RESUMEN
Dihydroquinolizinones (DHQs) that inhibit cellular polyadenylating polymerases 5 and 7 (PAPD5 & 7), such as RG7834, have been shown to inhibit both hepatitis A (HAV) and hepatitis B virus (HBV) in vitro and in vivo. In this report, we describe RG7834-based proteolysis-targeting chimeras (PROTACs), such as compound 12b, (6S)-9-((1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-21-oxo-3,6,9,12,15,18-hexaoxa-22-azapentacosan-25-yl)oxy)-6-isopropyl-10-methoxy-2-oxo-6,7-dihydro-2H-pyrido[2,1-a]isoquinoline-3-carboxylic acid. The PROTAC DHQs described here inhibited an HAV reporter virus in vitro with an IC50 of 277 nM. Although the PROTAC DHQs were also inhibitory to HBV, their activities were substantially less potent against HBV in vitro, being in the 10 to 20 µM range, based on the reduction of HBsAg and HBV mRNA levels. Importantly, unlike RG7834, the incubation of cells in vitro with PROTAC DHQ 12b resulted in the degradation of PAPD5, as expected for a PROTAC compound, but curiously not PAPD7. PAPD5 polypeptide degradation was prevented when a proteasome inhibitor, epoxomicin, was used, indicating that proteasome mediated proteolysis was associated with the observed activities of 12b. Taken together, these data show that 12b is the first example of a PROTAC that suppresses both HAV and HBV that is based on a small molecule warhead. The possibility that it has mechanisms that differ from its parent compound, RG7834, and has clinical value, is discussed.
Asunto(s)
Virus de la Hepatitis A , Virus de la Hepatitis B , Proteolisis , Complejo de la Endopetidasa ProteasomalRESUMEN
Chronic hepatitis B (CHB) is characterized by high levels of hepatitis B virus (HBV) surface antigen (HBsAg) in blood circulation. A major goal of CHB interventions is reducing or eliminating this antigenemia; however, there are currently no approved methods that can do this. A novel family of compounds with a dihydroquinolizinone (DHQ) scaffold has been shown to reduce circulating levels of HBsAg in animals, representing a first for a small molecule. Reductions of HBsAg were a result of the compound's effect on HBsAg mRNA levels. However, commercial development by Roche of a DHQ lead compound, RG-7834, was stopped due to undisclosed toxicity issues. Herein we report our effort to convert the systemic RG7834 compound to a hepatoselective DHQ analog to limit its distribution to the bloodstream and thus to other body tissues.
RESUMEN
Upon infection of hepatocyte, Hepatitis B virus (HBV) genomic DNA in nucleocapsid is transported into the nucleus and converted into a covalently closed circular (ccc) DNA to serve as the template for transcription of viral RNAs. Viral DNA in the cytoplasmic progeny nucleocapsid is another resource to fuel cccDNA amplification. Apparently, nucleocapsid disassembly, or viral genomic DNA uncoating, is an essential step for cccDNA synthesis from both de novo infection and intracellular amplification pathways, and has a potential to activate DNA sensors and induce an innate immune response in infected hepatocytes. However, where and how the nucleocapsid disassembly occurs is not well understood. The work reported herein showed that the enhanced disassembly of progeny mature nucleocapsids in the cytoplasm supported cccDNA intracellular amplification, but failed to activate the cGAS-STING-mediated innate immune response in hepatocytes. Interestingly, while expression of a cytoplasmic exonuclease TREX1 in human hepatoma cells supporting HBV replication significantly reduced the amounts of cccDNA as well as its precursor, deproteinized relaxed circular (rc) DNA, expression of TREX1 in sodium taurocholate cotransporting polypeptide-expressing human hepatoma cells did not inhibit cccDNA synthesis from de novo HBV infection. The results from this cytoplasmic nuclease protection assay imply that the disassembly of progeny mature nucleocapsids and removal of viral DNA polymerase covalently linked to the 5' end of minus strand of rcDNA take place in the cytoplasm. On the contrary, the disassembly of virion-derived nucleocapsids during de novo infection may occur at a different subcellular compartment and possibly via distinct mechanisms.
