RESUMEN
HBV DNA integration originally recognized as a non-functional byproduct of the HBV life cycle has now been accepted as a significant contributor to HBV pathogenesis and HDV persistence. Integrated HBV DNA is derived from linear genomic DNA present in virus particles or produced from aberrantly processed relaxed circular genomic DNA following an infection, and can drive expression of HBsAg and HBx. DNA integration events accumulate over the course of viral infection ranging from a few percent during early phases to nearly 100 percent of infected cells after prolonged chronic infection. HBV DNA integration events have primarily been investigated in the context of HCC development where they can activate known oncogenes and other growth promoting genes, cause chromosomal instability and presumably epigenetic alterations promoting tumor growth. More recent evidence suggests that HBsAg expression from integrated DNA might contribute to HBV pathogenesis by attenuating the immune response. Integrated DNA provides a source for envelope proteins required for HDV replication and hence represents a means for HDV persistence. Because integrated DNA is responsible for persistence of HBsAg in the absence of viral replication it impacts established criteria for the resolution of HBV infection which relies on HBsAg as a diagnostic marker. Integrated HBV DNA has been useful in assessing the turnover of infected hepatocytes which occurs during all phases of chronic hepatitis B including the initial phase of infection historically termed immune tolerant. HBV DNA integration was also shown to impact the development of novel therapies targeting viral RNAs.
RESUMEN
Polyoxometalates (POM) are anionic oxoclusters of early transition metals that are of great interest for a variety of applications, including the development of sensors and catalysts. A crucial step in the use of POM in functional materials is the production of composites that can be further processed into complex materials, e.g. by printing on different substrates. In this work, we present an immobilization approach for POMs that involves two key processes: first, the stable encapsulation of POMs in the pores of mesoporous silica nanoparticles (MSPs) and, second, the formation of microstructured arrays with these POM-loaded nanoparticles. Specifically, we have developed a strategy that leads to water-stable, POM-loaded mesoporous silica that can be covalently linked to alkene-bearing surfaces by amine-Michael addition and patterned into microarrays by scanning probe lithography (SPL). The immobilization strategy presented facilitates the printing of hybrid POM-loaded nanomaterials onto different surfaces and provides a versatile method for the fabrication of POM-based composites. Importantly, POM-loaded MSPs are useful in applications such as microfluidic systems and sensors that require frequent washing. Overall, this method is a promising way to produce surface-printed POM arrays that can be used for a wide range of applications.
RESUMEN
IMPORTANCE: Hepatitis B virus cccDNA is the key target for the necessary development of antiviral therapies aimed at curing chronic hepatitis B. The CRISPR-based system to produce covalently closed circular (cccDNA)-like extrachromosomal DNAs described in this report enables large-scale screens of chemical libraries to identify drug candidates with the potential to permanently inactivate cccDNA. Moreover, this approach permits investigations on unresolved problems as described in this report concerning cccDNA biology including mechanisms of SMC5/6-dependent transcriptional silencing and the contributions of the SMC5/6 complex to cccDNA stability in resting and dividing hepatocytes.
Asunto(s)
Sistemas CRISPR-Cas , ADN Circular , Virus de la Hepatitis B , Humanos , ADN Circular/genética , ADN Viral/genética , Virus de la Hepatitis B/genética , Hepatitis B Crónica , Replicación Viral/genéticaRESUMEN
Chronic hepatitis B virus infections affect over 250 million people world-wide, and, at present, are not curable. Of those, over 800000 are expected to die yearly from complications including cirrhosis and primary hepatocellular carcinoma (HCC). A viral episomal DNA intermediate, covalently closed circular DNA (cccDNA) can persist in nuclei of infected hepatocytes and trigger production of infectious virus. Current standard of care treatments against chronic HBV infections primarily rely on nucleoside analogs (NA) that inhibit de novo virus production by inhibiting the viral reverse transcriptase and, as a consequence, reducing virus titers. However, they cannot cure infections, because they do not directly target cccDNA persistence. Nevertheless, NA therapies can halt progression of liver disease including cirrhosis and can reduce the development of hepatocellular carcinoma (HCC). A cure for chronic hepatitis B (CHB) must reduce the load of cccDNA or permanently silence transcription from cccDNA, and ensure sustained activation of an adaptive immune response that prohibits reactivation and spread of residual virus in the liver. As discussed in this review, novel technologies enabling genetic destruction of cccDNA and advances in our understanding of HBV transcriptional control provide exciting opportunities for the future development of curative therapies desperately needed to reduce the burden of chronic HBV infections.
