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Hepatitis B virus (HBV) is an obligate human hepatotropic DNA virus causing both transient and chronic infection. The livers of chronic hepatitis B patients have a high risk of developing liver fibrosis, cirrhosis, and hepatocellular carcinoma. The nuclear episomal viral DNA intermediate, covalently closed circular DNA (cccDNA), forms a highly stable complex with host and viral proteins to serve as a transcription template and support HBV infection chronicity. Thus, characterization of the composition and dynamics of cccDNA nucleoprotein complexes providing cccDNA stability and gene regulation is of high importance for both basic and medical research. The presented method for chromatin immunoprecipitation coupled with qPCR (ChIP-qPCR) allows to assess provisional physical interaction of the protein of interest (POI) with cccDNA using POI-specific antibody, the level of enrichment of a POI on cccDNA versus control/background is characterized quantitatively using qPCR.
Assuntos
Imunoprecipitação da Cromatina , DNA Circular , DNA Viral , Vírus da Hepatite B , Vírus da Hepatite B/genética , DNA Circular/genética , DNA Circular/metabolismo , Humanos , DNA Viral/genética , Imunoprecipitação da Cromatina/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Hepatite B/virologia , Hepatite B/genéticaRESUMO
Immunotherapies have demonstrated limited clinical efficacy in malignant mesothelioma treatment. We conducted multiplex immunofluorescence analyses on tissue microarrays (n = 3) from patients with malignant pleural mesothelioma (MPM, n = 88) and malignant peritoneal mesothelioma (MPeM, n = 25). Our study aimed to elucidate spatial distributions of key immune cell populations and their association with lymphocyte activation gene 3 (LAG3), BRCA1-associated protein 1 (BAP1), neurofibromatosis type 2 (NF2), and methylthioadenosine phosphorylase (MTAP), with MTAP serving as a cyclin-dependent kinase inhibitor 2A/2B (CDKN2A/B) surrogate marker. Additionally, we examined the relationship between the spatial distribution of major immune cell types and prognosis and clinical characteristics of patients with malignant mesothelioma. We observed a higher degree of interaction between immune cells and tumor cells in MPM compared with MPeM. Notably, within MPM tumors, we detected a significantly increased interaction between tumor cells and CD8+ T cells in tumors with low BAP1 expression compared with those with high BAP1 expression. To support the broader research community, we have developed The Human Spatial Atlas of Malignant Mesothelioma, containing hematoxylin and eosin and multiplex immunofluorescence images with corresponding metadata. SIGNIFICANCE: Considering the limited therapeutic options available to patients with malignant mesothelioma, there is substantial translational potential in understanding the correlation between the spatial architecture of the malignant mesothelioma tumor immune microenvironment and tumor biology. Our investigation reveals critical cell-cell interactions that may influence the immune response against malignant mesothelioma tumors, potentially contributing to the differential behaviors observed in MPM and MPeM. These findings represent a valuable resource for the malignant mesothelioma cancer research community.
Assuntos
Mesotelioma Maligno , Mesotelioma , Neoplasias Peritoneais , Neoplasias Pleurais , Purina-Núcleosídeo Fosforilase , Microambiente Tumoral , Ubiquitina Tiolesterase , Humanos , Microambiente Tumoral/imunologia , Mesotelioma Maligno/imunologia , Mesotelioma Maligno/patologia , Neoplasias Pleurais/imunologia , Neoplasias Pleurais/patologia , Masculino , Feminino , Neoplasias Peritoneais/imunologia , Neoplasias Peritoneais/patologia , Purina-Núcleosídeo Fosforilase/metabolismo , Purina-Núcleosídeo Fosforilase/genética , Pessoa de Meia-Idade , Ubiquitina Tiolesterase/metabolismo , Mesotelioma/imunologia , Mesotelioma/patologia , Idoso , Proteínas Supressoras de Tumor/metabolismo , Neoplasias Pulmonares/imunologia , Neoplasias Pulmonares/patologia , Neurofibromina 2/genética , Biomarcadores Tumorais , Prognóstico , Antígenos CD , Linfócitos T CD8-Positivos/imunologiaRESUMO
Performing cardiac surgery under cardiopulmonary bypass (CPB) and circulatory arrest (CA) provokes the development of complications caused by tissue metabolism, microcirculatory disorders, and endogenous nitric oxide (NO) deficiency. This study aimed to investigate the potential mechanisms for systemic organoprotective effects of exogenous NO during CPB and CA based on the assessment of dynamic changes in glycocalyx degradation markers, deformation properties of erythrocytes, and tissue metabolism in the experiment. A single-center prospective randomized controlled study was conducted on sheep, n = 24, comprising four groups of six in each. In two groups, NO was delivered at a dose of 80 ppm during CPB ("CPB + NO" group) or CPB and CA ("CPB + CA + NO"). In the "CPB" and "CPB + CA" groups, NO supply was not carried out. NO therapy prevented the deterioration of erythrocyte deformability. It was associated with improved tissue metabolism, lower lactate levels, and higher ATP levels in myocardial and lung tissues. The degree of glycocalyx degradation and endothelial dysfunction, assessed by the concentration of heparan sulfate proteoglycan and asymmetric dimethylarginine, did not change when exogenous NO was supplied. Intraoperative delivery of NO provides systemic organoprotection, which results in reducing the damaging effects of CPB on erythrocyte deformability and maintaining normal functioning of tissue metabolism.