Asunto(s)
ADN Circular/genética , Exodesoxirribonucleasas/metabolismo , Virus de la Hepatitis B/genética , Hepatocitos/virología , Nucleocápside/genética , Fosfoproteínas/metabolismo , Línea Celular , Citoplasma/genética , ADN Circular/inmunología , ADN Viral/genética , ADN Viral/inmunología , Exodesoxirribonucleasas/genética , Células Hep G2 , Virus de la Hepatitis B/inmunología , Hepatocitos/citología , Hepatocitos/inmunología , Humanos , Inmunidad Innata , Mutación , Nucleocápside/inmunología , Nucleotidiltransferasas/metabolismo , Fosfoproteínas/genéticaRESUMEN
Hepatitis B virus (HBV) small (S) envelope protein has the intrinsic ability to direct the formation of small spherical subviral particles (SVPs) in eukaryotic cells. However, the molecular mechanism underlying the morphogenesis of SVPs from the monomeric S protein initially synthesized at the endoplasmic reticulum (ER) membrane remains largely elusive. Structure prediction and extensive mutagenesis analysis suggested that the amino acid residues spanning W156 to R169 of S protein form an amphipathic alpha helix and play essential roles in SVP production and S protein metabolic stability. Further biochemical analyses showed that the putative amphipathic alpha helix was not required for the disulfide-linked S protein oligomerization, but was essential for SVP morphogenesis. Pharmacological disruption of vesicle trafficking between the ER and Golgi complex in SVP producing cells supported the hypothesis that S protein-directed SVP morphogenesis takes place at the ER-Golgi intermediate compartment (ERGIC). Moreover, it was demonstrated that S protein is degraded in hepatocytes via a 20S proteasome-dependent, but ubiquitination-independent non-classic ER-associated degradation (ERAD) pathway. Taken together, the results reported herein favor a model in which the amphipathic alpha helix at the antigenic loop of S protein attaches to the lumen leaflet to facilitate SVP budding from the ERGIC compartment, whereas the failure of budding process may result in S protein degradation by 20S proteasome in an ubiquitination-independent manner.Importance Subviral particles are the predominant viral product produced by HBV-infected hepatocytes. Their levels exceed the virion particles by 10,000 to 100,000-fold in the blood of HBV infected individuals. The high levels of SVPs, or HBV surface antigen (HBsAg), in the circulation induces immune tolerance and contributes to the establishment of persistent HBV infection. The loss of HBsAg, often accompanied by appearance of anti-HBs antibodies, is the hallmark of durable immune control of HBV infection. Therapeutic induction of HBsAg loss is, therefore, considered to be essential for the restoration of host antiviral immune response and functional cure of chronic hepatitis B. Our findings on the mechanism of SVP morphogenesis and S protein metabolism will facilitate the rational discovery and development of antiviral drugs to achieve this therapeutic goal.
RESUMEN
Inhibition of the host RNA polyadenylating polymerases, PAPD5 and PAPD7 (PAPD5/7), with dihydroquinolizinone, a small orally available, molecule, results in a rapid and selective degradation of hepatitis B virus (HBV) RNA, and hence reduction in the amounts of viral gene products. DHQ, is a first in class investigational agent and could represent an entirely new category of HBV antivirals. PAPD5 and PAPD7 are non-canonical, cell specified, polyadenylating polymerases, also called terminal nucleotidyl transferases 4B and 4A (TENT4B/A), respectively. They are involved in the degradation of poor-quality cell transcripts, mostly non-coding RNAs and in the maturation of a sub-set of transcripts. They also appear to play a role in shielding some mRNA from degradation. The results of studies with DHQ, along with other recent findings, provide evidence that repression of the PAPD5/7 arm of the cell "RNA quality control" pathway, causes a profound (multi-fold) reduction rather than increase, in the amount of HBV pre-genomic, pre-core and HBsAg mRNA levels in tissue culture and animal models, as well. In this review we will briefly discuss the need for new HBV therapeutics and provide background about HBV transcription. We also discuss cellular degradation of host transcripts, as it relates to a new family of anti-HBV drugs that interfere with these processes. Finally, since HBV mRNA maturation appears to be selectively sensitive to PAPD5/7 inhibition in hepatocytes, we discuss the possibility of targeting host RNA "quality control" as an antiviral strategy.