Asunto(s)
Antivirales/farmacología , Carcinoma Hepatocelular/prevención & control , Terapia Genética/métodos , Hepatitis B Crónica/tratamiento farmacológico , Hepatitis B Crónica/virología , Factores Inmunológicos/farmacología , Cirrosis Hepática/prevención & control , Animales , Antivirales/uso terapéutico , Modelos Animales de Enfermedad , Descubrimiento de Drogas/tendencias , Hepatitis B Crónica/complicaciones , Humanos , Factores Inmunológicos/uso terapéuticoRESUMEN
Chronic hepatitis B affects over 300 million people who are at risk of developing liver cancer. The basis for the persistence of hepatitis B virus (HBV) in hepatocytes, even in the presence of available antiviral therapies, lies in the accumulation of covalently closed circular DNA (cccDNA) in nuclei of infected cells. While methods for cccDNA quantification from liver biopsy specimens and cell lines expressing the virus are known, information about cccDNA formation, stability, and turnover is lacking. In particular, little is known about the fate of cccDNA during cell division. To fill the gaps in knowledge concerning cccDNA biology, we have developed a fluorescence imaging in situ hybridization (FISH)-based assay for the detection of duck hepatitis B virus (DHBV) cccDNA and HBV nuclear DNA in established cell lines. Using FISH, we determined the distribution of cccDNA under conditions mimicking chronic infections with and without antiviral therapy, which prevents de novo viral replication. Our results showed that the copy numbers of viral nuclear DNA can vary by as much as 1.8 orders of magnitude among individual cells and that antiviral therapy leads to a reduction in nuclear DNA in a manner consistent with symmetrical distribution of viral DNA to daughter cells.IMPORTANCE A mechanistic understanding of the stability of HBV cccDNA in the presence of antiviral therapy and during cell division induced by immune-mediated lysis of infected hepatocytes will be critical for the future design of curative antiviral therapies against chronic hepatitis B. Current knowledge about cccDNA stability was largely derived from quantitative analyses of cccDNA levels present in liver samples, and little was known about the fate of cccDNA in individual cells. The development of a FISH-based assay for cccDNA tracking provided the first insights into the fate of DHBV cccDNA and nuclear HBV DNA under conditions mimicking antiviral therapy.
