RESUMEN

Hepatitis B virus (HBV) is an enveloped DNA human virus belonging to the Hepadnaviridae family. Perhaps its main distinguishable characteristic is the replication of its genome through a reverse transcription process. The HBV circular genome encodes only four overlapping reading frames, encoding for the main canonical proteins named core, P, surface, and X (or HBx protein). However, pre- and post-transcriptional gene regulation diversifies the full HBV proteome into diverse isoform proteins. In line with this, hepatitis B virus X protein (HBx) is a viral multifunctional and regulatory protein of 16.5 kDa, whose canonical reading frame presents two phylogenetically conserved internal in-frame translational initiation codons, and which results as well in the expression of two divergent N-terminal smaller isoforms of 8.6 and 5.8 kDa, during translation. The canonical HBx, as well as the smaller isoform proteins, displays different roles during viral replication and subcellular localizations. In this article, we reviewed the different mechanisms of pre- and post-transcriptional regulation of protein expression that take place during viral replication. We also investigated all the past and recent evidence about HBV HBx gene regulation and its divergent N-terminal isoform proteins. Evidence has been collected for over 30 years. The accumulated evidence simply strengthens the concept of a new paradigm of the canonical HBx, and its smaller divergent N-terminal isoform proteins, not only during viral replication, but also throughout cell pathogenesis.

RESUMEN

Hepatitis B virus (HBV) X protein (HBx) is a viral regulatory and multifunctional protein. It is well-known that the canonical HBx reading frame bears two phylogenetically conserved internal in-frame translational initiation codons at Met2 and Met3, thus possibly generating divergent N-terminal smaller isoforms during translation. Here, we demonstrate that the three distinct HBx isoforms are generated from the ectopically expressed HBV HBx gene, named XF (full-length), XM (medium-length), and XS (short-length); they display different subcellular localizations when expressed individually in cultured hepatoma cells. Particularly, the smallest HBx isoform, XS, displayed a predominantly cytoplasmic localization. To study HBx proteins during viral replication, we performed site-directed mutagenesis to target the individual or combinatorial expression of the HBx isoforms within the HBV viral backbone (full viral genome). Our results indicate that of all HBx isoforms, only the smallest HBx isoform, XS, can restore WT levels of HBV replication, and bind to the viral mini chromosome, thereby establishing an active chromatin state, highlighting its crucial activities during HBV replication. Intriguingly, we found that sequences of HBV HBx genotype H are devoid of the conserved Met3 position, and therefore HBV genotype H infection is naturally silent for the expression of the HBx XS isoform. Finally, we found that the HBx XM (medium-length) isoform shares significant sequence similarity with the N-terminus domain of the COMMD8 protein, a member of the copper metabolism MURR1 domain-containing (COMMD) protein family. This novel finding might facilitate studies on the phylogenetic origin of the HBV X protein. The identification and functional characterization of its isoforms will shift the paradigm by changing the concept of HBx from being a unique, canonical, and multifunctional protein toward the occurrence of different HBx isoforms, carrying out different overlapping functions at different subcellular localizations during HBV genome replication. Significantly, our current work unveils new crucial HBV targets to study for potential antiviral research, and human virus pathogenesis.

RESUMEN

Hepatitis B virus (HBV) is a circular, and partially double-stranded DNA virus. Upon infection, the viral genome is translocated into the cell nucleus, generating the covalently closed circular DNA (cccDNA) intermediate, and forming a mini chromosome. HBV HBx is a small protein displaying multiple roles in HBV-infected cells, and in different subcellular locations. In the nucleus, the HBx protein is required to initiate and maintain viral transcription from the viral mini chromosome. In contrast, HBx also functions in the cytoplasm, where it is able to alter multiple cellular functions such as mitochondria metabolism, apoptosis and signal transduction pathways. It has been reported that in cultured cells, at low expression levels, the HBx protein is localized in the nucleus, whereas at high expression levels, it accumulates in the cytoplasm. This dynamic subcellular distribution of HBx might be essential to exert its multiple roles during viral infection. However, the mechanism that regulates different subcellular localizations of the HBx protein is unknown. We have previously taken a bioinformatics approach to investigate whether HBx might be regulated via post-translational modification, and we have proposed that the multiple nucleocytoplasmic functions of HBx might be regulated by an evolutionarily conserved mechanism via phosphorylation. In the current study, phylogenetically conserved amino acids of HBx with a high potential of phosphorylation were targeted for site-directed mutagenesis. Two conserved serine (Ser25 and Ser41), and one conserved threonine (Thr81) amino acids were replaced by either alanine or aspartic acid residues to simulate an unphosphorylated or phosphorylated state, respectively. Human hepatoma cells were transfected with increasing amounts of the HBx DNA constructs, and the cells were analyzed by fluorescence microscopy. Together, our results show that the nucleocytoplasmic distribution of the HBx protein could be regulated by phosphorylation since some of the modified proteins were mainly confined to distinct subcellular compartments. Remarkably, both HBx Ser41A, and HBx Thr81D proteins were predominantly localized within the nuclear compartment throughout the different expression levels of HBx mutants.