Asunto(s)
Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Interacciones Microbiota-Huesped/efectos de los fármacos , Interacciones Microbiota-Huesped/genética , Estabilidad del ARN/efectos de los fármacos , Regulación Viral de la Expresión Génica , Hepatitis B/virología , Hepatocitos/virología , Humanos , Estabilidad del ARN/fisiología , Replicación ViralRESUMEN
Standard literature procedures for the chemical synthesis of l-threose nucleosides generally employ l-ascorbic acid as starting material. Herein, we have explored two alternative routes that start from either l-arabitol or l-diethyl tartrate, both affording 2-O-methyl-l-threofuranose as a key building block for nucleobase incorporation. The access to multigram quantities of this glycosyl donor in a reproducible fashion allows for the preparation of 2'-deoxy-α-l-threofuranosyl phosphonate nucleosides on a large scale. This methodology was applied to the gram scale synthesis of an aryloxy amidate prodrug of phosphonomethoxydeoxythreosyl adenine. This prodrug exerted potent activity against an entecavir-resistant hepatitis B virus (HBV) strain, while leading to a significant reduction in the levels of HBV covalently closed circular DNA in a cellular assay. Furthermore, its remarkable anti-HBV efficacy was also confirmed in vivo using a hydrodynamic injection-based HBV mouse model, without relevant toxicity and systemic exposure occurring.
Asunto(s)
Antivirales/farmacología , ADN Circular/genética , Farmacorresistencia Viral/efectos de los fármacos , Guanina/análogos & derivados , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B/tratamiento farmacológico , Profármacos/farmacología , Adenina/química , Animales , ADN Circular/análisis , ADN Viral/análisis , ADN Viral/genética , Guanina/farmacología , Hepatitis B/virología , Virus de la Hepatitis B/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Nucleósidos/química , Replicación ViralRESUMEN
Hepatitis B virus (HBV) mRNA metabolism is dependent upon host proteins PAPD5 and PAPD7 (PAPD5/7). PAPD5/7 are cellular, noncanonical, poly(A) polymerases (PAPs) whose main function is to oligoadenylate the 3' end of noncoding RNA (ncRNA) for exosome degradation. HBV seems to exploit these two ncRNA quality-control factors for viral mRNA stabilization, rather than degradation. RG7834 is a small-molecule compound that binds PAPD5/7 and inhibits HBV gene production in both tissue culture and animal study. We reported that RG7834 was able to destabilize multiple HBV mRNA species, ranging from the 3.5-kb pregenomic/precore mRNAs to the 2.4/2.1-kb hepatitis B virus surface protein (HBs) mRNAs, except for the smallest 0.7-kb X protein (HBx) mRNA. Compound-induced HBV mRNA destabilization was initiated by a shortening of the poly(A) tail, followed by an accelerated degradation process in both the nucleus and cytoplasm. In cells expressing HBV mRNA, both PAPD5/7 were found to be physically associated with the viral RNA, and the polyadenylating activities of PAPD5/7 were susceptible to RG7834 repression in a biochemical assay. Moreover, in PAPD5/7 double-knockout cells, viral transcripts with a regular length of the poly(A) sequence could be initially synthesized but became shortened in hours, suggesting that participation of PAPD5/7 in RNA 3' end processing, either during adenosine oligomerization or afterward, is crucial for RNA stabilization.
Asunto(s)
Virus de la Hepatitis B , Hepatitis B , Animales , Virus de la Hepatitis B/genética , Proteínas de la Membrana , ARN Mensajero/genética , ARN Viral/genética , Ribonucleasas , Replicación ViralRESUMEN
Hepatitis B virus (HBV) has infected one-third of world population, and 240 million people are chronic carriers, to whom a curative therapy is still not available. Similar to other viruses, persistent HBV infection relies on the virus to exploit host cell functions to support its replication and efficiently evade host innate and adaptive antiviral immunity. Understanding HBV replication and concomitant host cell interactions is thus instrumental for development of therapeutics to disrupt the virus-host interactions critical for its persistence and cure chronic hepatitis B. Although the currently available cell culture systems of HBV infection are refractory to genome-wide high throughput screening of key host cellular factors essential for and/or regulating HBV replication, classic one-gene (or pathway)-at-a-time studies in the last several decades have already revealed many aspects of HBV-host interactions. An overview of the landscape of HBV-hepatocyte interaction indicates that, in addition to more tightly suppressing viral replication by directly targeting viral proteins, disruption of key viral-host cell interactions to eliminate or inactivate the covalently closed circular (ccc) DNA, the most stable HBV replication intermediate that exists as an episomal minichromosome in the nucleus of infected hepatocyte, is essential to achieve a functional cure of chronic hepatitis B. Moreover, therapeutic targeting of integrated HBV DNA and their transcripts may also be required to induce hepatitis B virus surface antigen (HBsAg) seroclearance and prevent liver carcinogenesis.