Asunto(s)
ADN Circular/metabolismo , Virus de la Hepatitis B del Pato/genética , Virus de la Hepatitis B/genética , Animales , Antivirales/farmacología , Antivirales/uso terapéutico , División Celular/genética , Replicación del ADN/efectos de los fármacos , ADN Circular/aislamiento & purificación , ADN Viral/efectos de los fármacos , ADN Viral/metabolismo , Hepatitis B Crónica/tratamiento farmacológico , Hepatocitos/virología , Hibridación Fluorescente in Situ/métodos , Replicación ViralRESUMEN
Hepatitis B virus (HBV) causes chronic infections that cannot yet be cured. The virus persists in infected hepatocytes, because covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs, is stable in nondividing cells. Antiviral therapies with nucleoside analogues inhibit HBV DNA synthesis in capsids in the cytoplasm of infected hepatocytes, but do not destroy nuclear cccDNA. Because over 200 million people are still infected, a cure for chronic hepatitis B (CHB) has become one of the major challenges in antiviral therapy. As a first step toward the development of curative therapies, we previously demonstrated that the CRISPR/Cas9 system can be used to functionally inactivate cccDNA derived from infectious HBV. Moreover, some evidence suggests that certain cytokines might induce an APOBEC-mediated cascade leading to the destruction of cccDNA. In this report we investigated whether a combination of the two mechanisms could act synergistically to inactivate cccDNA. Using next generation sequencing (NGS), we determined the complete spectrum of mutations in cccDNA following Cas9 cleavage and repair by nonhomologous end joining (NHEJ). We found that over 90% of HBV DNA was cleaved by Cas9. In addition our results showed that editing of HBV DNA after Cas9 cleavage is at least 15,000 times more efficient that APOBEC-mediated cytosine deamination following treatment of infected cells with interferon alpha (IFNα). We also found that a previously used method to detect cytosine deaminated DNA, termed 3D-PCR, overestimates the amount and frequency of edited HBV DNA. Taken together, our results demonstrated that the CRISPR/Cas9 system is so far the best method to functionally inactivate HBV cccDNA and provide a cure for CHB.
Asunto(s)
Sistemas CRISPR-Cas , ADN Circular , ADN Viral/genética , Virus de la Hepatitis B/genética , Mutación , Secuencia de Bases , Línea Celular , Edición Génica , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Mutación INDEL , Tasa de Mutación , ARN Guía de KinetoplastidaRESUMEN
This volume explores these and other issues of relevance to our understanding of the HBV life cycle and clinical management of chronic HBV infections. The ultimate goals of these studies is not just to obtain a more precise understanding of the HBV life cycle, but to also acquire an understanding that will lead to more effective treatments for an infection and pathogenic process that currently causes â¼500,000 to 1,000,000 deaths per year.
Asunto(s)
Antivirales/uso terapéutico , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/tratamiento farmacológico , Replicación Viral/efectos de los fármacos , Hepatitis B Crónica/virología , HumanosRESUMEN
Hallmarks of the hepadnavirus replication cycle are the formation of covalently closed circular DNA (cccDNA) and the reverse transcription of a pregenomic RNA (pgRNA) in core particles leading to synthesis of the relaxed circular DNA (rcDNA) genome. cccDNA, the template for viral RNA transcription, is the basis for the persistence of these viruses in infected hepatocytes. In this review, we summarize the current state of knowledge on the mechanisms of hepadnavirus reverse transcription and the biochemical and structural properties of the viral reverse transcriptase (RT). We highlight important gaps in knowledge regarding cccDNA biosynthesis and stability. In addition, we discuss the impact of current antiviral therapies on viral persistence, particularly on cccDNA.
Asunto(s)
Replicación del ADN/genética , ADN Circular/genética , ADN Viral/biosíntesis , Virus de la Hepatitis B/genética , Hepatocitos/microbiología , ADN Polimerasa Dirigida por ARN/genética , HumanosRESUMEN
Hepatitis B virus (HBV) replication and persistence are sustained by a nuclear episome, the covalently closed circular (CCC) DNA, which serves as the transcriptional template for all viral RNAs. CCC DNA is converted from a relaxed circular (RC) DNA in the virion early during infection as well as from RC DNA in intracellular progeny nucleocapsids via an intracellular amplification pathway. Current antiviral therapies suppress viral replication but cannot eliminate CCC DNA. Thus, persistence of CCC DNA remains an obstacle toward curing chronic HBV infection. Unfortunately, very little is known about how CCC DNA is formed. CCC DNA formation requires removal of the virally encoded reverse transcriptase (RT) protein from the 5' end of the minus strand of RC DNA. Tyrosyl DNA phosphodiesterase-2 (Tdp2) was recently identified as the enzyme responsible for cleavage of tyrosyl-5' DNA linkages formed between topoisomerase II and cellular DNA. Because the RT-DNA linkage is also a 5' DNA-phosphotyrosyl bond, it has been hypothesized that Tdp2 might be one of several elusive host factors required for CCC DNA formation. Therefore, we examined the role of Tdp2 in RC DNA deproteination and CCC DNA formation. We demonstrated Tdp2 can cleave the tyrosyl-minus strand DNA linkage using authentic HBV RC DNA isolated from nucleocapsids and using RT covalently linked to short minus strand DNA produced in vitro. On the other hand, our results showed that Tdp2 gene knockout did not block CCC DNA formation during HBV infection of permissive human hepatoma cells and did not prevent intracellular amplification of duck hepatitis B virus CCC DNA. These results indicate that although Tdp2 can remove the RT covalently linked to the 5' end of the HBV minus strand DNA in vitro, this protein might not be required for CCC DNA formation in vivo.