Asunto(s)

Carcinoma Hepatocelular/genética , Hepatitis B/genética , Neoplasias Hepáticas/genética , Transactivadores/genética , Proteínas Reguladoras y Accesorias Virales/genética , Secuencia de Aminoácidos/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/virología , Secuencia Conservada/genética , Regulación Viral de la Expresión Génica/genética , Genoma Viral/genética , Células Hep G2 , Hepatitis B/patología , Hepatitis B/virología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/patogenicidad , Humanos , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/virología , Fosforilación/genética , Filogenia

RESUMEN

INTRODUCTION AND OBJECTIVES: About 250 million people around the world are chronically infected with the hepatitis B virus (HBV). Those people are at risk of developing hepatocellular carcinoma. The HBV genome is organized as a minichromosome in the infected hepatocyte and is associated with histones and non-histone proteins. In recent years, many groups have investigated the transcriptional regulation of HBV mediated by post-translational modifications on the histones associated with the covalently closed circular DNA (cccDNA). Our aim is to investigate the role of the histone variant H3.3. MATERIALS AND METHODS: An in vitro HBV replication model system based on the transfection of linear HBV genome monomeric molecules was used. We then either ectopically expressed or reduced the levels of H3.3, and of its histone chaperone HIRA. Viral intermediates were quantified and the level of H3K4me3 using Chromatin immunoprecipitation (ChIP) assay was measured. RESULTS: Histone variant H3.3 ectopically expressed in cells assembles into the viral cccDNA, correlating with increasing levels of the active histone mark H3K4me3 and HBV transcription. The opposite results were found upon diminishing H3.3 levels. Furthermore, the assembly of H3.3 into the cccDNA is dependent on the histone chaperone HIRA. Diminishing HIRA levels causes a reduction in the HBV intermediates. CONCLUSIONS: Histone variant H3.3 positively regulates HBV transcription. Importantly, the characterization of the viral chromatin dynamics might allow the discovery of new therapeutic targets to develop drugs for the treatment of chronically-infected HBV patients.

Asunto(s)

ADN Viral/genética , Epigénesis Genética/genética , Virus de la Hepatitis B/fisiología , Hepatitis B Crónica/genética , Histonas/genética , Replicación Viral/genética , Células Cultivadas , ADN Circular/genética , Histonas/metabolismo , Humanos , Transcripción Genética

RESUMEN

Posttranslational modification (PTM) of proteins is critical to modulate protein function and to improve the functional diversity of polypeptides. In this report, we have analyzed the PTM of both hepatitis C virus NS3 and NS5B enzyme proteins, upon their individual expression in insect cells under the baculovirus expression system. Using mass spectrometry, we present evidence that these recombinant proteins exhibit diverse covalent modifications on certain amino acid side chains, such as phosphorylation, ubiquitination, and acetylation. Although the functional implications of these PTM must be further addressed, these data may prove useful toward the understanding of the complex regulation of these key viral enzymes and to uncover novel potential targets for antiviral design.