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Antivirales/farmacología , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B Crónica/tratamiento farmacológico , Interacciones Microbiota-Huesped , Animales , ADN Circular , ADN Viral , Virus de la Hepatitis B/genética , Hepatitis B Crónica/virología , Hepatocitos/virología , Humanos , Replicación Viral/efectos de los fármacosRESUMEN
In pursuit of novel therapeutics targeting the hepatitis B virus (HBV) infection, we evaluated a dihydroquinolizinone compound (DHQ-1) that in the nanomolar range reduced the production of virion and surface protein (HBsAg) in tissue culture. This compound also showed broad HBV genotype coverage, but was inactive against a panel of DNA and RNA viruses of other species. Oral administration of DHQ-1 in the AAV-HBV mouse model resulted in a significant reduction of serum HBsAg as soon as 4 days following the commencement of treatment. Reduction of HBV markers in both in vitro and in vivo experiments was related to the reduced amount of viral RNA including pre-genomic RNA (pgRNA) and 2.4/2.1 kb HBsAg mRNA. Nuclear run-on and subcellular fractionation experiments indicated that DHQ-1 mediated HBV RNA reduction was the result of accelerated viral RNA degradation in the nucleus, rather than the consequence of inhibition of transcription initiation. Through mutagenesis of HBsAg gene sequences, we found induction of HBsAg mRNA decay by DHQ-1 required the presence of the HBV posttranscriptional regulatory element (HPRE), with a 109 nucleotides sequence within the central region of the HPRE alpha sub-element being the most critical. Taken together, the current study shows that a small molecule can reduce the overall levels of HBV RNA, especially the HBsAg mRNA, and viral surface proteins. This may shed light on the development of a new class of HBV therapeutics.
Asunto(s)
Antivirales/farmacología , Antígenos de Superficie de la Hepatitis B/genética , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/genética , Procesamiento Postranscripcional del ARN/efectos de los fármacos , ARN Mensajero/genética , ARN Viral/genética , Elementos de Respuesta , Sitios de Unión , Genotipo , Humanos , Unión Proteica , Estabilidad del ARN/efectos de los fármacos , Transfección , Replicación ViralRESUMEN
Induction of interferon and proinflammatory cytokines is a hallmark of the infection of many different viruses. However, hepatitis B virus (HBV) does not elicit a detectable cytokine response in infected hepatocytes. In order to investigate the molecular mechanism underlying the innate immune evasion, a functional cyclic GMP-AMP (cGAMP) synthase (cGAS)-stimulator of interferon genes (STING) pathway was reconstituted in a human hepatoma cell line supporting tetracycline-inducible HBV replication. It was demonstrated that induction of HBV replication neither activated nor inhibited this cytosolic DNA sensing pathway. However, human hepatoma cells, as well as immortalized mouse hepatocytes, express low levels of STING, which upon activation by cGAMP, the natural ligand of STING, led to induction of a proinflammatory cytokine response. Treatment of immortalized mouse hepatocytes supporting HBV replication with either cGAMP or a small molecule pharmacologic STING agonist significantly reduced viral DNA in a STING- and Janus kinase 1-dependent manner. Moreover, cGAMP treatment was able to induce inflammatory cytokine gene expression and inhibit the transcription of covalently closed circular DNA in HBV-infected human hepatoma cells expressing sodium taurocholate cotransporting polypeptide, an essential receptor for HBV infection of hepatocytes. The studies reported here and previously (F. Guo et al., Antimicrob Agents Chemother 59:1273-1281, 2015, https://doi.org/10.1128/AAC.04321-14) thus support the notion that pharmacological activation of STING in macrophages and hepatocytes induces host innate responses that can efficiently control HBV replication. Hence, despite not playing a significant role in host innate immune response to HBV infection of hepatocytes, STING is potentially a valuable target for immunotherapy of chronic hepatitis B.