Asunto(s)
ADN Circular/metabolismo , ADN Viral/metabolismo , Virus de la Hepatitis B/fisiología , Hepatitis B/metabolismo , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , ADN Circular/genética , ADN Viral/genética , Proteínas de Unión al ADN , Técnicas de Silenciamiento del Gen , Técnicas de Inactivación de Genes , Genoma Viral , Células Hep G2 , Hepatitis B/genética , Virus de la Hepatitis B del Pato/genética , Virus de la Hepatitis B del Pato/fisiología , Virus de la Hepatitis B/genética , Virus de la Hepatitis del Pato/genética , Virus de la Hepatitis del Pato/metabolismo , Humanos , Proteínas Nucleares/genética , Hidrolasas Diéster Fosfóricas , ADN Polimerasa Dirigida por ARN/genética , ADN Polimerasa Dirigida por ARN/metabolismo , Factores de Transcripción/genética , Regulación hacia Arriba , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación ViralRESUMEN
Human hepatitis B virus (HBV) is the prototype of a family of small DNA viruses that productively infect hepatocytes, the major cell of the liver, and replicate by reverse transcription of a terminally redundant viral RNA, the pregenome. Upon infection, the circular, partially double-stranded virion DNA is converted in the nucleus to a covalently closed circular DNA (cccDNA) that assembles into a minichromosome, the template for viral mRNA synthesis. Infection of hepatocytes is non-cytopathic. Infection of the liver may be either transient (<6 months) or chronic and lifelong, depending on the ability of the host immune response to clear the infection. Chronic infections can cause immune-mediated liver damage progressing to cirrhosis and hepatocellular carcinoma (HCC). The mechanisms of carcinogenesis are unclear. Antiviral therapies with nucleoside analog inhibitors of viral DNA synthesis delay sequelae, but cannot cure HBV infections due to the persistence of cccDNA in hepatocytes.
Asunto(s)
Virus de la Hepatitis B/fisiología , Hepatitis B/patología , Hepatitis B/virología , Interacciones Huésped-Patógeno , Replicación Viral , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Enfermedad Crónica , ADN Viral/metabolismo , Hepatitis B/complicaciones , Hepatitis B/inmunología , Humanos , Cirrosis Hepática/patología , Cirrosis Hepática/virología , ARN Mensajero/metabolismo , ARN Viral/metabolismo , Transcripción ReversaRESUMEN
Hepatitis B virus persistence in infected hepatocytes is due to the presence of covalently closed circular DNA (cccDNA), the template for the transcription of viral RNAs. Antiviral therapies with nucleoside analogues inhibit replication of HBV DNA in capsids present in the cytoplasm of infected cells, but do not reduce or destroy nuclear cccDNA. To investigate whether cccDNA derived from infectious HBV could be directly targeted for destruction, we used the CRISPR/Cas9 system in HepG2 cells expressing the HBV receptor sodium taurocholate cotransporting polypeptide (NTCP). We tested different HBV-specific guide RNAs and demonstrated that they could inhibit HBV infections up to eightfold. Inhibition was due to mutations and deletions in cccDNA similar to those observed with chromosomal DNA cleaved by Cas9 and repaired by nonhomologous end joining (NHEJ). Interferon alpha (IFN-α) did not have a measurable effect on the antiviral activity of the CRISPR/Cas9 system, suggesting that Cas9 and NHEJ activities are not affected by induction of an innate immune response with the cytokine. Taken together, our results demonstrated that Cas9 can be recruited to cccDNA, opening the possibility for the development of future antiviral strategies aimed at targeting cccDNA for endonucleolytic cleavage with small molecules.