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Emerging evidence supports a high prevalence of cancer type-specific microbiota residing within tumor tissues. The intratumoral microbiome in hepatocellular carcinoma (HCC), especially in viral (hepatitis B virus [HBV]/hepatitis C virus [HCV]) HCC, has not been well characterized for their existence, composition, distribution, and biological functions. We report herein a finding of specific microbial signature in viral HCC as compared to non-HBV/non-HCV (NBNC) HCC. However, the significantly diverse tumor microbiome was only observed in HBV-related HCC, and Cutibacterium was identified as the representative taxa biomarker. Biological function of the unique tumor microbiota in modulating tumor microenvironment (TME) was characterized by using formalin-fixed paraffin-embedded (FFPE) tissue-based multiplex immunofluorescence histochemistry (mIFH) allowing simultaneous in situ detection of the liver cancer cells surrounded with high/low density of microbiota, and the infiltrating immune cells. In HBV_HCC, the intratumoral microbiota are positively associated with increased tumor-infiltrating CD8+ T lymphocytes, but not the CD56+ NK cells. Two subtypes of myeloid-derived suppressor cells (MDSCs): monocytic MDSCs and polymorphonuclear MDSCs, were also found to be positively correlated with the intratumoral microbiota in HBV_HCC, indicating an inhibitory role of these microbial species in antitumor immunity and the contribution to the liver TME in combination of chronic viral hepatitis during HCC development.
Assuntos
Carcinoma Hepatocelular , Hepatite B , Hepatite C , Neoplasias Hepáticas , Humanos , Vírus da Hepatite B , Hepatite C/complicações , Hepatite B/complicações , Hepatite B/patologia , Microambiente TumoralRESUMO
HBV cccDNA is the persistent form of viral genome, which exists in host cell nucleus as an episomal minichromosome decorated with histone and non-histone proteins. cccDNA is the authentic viral transcription template and resistant to current antivirals. Growing evidence shows that the transcriptional activity of cccDNA minichromosome undergoes epigenetic regulations, suggesting a new perspective for anti-cccDNA drug development through targeting histone modifications. In this study, we screened an epigenetic compound library in the cccDNA reporter cell line HepBHAe82, which produces the HA-tagged HBeAg in a cccDNA-dependent manner. Among the obtained hits, a bromodomain-containing protein 4 (BRD4) inhibitor MS436 exhibited marked inhibition of cccDNA transcription in both HBV stable cell line HepAD38 and HepG2-NTCP or primary human hepatocyte infection system under noncytotoxic concentrations. Chromatin immunoprecipitation (ChIP) assay demonstrated that MS436 dramatically reduced the enrichment of H3K27ac, an activating histone modification pattern, on cccDNA minichromosome. RNAseq differential analysis showed that MS436 does not drastically change host transcriptome or induce any known anti-HBV factors/pathways, indicating a direct antiviral effect of MS436 on cccDNA minichromosome. Interestingly, the MS436-mediated inhibition of cccDNA transcription is accompanied by cccDNA destabilization in HBV infection and a recombinant cccDNA system, indicating that BRD4 activity may also play a role in cccDNA maintenance. Furthermore, depletion of BRD4 by siRNA knockdown or PROTAC degrader resulted in cccDNA inhibition in HBV-infected HepG2-NTCP cells, further validating BRD4 as an antiviral target. Taken together, our study has demonstrated the practicability of HepBHAe82-based anti-HBV drug screening system and provided a proof-of-concept for targeting HBV cccDNA with epigenetic compounds.