Asunto(s)
Virus de la Hepatitis B/crecimiento & desarrollo , Hepatocitos/inmunología , Interferones/biosíntesis , Macrófagos/inmunología , Proteínas de la Membrana/metabolismo , Replicación Viral/genética , Animales , Antivirales/farmacología , Línea Celular Tumoral , Células Hep G2 , Virus de la Hepatitis B/inmunología , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Hepatocitos/metabolismo , Hepatocitos/virología , Humanos , Interferones/inmunología , Macrófagos/metabolismo , Macrófagos/virología , Proteínas de la Membrana/agonistas , Ratones , Nucleotidiltransferasas/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Simportadores/metabolismoRESUMEN
Is a cure for chronic hepatitis B virus (HBV) infection possible? Hepatitis C virus infection is now routinely cured medically. There is a growing expectation that new drugs for the management of chronic HBV infection should provide substantial benefit over and above that of current chronic HBV medications, if not be curative. Although the definition of medically induced cure for chronic HBV infection varies, most include sustained off-drug absence of viremia and negativity for other virologic markers. There are currently more than 29 drugs in the pipeline being tested for the management of chronic HBV infection. This article discusses the potential drugs with respect to their possible contributions to achieving medically induced cure.
RESUMEN
SETDB1 is a histone H3K9 methyltransferase that has a critical role in early development. It is located within a melanoma susceptibility locus and facilitates melanoma formation. However, the mechanism by which SETDB1 regulates tumorigenesis remains unknown. Here we report the molecular interplay between SETDB1 and the well-known hotspot gain-of-function (GOF) TP53 R249S mutation. We show that in hepatocellular carcinoma (HCC) SETDB1 is overexpressed with moderate copy number gain, and GOF TP53 mutations including R249S associate with this overexpression. Inactivation of SETDB1 in HCC cell lines bearing the R249S mutation suppresses cell growth. The TP53 mutation status renders cancer cells dependent on SETDB1. Moreover, SETDB1 forms a complex with p53 and catalyses p53K370 di-methylation. SETDB1 attenuation reduces the p53K370me2 level, which subsequently leads to increased recognition and degradation of p53 by MDM2. Together, we provide both genetic and biochemical evidence for a mechanism by which SETDB1 regulates cancer cell growth via methylation of p53.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Genes p53 , Neoplasias Hepáticas Experimentales/metabolismo , Proteína Metiltransferasas/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Variaciones en el Número de Copia de ADN , Células HCT116 , N-Metiltransferasa de Histona-Lisina , Humanos , Ratones DesnudosRESUMEN
The woodchuck model is an informative model for studies on hepadnaviral infection. In this study, woodchuck hepatitis virus (WHV) transgenic (Tg) mouse models based on C57BL/6 mice were established to study the pathogenesis associated with hepadnaviral infection. Two lineages of WHV Tg mice, harboring the WHV wild-type genome (lineage 1217) and a mutated WHV genome lacking surface antigen (lineage 1281), were generated. WHV replication intermediates were detected by Southern blotting. DNA vaccines against WHV proteins were applied by intramuscular injection. WHV-specific immune responses were analyzed by flow cytometry and enzyme-linked immunosorbent assays (ELISAs). The presence of WHV transgenes resulted in liver-specific but sex- and age-dependent WHV replication in Tg mice. Pathological changes in the liver, including hepatocellular dysplasia, were observed in aged Tg mice, suggesting that the presence of WHV transgenes may lead to liver diseases. Interestingly, Tg mice of lineage 1281 spontaneously developed T- and B-cell responses to WHV core protein (WHcAg). DNA vaccination induced specific immune responses to WHV proteins in WHV Tg mice, indicating a tolerance break. The magnitude of the induced WHcAg-specific immune responses was dependent on the effectiveness of different DNA vaccines and was associated with a decrease in WHV loads in mice. In conclusion, sex- and age-dependent viral replication, development of autoimmune responses to viral antigens, pathological changes in the liver in WHV Tg mice, and the possibility of breaking immune tolerance to WHV transgenes will allow future studies on pathogenesis related to hepadnaviral infection and therapeutic vaccines.