Asunto(s)

Hepacivirus/genética , Hepatitis C/virología , Proteínas no Estructurales Virales/genética , Regulación Viral de la Expresión Génica/genética , Hepacivirus/patogenicidad , Hepatitis C/genética , Humanos , Procesamiento Proteico-Postraduccional/genética

RESUMEN

With about 350 million people chronically infected around the world hepatitis B is a major health problem. Template for progeny HBV synthesis is the viral genome, organized as a minichromosome (cccDNA) inside the hepatocyte nucleus. How viral cccDNA gene expression is regulated by its chromatin structure; more importantly, how the modulation of this structure impacts on viral gene expression remains elusive. Here, we found that the enzyme SetDB1 contributes to setting up a repressed cccDNA chromatin state. This repressive state is activated by the histone lysine demethylase-1 (LSD1). Consistently, inhibiting or reducing LSD1 levels led to repression of viral gene expression. This correlates with the transcriptionally repressive mark H3K9 methylation and reduction on the activating marks H3 acetylation and H3K4 methylation on viral promoters. Investigating the importance of viral proteins we found that LSD1 recruitment to viral promoters was dependent on the viral transactivator protein HBx. Moreover, the histone methyltransferase Set1A and HBx are simultaneously bound to the core promoter, and Set1A expression correlates with cccDNA H3K4 methylation. Our results shed light on the mechanisms of HBV regulation mediated by the cccDNA chromatin structure, offering new therapeutic targets to develop drugs for the treatment of chronically infected HBV patients.

Asunto(s)

Cromatina/genética , Virus de la Hepatitis B/fisiología , Hepatitis B/metabolismo , Histona Demetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Transactivadores/metabolismo , Acetilación , Línea Celular , Cromatina/química , Cromatina/metabolismo , Regulación Viral de la Expresión Génica , Células Hep G2 , Hepatitis B/virología , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/metabolismo , Histonas/metabolismo , Humanos , Metilación , Regiones Promotoras Genéticas , Proteínas Reguladoras y Accesorias Virales , Replicación Viral

RESUMEN

Genotype F is one of the less-studied genotypes of human hepatitis B virus, although it is widely distributed in regions of Central and South American. Our previous studies have shown that HBV genotype F is prevalent in Chile, and phylogenetic analysis of its full-length sequence amplified from the sera of chronically infected patients identified it as HBV subgenotype F1b. We have previously reported the full-length sequence of a HBV molecular clone obtained from a patient chronically infected with genotype F1b. In this report, we established a system to study HBV replication based on hepatoma cell lines transfected with full-length monomers of the HBV genome. Culture supernatants were analyzed after transfection and found to contain both HBsAg and HBeAg viral antigens. Consistently, fractionated cell extracts revealed the presence of viral replication, with both cytoplasmic and nuclear DNA intermediates. Analysis of HBV-transfected cells by indirect immunofluorescence or immunoelectron microscopy revealed the expression of viral antigens and cytoplasmic viral particles, respectively. To test the functionality of the ongoing viral replication further at the level of chromatinized cccDNA, transfected cells were treated with a histone deacetylase inhibitor, and this resulted in increased viral replication. This correlated with changes posttranslational modifications of histones at viral promoters. Thus, the development of this viral replication system for HBV genotype F will facilitate studies on the regulation of viral replication and the identification of new antiviral drugs.

Asunto(s)

Genotipo , Virus de la Hepatitis B/fisiología , Hepatocitos/virología , Replicación Viral , Fraccionamiento Celular , Línea Celular Tumoral , Medios de Cultivo/química , ADN Viral/análisis , Antígenos de Superficie de la Hepatitis B/análisis , Antígenos e de la Hepatitis B/análisis , Virus de la Hepatitis B/clasificación , Virus de la Hepatitis B/genética , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/virología , Humanos , Microscopía Fluorescente , Microscopía Inmunoelectrónica

RESUMEN

The RNA-dependent RNA polymerase (RdRP) of the Hepatitis C virus (HCV), named NS5B, is phosphorylated by the cellular protein kinase C-related kinase 2 (PRK2) at two serine residues (Ser29 and Ser42) of the finger subdomain (genotype 1b). Herein, using bioinformatics, we selected four potential phosphorylation residues (Ser46, Ser76, Ser96 and Ser112) of NS5B (genotype 2a) for study. Whereas the NS5B Ser46D and Ser76D substitutions seemed to improve polymerase activity, the Ser96D mutation decreased colony formation efficiency. Active WT NS5B was utilized in in vitro kinase assays, and phosphopeptides were analyzed by mass spectrometry. Interestingly, the data indicated that both the NS5B Ser29 and Ser76 residues resulted phosphorylated. Thus, as Ser76 is absolutely conserved across HCV genotypes, our results confirmed the relevance of these sites for both genotypes and suggested that Ser76 becomes phosphorylated by a cellular kinase different from PRK2. By molecular dynamic simulations, we show that new interactions between space-adjacent amino acid chains could be established by the presence of a di-anionic phosphate group on the analyzed serines to possibly modify RNA polymerase activity. Together, our data present novel evidence on the complex regulation at the finger subdomain of HCV NS5B via phosphorylation.