RESUMEN
Lucifora et al. (Research Articles, 14 March 2014, p. 1221) report that the hepatitis B virus (HBV) transcriptional template, a long-lived covalently closed circular DNA (cccDNA) molecule, is degraded noncytolytically by agents that up-regulate APOBEC3A and 3B. If these results can be independently confirmed, they would represent a critical first step toward development of a cure for the 400 million patients who are chronically infected by HBV.
Asunto(s)
Antivirales/farmacología , ADN Circular/metabolismo , ADN Viral/metabolismo , Virus de la Hepatitis B/efectos de los fármacos , Hepatitis B/tratamiento farmacológico , Hepatocitos/efectos de los fármacos , Interferón-alfa/farmacología , Receptor beta de Linfotoxina/agonistas , Animales , HumanosAsunto(s)
Virus de la Hepatitis B/metabolismo , Hepatitis B/metabolismo , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Receptores Virales/metabolismo , Simportadores/metabolismo , Secuencia de Aminoácidos , Animales , Humanos , Ratones , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Alineación de Secuencia , Simportadores/genéticaRESUMEN
Productive virus infection requires evasion, inhibition, or subversion of innate immune responses. West Nile virus (WNV), a human pathogen that can cause symptomatic infections associated with meningitis and encephalitis, inhibits the interferon (IFN) signal transduction pathway by preventing phosphorylation of Janus kinases and STAT transcription factors. Inhibition of the IFN signal cascade abrogates activation of IFN-induced genes, thus attenuating an antiviral response. We investigated the mechanism responsible for this inhibition and found that WNV infection prevents accumulation of the IFN-α receptor subunit 1 (IFNAR1). The WNV-induced depletion of IFNAR1 was conserved across multiple cell types. Our results indicated that expression of WNV nonstructural proteins resulted in activated lysosomal and proteasomal protein degradation pathways independent of the unfolded protein response (UPR). Furthermore, WNV infection did not induce serine phosphorylation, a modification on IFNAR1 that precedes its natural turnover. These data demonstrate that WNV infection results in a reduction of IFNAR1 protein through a non-canonical protein degradation pathway, and may participate in the inhibition of the IFN response.
Asunto(s)
Interacciones Huésped-Patógeno , Evasión Inmune , Receptor de Interferón alfa y beta/antagonistas & inhibidores , Virus del Nilo Occidental/inmunología , Virus del Nilo Occidental/patogenicidad , Animales , Línea Celular , Humanos , Receptor de Interferón alfa y beta/metabolismoRESUMEN
Hepadnavirus replication requires the synthesis of a covalently closed circular (CCC) DNA from the relaxed circular (RC) viral genome by an unknown mechanism. CCC DNA formation could require enzymatic activities of the viral reverse transcriptase (RT), or cellular DNA repair enzymes, or both. Physical mapping of the 5' and 3' ends of RC DNA and sequence analysis of CCC DNA revealed that CCC DNA synthesis requires the removal of the RT and an RNA oligomer from the 5' ends of minus and plus strand DNA, respectively, removal of sequences from the terminally redundant minus strand, completion of the less than full-length plus strand, and ligation of the ends. Two models have been proposed that could explain CCC DNA formation. The first (model 1) invokes a role for the RT to catalyze a cleavage-ligation reaction leading to the formation of a unit length minus strand in CCC DNA and a DNA repair reaction for the completion and ligation of plus strand DNA; the second (model 2) predicts that CCC DNA formation depends entirely on cellular DNA repair enzymes. To determine which mechanism is utilized, we developed cell lines expressing duck hepatitis B virus genomes carrying mutations permitting us to follow the fate of viral DNA sequences during their conversion from RC to CCC DNA. Our results demonstrated that the oligomer at the 5' end of minus strand DNA is completely or at least partially removed prior to CCC DNA synthesis. The results indicated that both RC DNA strands undergo DNA repair reactions carried out by the cellular DNA repair machinery as predicted by model 2. Thus, our study provided the basis for the identification of the cellular components required for CCC DNA formation.