Asunto(s)
Modelos Animales de Enfermedad , Virus de la Hepatitis B de la Marmota/fisiología , Hepatitis B/inmunología , Hepatitis B/virología , Proteínas Virales/inmunología , Replicación Viral , Animales , Femenino , Anticuerpos contra la Hepatitis B/inmunología , Virus de la Hepatitis B de la Marmota/genética , Virus de la Hepatitis B de la Marmota/inmunología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/fisiología , Humanos , Hígado/inmunología , Hígado/virología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Factores Sexuales , Proteínas Virales/genéticaRESUMEN
Synthesis of the covalently closed circular (ccc) DNA is a critical, but not well-understood step in the life cycle of hepadnaviruses. Our previous studies favor a model that removal of genome-linked viral DNA polymerase occurs in the cytoplasm and the resulting deproteinized relaxed circular DNA (DP-rcDNA) is subsequently transported into the nucleus and converted into cccDNA. In support of this model, our current study showed that deproteinization of viral double-stranded linear (dsl) DNA also took place in the cytoplasm. Furthermore, we demonstrated that Ku80, a component of non-homologous end joining DNA repair pathway, was essential for synthesis of cccDNA from dslDNA, but not rcDNA. In an attempt to identify additional host factors regulating cccDNA biosynthesis, we found that the DP-rcDNA was produced in all tested cell lines that supported DHBV DNA replication, but cccDNA was only synthesized in the cell lines that accumulated high levels of DP-rcDNA, except for NCI-H322M and MDBK cells, which failed to synthesize cccDNA despite of the existence of nuclear DP-rcDNA. The results thus imply that while removal of the genome-linked viral DNA polymerase is most likely catalyzed by viral or ubiquitous host function(s), nuclear factors required for the conversion of DP-rcDNA into cccDNA and/or its maintenance are deficient in the above two cell lines, which could be useful tools for identification of the elusive host factors essential for cccDNA biosynthesis or maintenance.
Asunto(s)
Antígenos Nucleares/metabolismo , ADN Circular/biosíntesis , ADN Viral/biosíntesis , Proteínas de Unión al ADN/metabolismo , Hepadnaviridae/genética , Transporte Activo de Núcleo Celular/fisiología , Animales , Antígenos Nucleares/genética , Western Blotting , Células CHO , Cricetinae , Cricetulus , Citoplasma/metabolismo , ADN Viral/metabolismo , Proteínas de Unión al ADN/genética , Células Hep G2 , Humanos , Autoantígeno Ku , Modelos Biológicos , Plásmidos/genética , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Hepatitis B virus (HBV) is a major cause of liver infection in human. Because of the lack of an appropriate cell culture system for supporting HBV infection efficiently, the cellular and molecular mechanisms of hepadnavirus infection remain incompletely understood. Duck heptatitis B virus (DHBV) can naturally infect primary duck hepatocytes (PDHs) that provide valuable model systems for studying hepadnavirus infection in vitro. In this report, we explored global changes in cellular protein expression in DHBV infected PDHs by two-dimension gel electrophoresis (2-DE) combined with MALDI-TOF/TOF tandem mass spectrometry (MS/MS). RESULTS: The effects of hepadnavirus infection on hepatocytes were investigated in DHBV infected PDHs by the 2-DE analysis. Proteomic profile of PDHs infected with DHBV were analyzed at 24, 72 and 120 h post-infection by comparing with uninfected PDHs, and 75 differentially expressed protein spots were revealed by 2-DE analysis. Among the selected protein spots, 51 spots were identified corresponding to 42 proteins by MS/MS analysis; most of them were matched to orthologous proteins of Gallus gallus, Anas platyrhynchos or other avian species, including alpha-enolase, lamin A, aconitase 2, cofilin-2 and annexin A2, etc. The down-regulated expression of beta-actin and annexin A2 was confirmed by Western blot analysis, and potential roles of some differentially expressed proteins in the virus-infected cells have been discussed. CONCLUSIONS: Differentially expressed proteins of DHBV infected PDHs revealed by 2-DE, are involved in carbohydrate metabolism, amino acid metabolism, stress responses and cytoskeleton processes etc, providing the insight to understanding of interactions between hepadnavirus and hepatocytes and molecular mechanisms of hepadnavirus pathogenesis.