Assuntos
Vírus da Hepatite B , Hepatite B , Humanos , Antivirais/farmacologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Replicação Viral , DNA Viral/genética , DNA Circular/metabolismo , Histonas/metabolismo , Epigênese Genética , Proteínas de Ciclo Celular/metabolismoRESUMO
Hepatitis B virus (HBV) covalently closed circular DNA (cccDNA), serving as the viral persistence form and transcription template of HBV infection, hijacks host histone and non-histone proteins to form a minichromosome and utilizes posttranslational modifications (PTMs) "histone code" for its transcriptional regulation. HBV X protein (HBx) is known as a cccDNA transcription activator. In this study we established a dual system of the inducible reporter cell lines modelling infection with wildtype (wt) and HBx-null HBV, both secreting HA-tagged HBeAg as a semi-quantitative marker for cccDNA transcription. The cccDNA-bound histone PTM profiling of wt and HBx-null systems, using chromatin immunoprecipitation coupled with quantitative PCR (ChIP-qPCR), confirmed that HBx is essential for maintenance of cccDNA at transcriptionally active state, characterized by active histone PTM markers. Differential proteomics analysis of cccDNA minichromosome established in wt and HBx-null HBV cell lines revealed group-specific hits. One of the hits in HBx-deficient condition was a non-histone host DNA-binding protein high mobility group box 1 (HMGB1). Its elevated association to HBx-null cccDNA was validated by ChIP-qPCR assay in both the HBV stable cell lines and infection systems in vitro. Furthermore, experimental downregulation of HMGB1 in HBx-null HBV inducible and infection models resulted in transcriptional re-activation of the cccDNA minichromosome, accompanied by a switch of the cccDNA-associated histones to euchromatic state with activating histone PTMs landscape and subsequent upregulation of cccDNA transcription. Mechanistically, HBx interacts with HMGB1 and prevents its binding to cccDNA without affecting the steady state level of HMGB1. Taken together, our results suggest that HMGB1 is a novel host restriction factor of HBV cccDNA with epigenetic silencing mechanism, which can be counteracted by viral transcription activator HBx.
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Proteína HMGB1 , Hepatite B , DNA Circular/genética , DNA Circular/metabolismo , DNA Viral/genética , DNA Viral/metabolismo , Epigênese Genética , Proteína HMGB1/genética , Células Hep G2 , Vírus da Hepatite B/metabolismo , Histonas/metabolismo , Humanos , Transativadores , Fatores de Transcrição/metabolismo , Proteínas Virais Reguladoras e Acessórias , Replicação Viral/genéticaRESUMO
This study aimed to gain a deeper understanding of spiritual change processes by conducting an empirical investigation of clinically meaningful events occurring within the context of a Christian-Integrated Psychotherapy Framework. The discovery phase of task analysis was used to build a rational-empirical model that explicated how clients developed stronger attachments to their God images. A rational model was specified, and five cases were selected for further analysis from a pool of 27 client-participants and 423 video-recorded psychotherapy sessions. Clinical observations and the coding of in-session measures were used to select resolved and unresolved cases, which were then contrasted to create a 10-step rational-empirical model. In this model, clients initially presented with intense feelings of shame, guilt, or helplessness but concluded with heightened levels of love and joy. Clients who reached this resolution all underwent an experience whereby their God images became dynamic, alive, intimate, and authentic. Results are discussed, and implications for the existence of an in vivo God image and corrective emotional spiritual experiences are introduced. Specifically, corrective emotional spiritual experiences appear to have influenced clients to transition from being insecurely attached to more securely attached to their in vivo God images. (PsycInfo Database Record (c) 2022 APA, all rights reserved).