Asunto(s)

Hepacivirus/enzimología , Hepatitis C/virología , ARN Polimerasa Dependiente del ARN/metabolismo , Proteínas no Estructurales Virales/metabolismo , Línea Celular , Hepacivirus/genética , Hepacivirus/fisiología , Humanos , Simulación de Dinámica Molecular , Fosforilación , Mutación Puntual , Estructura Terciaria de Proteína , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/genética , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/genética , Replicación Viral

RESUMEN

BACKGROUND: The relevance of HBV genotype diversity on interferon (IFN) therapy outcome in chronic hepatitis B patients has recently been highlighted. Data available for genotype F is poor. The aim of this work was to analyse the response of HBV genotype F to treatment with IFN. Additionally, response was analysed according to the role of single nucleotide polymorphisms (SNPs) near to the IL28B gene. METHODS: A total of 29 HBeAg-positive patients with chronic infection were included with a median age 47 (18-68) years. Of them, 27 were male. One patient was treated with standard IFN-α for 16 weeks, 6 patients received PEG-IFN-α2a 180 µg weekly for 24 weeks and 22 patients for 48 weeks. Response to treatment was defined as loss of HBeAg, anti-HBe seroconversion and decline of HBV DNA level to below 3 log of baseline (IU/ml) at the 6-month of follow-up. The SNPs rs12979860, rs12980275 and rs8099917 were studied by PCR-RFLP. RESULTS: The overall response was obtained in 18 (62%) patients, including one patient who was treated with standard IFN. Additionally, a total of 9 (31%) patients cleared HBsAg, with appearance of anti-HBs. The viral load was undetectable in all of these patients. The same IL28B variants associated with IFN response in HCV infections were also more frequently found in HBV patients compared with non-responders. CONCLUSIONS: Our study indicates that treatment with IFN is effective in patients with HBV genotype F.

Asunto(s)

Antivirales/uso terapéutico , ADN Viral/genética , Genotipo , Virus de la Hepatitis B/genética , Hepatitis B Crónica/tratamiento farmacológico , Interferón-alfa/uso terapéutico , Polietilenglicoles/uso terapéutico , Adolescente , Adulto , Anciano , Femenino , Expresión Génica , Antígenos de Superficie de la Hepatitis B/inmunología , Antígenos e de la Hepatitis B/inmunología , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/crecimiento & desarrollo , Hepatitis B Crónica/inmunología , Hepatitis B Crónica/virología , Humanos , Interferones , Interleucinas/genética , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Proteínas Recombinantes/uso terapéutico , Estudios Retrospectivos , Resultado del Tratamiento

RESUMEN

The hepatitis B virus (HBV) is a DNA virus belonging to the Hepadnaviridae family. Viral isolates have been classified into 10 genotypes, named from A to J, and several subtypes. We report the full-genome sequence from a single molecular clone of HBV genotype F1b, amplified from a chronically infected Chilean patient.

RESUMEN

Hepatitis C virus (HCV) is an important global health problem with an estimated prevalence of more than 170 million infected individuals worldwide. Currently, the standard antiviral therapy, based on pegylated interferon alpha and ribavirin, can achieve a virological response in only nearly 50% of the patients infected with HCV genotype 1, the most widely distributed globally. During the last years, relevant data from genome-wide association studies (GWAS) about the impact and contribution of the patient genomics on viral infection outcomes has suggested the possibility that an individualized antiviral therapy can be considered. In this review, we analyze the existing information on single nucleotide polymorphisms (SNPs) of several host genes and viral factors that influence, as a whole, the outcome of the standard antiviral therapy, and that might be used to predict an individualized antiviral response. We also discuss the clinical data within the most recent context of the triple antiviral therapy.