RESUMEN
The 1762T/1764A double mutation of the hepatitis B virus (HBV) basal core promoter has been suggested to be a potential biomarker for hepatocellular carcinoma (HCC) among individuals with chronic HBV infection. In this study, a real-time PCR assay is established using the hybridization probes and an oligonucleotide clamp containing locked nucleic acids (LNAs). The LNA-containing oligonucleotide clamp specific for the wild type HBV is able to suppress the amplification of the wild type HBV templates. In addition, the clamp can inhibit the binding of the WT templates to the fluorescence probes thereby suppress the wild type HBV signals during the melting curve analyses. These effects facilitated the detection of HBV double mutation in the presence of 3000-fold excess of the wild type genome. Thus PCR amplification coupled with the melting curve analyses provides a quick, simple, and highly sensitive tool for the detection of this HBV double mutation.
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Carcinoma Hepatocelular/virología , Sondas de ADN , ADN Viral/genética , Virus de la Hepatitis B/genética , Mutación Puntual , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases , Humanos , Datos de Secuencia Molecular , Sensibilidad y EspecificidadRESUMEN
Although alpha interferon (IFN-alpha) is of benefit in the treatment of viral hepatitis B, HBV replication has been refractory to the cytokine in commonly used hepatocyte-derived cell lines. In search for a cell culture system to study the mechanism by which IFN-alpha inhibits HBV replication, we infected a variety of cell lines with an adenoviral vector containing a replication competent 1.3-fold genome length HBV DNA (AdHBV) and followed by incubation with IFN-alpha. We found that IFN-alpha efficiently decreased the level of HBV DNA replicative intermediates in AdHBV infected Madin-Darby bovine kidney (MDBK) cells. Further analysis revealed, surprisingly, that IFN-alpha did not directly inhibit HBV replication, rather the amount of adenovirus DNA in the nuclei of MDBK cells was reduced. As a consequence, HBV RNA transcription and DNA replication were inhibited. Experiments with adenoviral vector expressing a green fluorescent protein (GFP) further supported the notion that IFN-alpha treatment noncytolytically eliminated adenovirus DNA, but did not kill the vector infected MDBK cells. Our data suggest that IFN-alpha-induced antiviral program is able to discriminate host cellular DNA from episomal viral DNA and might represent a novel pathway of interferon mediate innate defense against DNA virus infections.
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Adenoviridae/inmunología , ADN Viral/biosíntesis , Interferón-alfa/inmunología , Adenoviridae/genética , Animales , Bovinos , Línea Celular , Núcleo Celular/virología , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/inmunología , ARN Viral/biosíntesis , Replicación Viral/genética , Replicación Viral/inmunologíaRESUMEN
Covalently closed circular DNA (cccDNA) of hepatitis B virus (HBV) is formed by conversion of capsid-associated relaxed circular DNA (rcDNA) via unknown mechanisms and exists in the nucleus of the infected hepatocyte as a minichromosome that serves as the transcription template for viral RNAs. To study the molecular pathway of cccDNA formation and its regulation by viral and cellular factors, we have established a cell line that supports the replication of an envelope protein-deficient HBV genome in a tetracycline-inducible manner. Following induction of HBV replication, the cells accumulate higher levels of cccDNA as well as larger amounts of deproteinized rcDNA (DP-rcDNA) than cells that replicate wild-type HBV genomes. These results indicate that HBV envelope proteins negatively regulate cccDNA formation, and conversion of DP-rcDNA into cccDNA is a rate-limiting step of cccDNA formation in HepG2 cells. Detailed analyses reveal the following: (i) DP-rcDNA exists in both cytoplasm and nucleus; (ii) while nuclear DP-rcDNA is sensitive to DNase I digestion, a small fraction of cytoplasmic DP-rcDNA is DNase I resistant; (iii) both DNase I-sensitive and -resistant cytoplasmic DP-rcDNAs cosediment with capsids and can be immunoprecipitated with HBV core antibody; and (iv) a primer extension assay maps the 5' end of the minus strand of DP-rcDNA at the authentic end of virion rcDNA. Hence, our results favor a hypothesis that the removal of viral polymerase protein covalently linked to the 5' end of the minus-strand DNA occurs inside the capsid in the cytoplasm and most possibly via a reaction that cleaves the phosphodiester bond between the tyrosine of the polymerase and the 5' phosphoryl group of minus-strand DNA.