Assuntos
Relações Profissional-Paciente , Psicoterapia , Emoções , Humanos , Psicoterapia/métodosRESUMO
Hepatitis B virus (HBV) utilizes host DNA repair mechanisms to convert viral relaxed circular DNA (rcDNA) into a persistent viral genome, the covalently closed circular DNA (cccDNA). To identify host factors involved in cccDNA formation, we developed an unbiased approach to discover proteins involved in cccDNA formation by precipitating nuclear rcDNA from induced HepAD38 cells and identifying the coprecipitated proteins by mass spectrometry. DNA damage binding protein 1 (DDB1) surfaced as a hit, coinciding with our previously reported short hairpin RNA (shRNA) screen in which shRNA-DDB1 in HepDES19 cells reduced cccDNA production. DDB1 binding to nuclear rcDNA was confirmed in HepAD38 cells via ChIP-qPCR. DDB1 and DNA damage binding protein 2 (DDB2) form the UV-DDB complex, and the latter senses DNA damage to initiate the global genome nucleotide excision repair (GG-NER) pathway. To investigate the role of the DDB complex in cccDNA formation, DDB2 was knocked out in HepAD38 and HepG2-NTCP cells. In both knockout cell lines, cccDNA formation was stunted significantly, and in HepG2-NTCP-DDB2 knockout cells, downstream indicators of cccDNA such as HBV RNA, HBcAg, and HBeAg were similarly reduced. Knockdown of DDB2 in HBV-infected HepG2-NTCP cells and primary human hepatocytes (PHH) also resulted in cccDNA reduction. Transcomplementation of wild-type DDB2 in HepG2-NTCP-DDB2 knockout cells rescued cccDNA formation and its downstream indicators. However, ectopic expression of DDB2 mutants deficient in DNA binding, DDB1 binding, or ubiquitination failed to rescue cccDNA formation. Our study thus suggests an integral role of UV-DDB, specifically DDB2, in the formation of HBV cccDNA. IMPORTANCE Serving as a key viral factor for chronic hepatitis B virus (HBV) infection, HBV covalently closed circular DNA (cccDNA) is formed in the cell nucleus from viral relaxed circular DNA (rcDNA) by hijacking host DNA repair machinery. Previous studies have identified several host DNA repair factors involved in cccDNA formation through hypothesis-driven research with some help from RNA interference (RNAi) screening and/or biochemistry approaches. To enrich the landscape of tools for discovering host factors responsible for rcDNA-to-cccDNA conversion, we developed an rcDNA immunoprecipitation paired mass spectrometry assay, which allowed us to pull down nuclear rcDNA in its transitional state to cccDNA and observe the associated host factors. From this assay, we discovered a novel relationship between the UV-DDB complex and cccDNA formation, providing a proof of concept for a more direct discovery of novel HBV DNA-host interactions that can be exploited to develop new cccDNA-targeting antivirals.
Assuntos
DNA Circular/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Vírus da Hepatite B/fisiologia , Linhagem Celular , Núcleo Celular/metabolismo , Núcleo Celular/virologia , Replicação do DNA , Proteínas de Ligação a DNA/genética , Antígenos da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Humanos , Ligação Proteica , Proteômica , RNA Viral/metabolismo , Ubiquitinação , Replicação ViralRESUMO
This article analyzes contradictory practices carried out in Kyrgyzstani crisis centers for victims of gender violence resulting in women-clients failing to obtain the protection they seek. These problematic dynamics are shaped by a global apparatus on women's human rights protection and international standards of practice. Crisis center professionals perform the final activation of this ruling apparatus through textual work driven not by the women's needs but by the goal of bringing local actions into accord with the "legal framework" organized and expressed by the national anti-violence law and the government's need to report on it to international treaty bodies.
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Intervenção em Crise , Violência , Antropologia Cultural , Feminino , Humanos , Quirguistão , Violência/prevenção & controle , Direitos da MulherRESUMO
Climate change is impacting crop performance and agricultural systems around the world with implications for farmers and consumers. We carried out a systematic review to synthesize evidence regarding the effects of environmental factors associated with climate change and management conditions associated with climate adaptation on the crop quality of a culturally-relevant perennial crop, coffee (Coffea arabica and Coffea canephora). Seventy-three articles were identified that addressed the study's research question including 42 articles on environmental factors, 20 articles on management conditions, and 11 articles on both. While variation was found between studies, findings highlight that coffee quality is vulnerable to changes in light exposure, altitude, water stress, temperature, carbon dioxide, and nutrient management. Both increases as well as decreases were found in secondary metabolites and sensory attributes that determine coffee quality in response to shifts in environmental and management conditions. The most consistent evidence identified through this systematic review includes the following two trends: (1) increased altitude is associated with improved sensory attributes of coffee and; (2) increased light exposure is associated with decreased sensory attributes of coffee. Research gaps were found regarding the effects of shifts in carbon dioxide, water stress, and temperature on the directionality (increase, decrease, or non-linear) of coffee quality and how this varies with location, elevation, and management conditions. This systematic review further identified the following research needs: (1) long-term studies that examine the interactive effects of multiple environmental factors and management conditions on coffee quality; (2) studies that examine the interaction between sensory attributes and secondary metabolites that determine coffee quality and; (3) studies on the feasibility of various climate-adaptation strategies for mitigating the effects of climate change on coffee quality. Evidence-based innovations are needed to mitigate climate impacts on coffee quality toward enhanced sustainability and resilience of the coffee sector from farm to cup.