Asunto(s)

Antivirales/uso terapéutico , Hepacivirus/efectos de los fármacos , Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Hepatitis C/genética , Polimorfismo de Nucleótido Simple , Medicina de Precisión , Quimioterapia Combinada , Genotipo , Hepatitis C/diagnóstico , Hepatitis C/epidemiología , Hepatitis C/virología , Humanos , Interferones , Interleucinas/genética , Selección de Paciente , Farmacogenética , Pirofosfatasas/genética , Receptores de LDL/genética , Resultado del Tratamiento

RESUMEN

BACKGROUND: Hepatitis B virus (HBV) encodes an oncogenic factor, HBx, which is a multifunctional protein that can induce dysfunctional regulation of signaling pathways, transcription, and cell cycle progression, among other processes, through interactions with target host factors. The subcellular localization of HBx is both cytoplasmic and nuclear. This dynamic distribution of HBx could be essential to the multiple roles of the protein at different stages during HBV infection. Transactivational functions of HBx may be exerted both in the nucleus, via interaction with host DNA-binding proteins, and in the cytoplasm, via signaling pathways. Although there have been many studies describing different pathways altered by HBx, and its innumerable binding partners, the molecular mechanism that regulates its different roles has been difficult to elucidate. METHODS: In the current study, we took a bioinformatics approach to investigate whether the viral protein HBx might be regulated via phosphorylation by an evolutionarily conserved mechanism. RESULTS: We found that the phylogenetically conserved residues Ser25 and Ser41 (both within the negative regulatory domain), and Thr81 (in the transactivation domain) are predicted to be phosphorylated. By molecular 3D modeling of HBx, we further show these residues are all predicted to be exposed on the surface of the protein, making them easily accesible to these types of modifications. Furthermore, we have also identified Yin Yang sites that might have the potential to be phosphorylated and O-ß-GlcNAc interplay at the same residues. CONCLUSIONS: Thus, we propose that the different roles of HBx displayed in different subcellular locations might be regulated by an evolutionarily conserved mechanism of posttranslational modification, via phosphorylation.

RESUMEN

AIM: To analyze the association of three IL28B single nucleotide polymorphisms with response to therapy in Chilean patients infected with hepatitis C virus (HCV). METHODS: We studied two groups of patients with chronic HCV infection (genotype 1), under standard combined treatment with pegylated interferon plus ribavirin. One group consisted of 50 patients with sustained virological response, whereas the second group consisted of 49 null responders. In order to analyze the IL28B single nucleotide polymorphisms rs12979860, rs12980275 and rs8099917, samples were used for polymerase chain reaction amplification, and the genotyping was performed by restriction fragment length polymorphism. RESULTS: The IL28B rs12979860 CC, rs12980275 AA and rs8099917 TT genotypes were much more frequently found in patients with sustained virological response compared to null responders (38%, 44% and 50% vs 2%, 8.2% and 8.2%, respectively). These differences were highly significant in all three cases (P < 0.0001). CONCLUSION: The three IL28B polymorphisms studied are strongly associated with sustained virological response to therapy in Chilean patients with chronic HCV (genotype 1).

Asunto(s)

Antivirales/uso terapéutico , Hepatitis C Crónica/tratamiento farmacológico , Hepatitis C Crónica/genética , Hepatitis C Crónica/inmunología , Interleucinas/genética , Polimorfismo de Nucleótido Simple , Antivirales/farmacología , Chile , Genotipo , Hepacivirus/efectos de los fármacos , Hepacivirus/metabolismo , Humanos , Interferón-alfa/farmacología , Interferón-alfa/uso terapéutico , Interferones , Ribavirina/farmacología , Ribavirina/uso terapéutico , Resultado del Tratamiento

RESUMEN

Molecular epidemiological data concerning the hepatitis B virus (HBV) in Chile are not known completely. Since the HBV genotype F is the most prevalent in the country, the goal of this study was to obtain full HBV genome sequences from patients infected chronically in order to determine their subgenotypes and the occurrence of resistance-associated mutations. Twenty-one serum samples from antiviral drug-naive patients with chronic hepatitis B were subjected to full-length PCR amplification, and both strands of the whole genomes were fully sequenced. Phylogenetic analyses were performed along with reference sequences available from GenBank (n = 290). The sequences were aligned using Clustal X and edited in the SE-AL software. Bayesian phylogenetic analyses were conducted by Markov Chain Monte Carlo simulations (MCMC) for 10 million generations in order to obtain the substitution tree using BEAST. The sequences were also analyzed for the presence of primary drug resistance mutations using CodonCode Aligner Software. The phylogenetic analyses indicated that all sequences were found to be the HBV subgenotype F1b, clustered into four different groups, suggesting that diverse lineages of this subgenotype may be circulating within this population of Chilean patients.

