , ADN Polimerasa Dirigida por ARN/genética , Proteínas Virales/genética , Replicación Viral , Regulación Viral de la Expresión Génica , Antígenos de Superficie de la Hepatitis B/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Humanos , Mutación Missense , ADN Polimerasa Dirigida por ARN/metabolismo , Proteínas Virales/metabolismo , Replicación Viral/efectos de los fármacosRESUMEN
BACKGROUND & AIMS: Analysing the mutation pattern of multidrug resistance (MDR) is important in the treatment of chronic hepatitis B (CHB). In this study, the evolutionary pattern of MDR mutations was investigated in patients receiving entecavir (ETV) rescue therapy. METHODS: Eight CHB patients with lamivudine (LAM)- and adefovir (ADV)-resistant mutations showing suboptimal response to ETV and to subsequent ETV-plus-ADV therapy were enrolled. The clonal evolution of the mutation pattern was investigated through direct sequencing, multiplex restriction fragment mass polymorphism (RFMP), and clonal analysis and the utility of these methods was compared. RESULTS: Among 160 clones at baseline, wild-type hepatitis B virus (HBV) was present in 62 (38.8%), LAM-resistant mutations in 92 (57.6%) and ADV-resistant mutations in 55 (34.4%). LAM-resistant mutations increased to 70.6% at the end of ETV therapy and increased to 74.4% at the 12th month of ETV-plus-ADV therapy. During the same time periods, ETV-resistant mutations were present in 46.3% and 38.8%, and ADV-resistant mutations were present in 3.1% and 9.4% respectively. When 256 nucleotides from 32 samples were examined for mutations, clonal analysis detected 93 mutations (36.3%), direct sequencing detected 36 mutations (14.1%) and RFMP detected 73 mutations (28.5%). The sensitivity (73.1%, 95% CI; 64.1-82.1%) and specificity (96.9%, 95% CI; 94.4-99.4%) of RFMP were high, showing a concordance rate of 88.3% with the results from clonal analysis. All mutations exceeding 40% of the total clones detected by clonal analysis were also detected by RFMP. CONCLUSIONS: The clonal evolution of the mutation pattern in MDR HBV showed the selection of LAM-resistant (±ETV-resistant) HBV during ETV rescue therapy, which may be the primary reason for patients' suboptimal response. Multiplex RFMP is a useful method for detecting MDR mutations in clinical practice.