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An increase in new HIV infections among women in Kazakhstan has motivated efforts to improve access to comprehensive health services. This study estimates anxiety and depression frequency among women seeking HIV services. A cross-sectional survey was administered to women seen at the Almaty AIDS Center. Bivariable analyses (e.g., Chi-square, means with 95% confidence intervals) were performed to assess the relationship between anxiety (score of 10 or more on the Generalized Anxiety Disorder-7 Scale (GAD-7)), major depression (Patient Health Questionnaire 8 (PHQ-8)), and comorbid anxiety and major depression with sociodemographic characteristics, health functioning, and medication history. Of the 410 participants, 62 (15.1%) had a GAD-7 ≥ 10; 52 (12.7%) met major depression criteria; 35 (8.5%) met both criteria, and 79 (19.3%) met GAD-7, major depression, or both criteria. Women reporting depression or anxiety were more likely to lack food security (p < 0.01), not finish secondary school (p < 0.01), speak Russian at home (p < 0.01), perceive their health to be poor (p < 0.01), and report poorer physical and mental health functioning (p < 0.05). No medications approved for the treatment of anxiety or depression were reported. The considerable number of women reporting major depression and anxiety symptoms suggests a need for improving access to mental health care.
Assuntos
Fármacos Anti-HIV/uso terapêutico , Ansiedade/epidemiologia , Depressão/epidemiologia , Infecções por HIV/tratamento farmacológico , Infecções por HIV/psicologia , Adulto , Ansiedade/psicologia , Transtornos de Ansiedade/epidemiologia , Transtornos de Ansiedade/psicologia , Estudos Transversais , Depressão/psicologia , Feminino , Infecções por HIV/etnologia , Humanos , Cazaquistão/epidemiologiaRESUMO
HBV is an enveloped DNA virus that replicates its DNA genome via reverse transcription of a pregenomic (pg) RNA intermediate in hepatocytes. Interestingly, HBV RNA can be detected in virus-like particles in chronic hepatitis B (CHB) patient serum and has been utilized as a biomarker for intrahepatic cccDNA activity in treated patients. However, the biogenesis and molecular characteristics of serum HBV RNA remain to be fully defined. In this study, we found that the encapsidated serum HBV RNA predominately consists of pgRNA, which are detergent- and ribonuclease-resistant. Through blocking HBV DNA replication without affecting pgRNA encapsidation by using the priming-defective HBV mutant Y63D or 3TC treatment, we demonstrated that the cell culture supernatant contains a large amount of pgRNA-containing nonenveloped capsids and a minor population of pgRNA-containing virions. The formation of pgRNA-virion requires both capsid assembly and viral envelope proteins, which can be inhibited by capsid assembly modulators and an envelope-knockout mutant, respectively. Furthermore, the pgRNA-virion utilizes the multivesicular body pathway for egress, in a similar way as DNA-virion morphogenesis. Northern blotting, RT-PCR, and 3' RACE assays revealed that serum/supernatant HBV pgRNA are mainly spliced and devoid of the 3'-terminal sequences. Furthermore, pgRNA-virion collected from cells treated with a reversible HBV priming inhibitor L-FMAU was unable to establish infection in HepG2-NTCP cells. In summary, serum HBV RNA is secreted in noninfectious virion-like particle as spliced and poly(A)-free pgRNA. Our study will shed light on the molecular biology of serum HBV RNA in HBV life cycle, and aid the development of serum HBV RNA as a novel biomarker for CHB diagnosis and treatment prognosis.