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Replicación del ADN , ADN Circular/metabolismo , ADN Viral/metabolismo , Virus de la Hepatitis B/genética , Proteínas del Envoltorio Viral/metabolismo , Línea Celular , Núcleo Celular/metabolismo , Citoplasma/metabolismo , ADN Circular/química , ADN Viral/química , Virus de la Hepatitis B/metabolismo , Humanos , Transfección , Replicación ViralRESUMEN
To study the pathway of degradation of the hepatitis B virus (HBV) middle envelope protein (M), human hepatoblastoma cells were transfected with a plasmid that specifies production of M in the absence of other viral proteins. When expressed in HepG2 cells, 90% of M protein was secreted into the culture media within a 24-h period. However, quite surprisingly, 10% of this protein remained cell associated and was only slowly degraded over a 24-48-h period. Treatment with inhibitors of the cytosolic proteasome complex resulted in the accumulation of full-length HBV M protein and M derived HBV-specific polypeptides of 20 and 17 kDa. Treatment with the endoglycosidases PNGase F and Endo H, confirmed that the two species were derived from a similar polypeptide with a N-linked glycan modification. Evidence that this peptide was derived from a proteolytic processing event was determined through the detection of the C-terminal fragment using a C-terminal tagged HA tagged construct. The hypothesis that the 20 and 17 kDa polypeptide species are intermediates of M degradation was reinforced by their detection in cells transfected with vectors specifying M secretion defective mutants that accumulate intracellular M. Moreover, deletion of a putative cleavage sites prevented the detection of the 20 and 17 kDa species, consistent with the notion that they are generated by the action of a cellular protease prior to proteasomal degradation. Thus, these results highlight an important way in which large protein aggregates, such as the HBsAg can be processed for efficient degradation via the proteasomes and allow for proper antigen presentation via the MHC I pathway.
Asunto(s)
Citosol/metabolismo , Endopeptidasas/metabolismo , Hepatocitos/metabolismo , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas del Envoltorio Viral/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Hepatocitos/química , Humanos , Immunoblotting , Modelos Biológicos , Datos de Secuencia Molecular , Proteínas del Envoltorio Viral/químicaRESUMEN
The phosphatidylinositol 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway is one of the major oncogenic pathways and is activated in many types of human cancers, including hepatocellular carcinoma. It can also be activated by the hepatitis C virus (HCV) nonstructural 5A (NS5A) protein. In the present study, we set out to determine the regulatory effects of this pathway on the replication of hepatitis B virus (HBV). Our results demonstrate that the expression of a constitutively active Akt1 profoundly inhibited HBV RNA transcription and consequently reduced HBV DNA replication in HepG2 cells. This suppression of HBV gene transcription was apparently mediated by the activation of mTOR, as it was abolished by the mTOR inhibitor rapamycin. Moreover, treatment of HBV-expressing HepG2.2.15 cells with inhibitors of PI3K, Akt, and mTOR increased the transcription of 3.5-kb and 2.4-kb viral RNA as well as the replication of HBV DNA. This observation implies that the basal level activation of this pathway in HepG2 cells regulated HBV replication. Consistent with previous reports showing that the HCV NS5A protein could bind to the p85 subunit of PI3K and activate the PI3K-Akt signal transduction pathway, our results showed that expression of this protein could inhibit HBV RNA transcription and reduce HBV DNA replication in HepG2 cells. Taken together, our results suggest that the activation of the PI3K-Akt pathway during liver oncogenesis may be at least partially responsible for the elimination of HBV replication from tumor cells and may also provide an explanation for the observed suppression of HBV replication by HCV coinfection.