Asunto(s)
Replicación del ADN , ADN Circular/genética , Hepadnaviridae/genética , Animales , Pollos , Reparación del ADN , ADN Circular/análisis , ADN Viral/metabolismo , Genoma Viral/genética , Genotipo , Virus de la Hepatitis B/genética , Modelos Biológicos , Modelos Genéticos , Mutagénesis Sitio-Dirigida , ADN Polimerasa Dirigida por ARN/metabolismo , Análisis de Secuencia de ADN , Replicación ViralRESUMEN
BACKGROUND: Hepatitis C virus (HCV) is a plus-strand RNA virus that replicates by amplification of genomic RNA from minus strands leading to accumulation of almost one thousand copies per cell under in vitro cell culture conditions. In contrast, HCV RNA copy numbers in livers of infected patients appear to be much lower, estimated at a few copies per cell. METHODOLOGY/PRINCIPAL FINDINGS: To gain insights into mechanisms that control HCV replication in vivo, we analyzed HCV RNA levels as well as expression of interferon beta (IFNbeta) and several interferon stimulated genes (ISGs) from whole liver sections and micro-dissected subpopulations of hepatocytes in biopsy samples from 21 HCV-infected patients. The results showed that intrahepatic HCV RNA levels range form less than one copy per hepatocyte to a maximum of about eight. A correlation existed between viral RNA levels and IFNbeta expression, but not between viral RNA and ISG levels. Also, IFNbeta expression did not correlate with ISGs levels. Replication of HCV RNA occurred in focal areas in the liver in the presence of a general induction of ISGs. CONCLUSION/SIGNIFICANCE: The low average levels of HCV RNA in biopsy samples can be explained by focal distribution of infected hepatocytes. HCV replication directly induces IFNbeta, which then activates ISGs. The apparent lack of a correlation between levels of IFNbeta and ISG expression indicates that control of the innate immune response during HCV infections depends on multiple factors.
Asunto(s)
Hepacivirus/genética , Hígado/metabolismo , ARN Viral/metabolismo , Western Blotting , Estudios de Cohortes , Dosificación de Gen , Hepatitis C/tratamiento farmacológico , Hepatitis C/virología , Humanos , Interferones/uso terapéutico , Hígado/virologíaRESUMEN
West Nile virus (WNV) is a human pathogen that can cause symptomatic infections associated with meningitis and encephalitis. Previously, we demonstrated that replication of WNV inhibits the interferon (IFN) signal transduction pathway by preventing the accumulation of phosphorylated Janus kinase 1 (JAK1) and tyrosine kinase 2 (Tyk2) (J. T. Guo et al., J. Virol. 79:1343-1350, 2005). Through a genetic analysis, we have now identified a determinant on the nonstructural protein 4B (NS4B) that controls IFN resistance in HeLa cells expressing subgenomic WNV replicons lacking the structural genes. However, in the context of infectious genomes, the same determinant did not influence IFN signaling. Thus, our results indicate that NS4B may be sufficient to inhibit the IFN response in replicon cells and suggest a role for structural genes, or as yet unknown interactions, in the inhibition of the IFN signaling pathway during WNV infections.