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Vírus da Hepatite B/patogenicidade , Hepatócitos/virologia , RNA Viral/genética , Replicação Viral/genética , Capsídeo/metabolismo , Proteínas do Capsídeo/metabolismo , DNA Viral/genética , Vírus da Hepatite B/metabolismo , Humanos , Transcrição Reversa/genética , Montagem de Vírus/genéticaRESUMO
The biosynthesis of hepatitis B virus (HBV) covalently closed circular DNA (cccDNA) requires the removal of the covalently linked viral polymerase from the 5' end of the minus strand [(-)strand] of viral relaxed circular DNA (rcDNA), which generates a deproteinated rcDNA (DP-rcDNA) intermediate. In the present study, we systematically characterized the four termini of cytoplasmic HBV DP-rcDNA by 5'/3' rapid amplification of cDNA ends (RACE), 5' radiolabeling, and exonuclease digestion, which revealed the following observations: (i) DP-rcDNA and rcDNA possess an identical 3' end of (-)strand DNA; (ii) compared to rcDNA, DP-rcDNA has an extended but variable 3' end of plus strand [(+)strand] DNA, most of which is in close proximity to direct repeat 2 (DR2); (iii) DP-rcDNA exhibits an RNA primer-free 5' terminus of (+)strand DNA with either a phosphate or hydroxyl group; and (iv) the 5' end of the DP-rcDNA (-)strand is unblocked at nucleotide G1828, bearing a phosphate moiety, indicating the complete removal of polymerase from rcDNA via unlinking the tyrosyl-DNA phosphodiester bond during rcDNA deproteination. However, knockout of cellular 5' tyrosyl-DNA phosphodiesterase 2 (TDP2) did not markedly affect rcDNA deproteination or cccDNA formation. Thus, our work sheds new light on the molecular mechanisms of rcDNA deproteination and cccDNA biogenesis.IMPORTANCE The covalently closed circular DNA (cccDNA) is the persistent form of the hepatitis B virus (HBV) genome in viral infection and an undisputed antiviral target for an HBV cure. HBV cccDNA is converted from viral genomic relaxed circular DNA (rcDNA) through a complex process that involves removing the covalently bound viral polymerase from rcDNA, which produces a deproteinated-rcDNA (DP-rcDNA) intermediate for cccDNA formation. In this study, we characterized the four termini of cytoplasmic DP-rcDNA and compared them to its rcDNA precursor. While rcDNA and DP-rcDNA have an identical 3' terminus of (-)strand DNA, the 3' terminus of (+)strand DNA on DP-rcDNA is further elongated. Furthermore, the peculiarities on rcDNA 5' termini, specifically the RNA primer on the (+)strand and the polymerase on the (-)strand, are absent from DP-rcDNA. Thus, our study provides new insights into a better understanding of HBV rcDNA deproteination and cccDNA biosynthesis.
Assuntos
Citoplasma/virologia , DNA Circular/genética , DNA Viral/genética , Vírus da Hepatite B/genética , Linhagem Celular , Citoplasma/metabolismo , Replicação do DNA , DNA Circular/metabolismo , DNA Viral/metabolismo , Proteínas de Ligação a DNA/metabolismo , Exonucleases/metabolismo , Vírus da Hepatite B/metabolismo , Humanos , Diester Fosfórico Hidrolases/metabolismo , Replicação ViralRESUMO
Antagonism of host immune defenses against hepatitis B virus (HBV) infection by the viral proteins is speculated to cause HBV persistence and the development of chronic hepatitis. The circulating hepatitis B e antigen (HBeAg, p17) is known to manipulate host immune responses to assist in the establishment of persistent viral infection, and HBeAg-positive (HBeAg+) patients respond less effectively to IFN-α therapy than do HBeAg-negative (HBeAg-) patients in clinical practice. However, the function(s) of the intracellular form of HBeAg, previously reported as the precore protein intermediate (p22) without the N-terminal signal peptide, remains elusive. Here, we report that the cytosolic p22 protein, but not the secreted HBeAg, significantly reduces interferon-stimulated response element (ISRE) activity and the expression of interferon-stimulated genes (ISGs) upon alpha interferon (IFN-α) stimulation in cell cultures. In line with this, HBeAg+ patients exhibit weaker induction of ISGs in their livers than do HBeAg- patients upon IFN-α therapy. Mechanistically, while p22 does not alter the total STAT1 or pSTAT1 levels in cells treated with IFN-α, it blocks the nuclear translocation of pSTAT1 by interacting with the nuclear transport factor karyopherin α1 through its C-terminal arginine-rich domain. In summary, our study suggests that HBV precore protein, specifically the p22 form, impedes JAK-STAT signaling to help the virus evade the host innate immune response and, thus, causes resistance to IFN therapy.IMPORTANCE Chronic hepatitis B virus (HBV) infection continues to be a major global health concern, and patients who fail to mount an efficient immune response to clear the virus will develop a life-long chronic infection that can progress to chronic active hepatitis, cirrhosis, and primary hepatocellular carcinoma. There is no definite cure for chronic hepatitis B, and alpha interferon (IFN-α) is the only available immunomodulatory drug, to which only a minority of chronic patients are responsive, with hepatitis B e antigen (HBeAg)-negative patients responding better than HBeAg-positive patients. We herein report that the intracellular HBeAg, also known as precore or p22, inhibits the antiviral signaling of IFN-α, which sheds light on the enigmatic function of precore protein in shaping HBV chronicity and provides a perspective toward areas that need to be further studied to make the current therapy better until a cure is achieved.
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Antígenos do Núcleo do Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/patogenicidade , Hepatite B/virologia , Interferon-alfa/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas do Core Viral/metabolismo , Adolescente , Adulto , Antivirais/farmacologia , Carcinoma Hepatocelular/metabolismo , Feminino , Células HEK293 , Células Hep G2 , Antígenos E da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Hepatite B Crônica , Humanos , Imunidade Inata , Fígado , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Transporte Proteico , Fator de Transcrição STAT1/metabolismo , Adulto JovemRESUMO
Hepadnavirus covalently closed circular (ccc) DNA is the bona fide viral transcription template, which plays a pivotal role in viral infection and persistence. Upon infection, the non-replicative cccDNA is converted from the incoming and de novo synthesized viral genomic relaxed circular (rc) DNA, presumably through employment of the host cell's DNA repair mechanisms in the nucleus. The conversion of rcDNA into cccDNA requires preparation of the extremities at the nick/gap regions of rcDNA for strand ligation. After screening 107 cellular DNA repair genes, we herein report that the cellular DNA ligase (LIG) 1 and 3 play a critical role in cccDNA formation. Ligase inhibitors or functional knock down/out of LIG1/3 significantly reduced cccDNA production in an in vitro cccDNA formation assay, and in cccDNA-producing cells without direct effect on viral core DNA replication. In addition, transcomplementation of LIG1/3 in the corresponding knock-out or knock-down cells was able to restore cccDNA formation. Furthermore, LIG4, a component in non-homologous end joining DNA repair apparatus, was found to be responsible for cccDNA formation from the viral double stranded linear (dsl) DNA, but not rcDNA. In conclusion, we demonstrate that hepadnaviruses utilize the whole spectrum of host DNA ligases for cccDNA formation, which sheds light on a coherent molecular pathway of cccDNA biosynthesis, as well as the development of novel antiviral strategies for treatment of hepatitis B.
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DNA Ligases/metabolismo , DNA Circular/biossíntese , DNA Viral/biossíntese , Hepadnaviridae/metabolismo , Linhagem Celular , DNA Ligase Dependente de ATP/antagonistas & inibidores , DNA Ligase Dependente de ATP/genética , DNA Ligase Dependente de ATP/metabolismo , DNA Ligases/antagonistas & inibidores , DNA Ligases/genética , Reparo do DNA/genética , Técnicas de Silenciamento de Genes , Técnicas de Inativação de Genes , Células HEK293 , Células Hep G2 , Hepadnaviridae/genética , Hepadnaviridae/patogenicidade , Vírus da Hepatite B/genética , Vírus da Hepatite B/metabolismo , Vírus da Hepatite B/patogenicidade , Hepatócitos/metabolismo , Hepatócitos/virologia , Interações Hospedeiro-Patógeno/genética , Interações Hospedeiro-Patógeno/fisiologia , Humanos , Redes e Vias Metabólicas , Proteínas de Ligação a Poli-ADP-Ribose/antagonistas & inibidores , Proteínas de Ligação a Poli-ADP-Ribose/genética , Proteínas de Ligação a Poli-ADP-Ribose/metabolismoRESUMO
Hepatitis B virus (HBV) infection represents a significant public health burden worldwide. Although current therapeutics manage to control the disease progression, lifelong treatment and surveillance are required because drug resistance develops during treatment and reactivations frequently occur following medication cessation. Thus, the occurrence of hepatocellular carcinoma is decreased, but not eliminated. One major reason for failure of HBV treatment is the inability to eradicate or inactivate the viral covalently closed circular DNA (cccDNA), which is a stable episomal form of the viral genome decorated with host histones and nonhistone proteins. Accumulating evidence suggests that epigenetic modifications of cccDNA contribute to viral replication and the outcome of chronic HBV infection. Here, we summarize current progress on HBV epigenetics research and the therapeutic implications for chronic HBV infection by learning from the epigenetic therapies for cancer and other viral diseases, which may open a new venue to cure chronic hepatitis B. (Hepatology 2017;66:2066-2077).
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DNA Circular/metabolismo , DNA Viral/metabolismo , Epigênese Genética , Vírus da Hepatite B/genética , Hepatite B Crônica/virologia , Vírus da Hepatite B/metabolismo , Hepatite B Crônica/terapia , HumanosRESUMO
Facioscapulohumeral dystrophy (FSHD) is an epi/genetic satellite disease associated with at least two satellite sequences in 4q35: (i) D4Z4 macrosatellite and (ii) ß-satellite repeats (BSR), a prevalent part of the 4qA allele. Most of the recent FSHD studies have been focused on a DUX4 transcript inside D4Z4 and its tandem contraction in FSHD patients. However, the D4Z4-contraction alone is not pathological, which would also require the 4qA allele. Since little is known about BSR, we investigated the 4qA BSR functional role in the transcriptional control of the FSHD region 4q35. We have shown that an individual BSR possesses enhancer activity leading to activation of the Adenine Nucleotide Translocator 1 gene (ANT1), a major FSHD candidate gene. We have identified ZNF555, a previously uncharacterized protein, as a putative transcriptional factor highly expressed in human primary myoblasts that interacts with the BSR enhancer site and impacts the ANT1 promoter activity in FSHD myoblasts. The discovery of the functional role of the 4qA allele and ZNF555 in the transcriptional control of ANT1 advances our understanding of FSHD pathogenesis and provides potential therapeutic targets.
Assuntos
Translocador 1 do Nucleotídeo Adenina/genética , Cromossomos Humanos Par 4 , Distrofia Muscular Facioescapuloumeral/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Translocador 1 do Nucleotídeo Adenina/biossíntese , Alelos , Sítios de Ligação , Células Cultivadas , DNA Satélite , Elementos Facilitadores Genéticos , Loci Gênicos , Humanos , Proteínas dos Microfilamentos , Mioblastos/metabolismo , Proteínas Nucleares/biossíntese , Proteínas Nucleares/genética , Proteínas de Ligação a RNA , Fatores de Transcrição/antagonistas & inibidoresRESUMO
The increased use of inhaled nicotine via e-cigarettes has unknown risks to lung health. Having previously shown that cigarette smoke (CS) extract disrupts the lung microvasculature barrier function by endothelial cell activation and cytoskeletal rearrangement, we investigated the contribution of nicotine in CS or e-cigarettes (e-Cig) to lung endothelial injury. Primary lung microvascular endothelial cells were exposed to nicotine, e-Cig solution, or condensed e-Cig vapor (1-20 mM nicotine) or to nicotine-free CS extract or e-Cig solutions. Compared with nicotine-containing extract, nicotine free-CS extract (10-20%) caused significantly less endothelial permeability as measured with electric cell-substrate impedance sensing. Nicotine exposures triggered dose-dependent loss of endothelial barrier in cultured cell monolayers and rapidly increased lung inflammation and oxidative stress in mice. The endothelial barrier disruptive effects were associated with increased intracellular ceramides, p38 MAPK activation, and myosin light chain (MLC) phosphorylation, and was critically mediated by Rho-activated kinase via inhibition of MLC-phosphatase unit MYPT1. Although nicotine at sufficient concentrations to cause endothelial barrier loss did not trigger cell necrosis, it markedly inhibited cell proliferation. Augmentation of sphingosine-1-phosphate (S1P) signaling via S1P1 improved both endothelial cell proliferation and barrier function during nicotine exposures. Nicotine-independent effects of e-Cig solutions were noted, which may be attributable to acrolein, detected along with propylene glycol, glycerol, and nicotine by NMR, mass spectrometry, and gas chromatography, in both e-Cig solutions and vapor. These results suggest that soluble components of e-Cig, including nicotine, cause dose-dependent loss of lung endothelial barrier function, which is associated with oxidative stress and brisk inflammation.
Assuntos
Sistemas Eletrônicos de Liberação de Nicotina/efeitos adversos , Endotélio Vascular/efeitos dos fármacos , Nicotina/efeitos adversos , Agonistas Nicotínicos/efeitos adversos , Estresse Oxidativo/efeitos dos fármacos , Pneumonia/patologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Ceramidas/metabolismo , Impedância Elétrica , Endotélio Vascular/metabolismo , Endotélio Vascular/patologia , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Immunoblotting , Lisofosfolipídeos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Pneumonia/induzido quimicamente , Pneumonia/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Esfingosina/análogos & derivados , Esfingosina/metabolismoRESUMO
Mechanisms that regulate attachment of the scaffold/matrix attachment regions (S/MARs) to the nuclear matrix remain largely unknown. We have studied the effect of simple sequence length polymorphism (SSLP), DNA methylation and chromatin organization in an S/MAR implicated in facioscapulohumeral dystrophy (FSHD), a hereditary disease linked to a partial deletion of the D4Z4 repeat array on chromosome 4q. This FSHD-related nuclear matrix attachment region (FR-MAR) loses its efficiency in myoblasts from FSHD patients. Three criteria were found to be important for high-affinity interaction between the FR-MAR and the nuclear matrix: the presence of a specific SSLP haplotype in chromosomal DNA, the methylation of one specific CpG within the FR-MAR and the absence of histone H3 acetylated on lysine 9 in the relevant chromatin fragment.