Asunto(s)

ADN Viral/genética , Genoma Viral , Virus de la Hepatitis B/genética , Hepatitis B Crónica/virología , Adolescente , Adulto , Anciano , Secuencia de Bases , Niño , Chile , Mapeo Cromosómico , Farmacorresistencia Viral Múltiple/genética , Genotipo , Hepatitis B Crónica/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Mutación , Filogenia , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN

RESUMEN

miR-122 is an abundant, liver-specific microRNA that is required for efficient amplification of hepatitis C virus (HCV) RNA. Recent studies with a miR-122-specific locked nucleic acid antagomir have shown it to be an important host target for therapeutic intervention. However, considerable controversy exists concerning the mechanisms underlying the dependence of HCV replication on miR-122. We studied the impact of miR-122 on the rate of [(32)P]-incorporation into positive-strand viral RNA by membrane-bound replicase complexes isolated from cells containing HCV RNA replicons. [(32)P]-incorporation in this cell-free system represents primarily the elongation phase of RNA synthesis, with little or no de novo initiation, and was not affected by the addition of either excess miR-122 or a miR-122-specific antisense oligonucleotide that suppresses replication in vivo. We also found no evidence that detectable quantities of miR-122 are specifically associated with replicase complexes in vivo. These results are consistent with miR-122 acting at an alternative step in the viral life cycle, promoting cap-independent viral translation, enhancing viral RNA stability, or facilitating de novo initiation of viral RNA synthesis.

Asunto(s)

Hepacivirus/genética , Hepacivirus/metabolismo , MicroARNs/metabolismo , ARN Viral/biosíntesis , Replicación Viral , Antivirales/farmacología , Línea Celular Tumoral , Sistema Libre de Células , Células Cultivadas , Regulación Viral de la Expresión Génica , Silenciador del Gen , Hepatocitos , Humanos , MicroARNs/genética , Inhibidores de la Síntesis del Ácido Nucleico/farmacología , Mutación Puntual , Estabilidad del ARN , ARN Polimerasa Dependiente del ARN/metabolismo , Replicón , Transcripción Genética , Proteínas Virales/biosíntesis , Replicación Viral/genética

RESUMEN

Infectious Pancreatic Necrosis Virus (IPNV) is a bisegmented, double-stranded RNA virus, which belongs to the Birnaviridae family. In the current study, we have analyzed the RNA replication intermediates (RI) purified throughout the viral replication cycle in cultured cells. Equilibrium ultracentrifugation of infected cellular lysates resulted in two major peaks of viral components. The first peak, at a buoyant density of 1.33 g/cm(3), contained assembled IPNV viral particles A and B, whereas the second peak, located at buoyant densities >1.4 g/cm(3), contained a higher molecular weight viral ribonucleoprotein complex composed of, at least, VPg/VP1 and a heterogeneous population of single- and double-stranded viral RNA species. Interestingly, analyses of these dsRNA RI indicated that they contain single-stranded segments of incompletely synthesized positive-strands of RNA. Northern blot experiments of total RNA isolated from infected cells confirmed our proposed configuration of the RNA RI, where the full-length negative-strand of RNA is used as the template for the synthesis of several 3'-truncated forms of the positive-strand of the viral RNA. Together, our results indicate that IPNV utilizes the negative-strand of RNA as template for genome replication.

Asunto(s)

Virus de la Necrosis Pancreática Infecciosa/fisiología , ARN Viral/aislamiento & purificación , ARN Viral/metabolismo , Replicación Viral , Animales , Línea Celular , Centrifugación Isopicnica , Sustancias Macromoleculares/aislamiento & purificación , Salmón

RESUMEN

The eight genotypes (designated A-H) of hepatitis B virus (HBV) display distinctive geographical distribution worldwide, with genotypes A, D and F frequently detected in South America. To determine the prevalence of HBV genotypes in Santiago, Chile, 131 samples from chronic carriers were used for PCR amplification, and genotyping was performed by RFLP. The results indicated that genotype F was the most prevalent among HBV carries (84% of the cases), whereas genotypes A, B, C and D were found at a prevalence of 3.8, 3.8, 6.1, and 2.3%, respectively. We discuss these data in the complex scenario of HBV epidemiology.

Asunto(s)

Portador Sano/virología , Virus de la Hepatitis B/genética , Hepatitis B Crónica/virología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Portador Sano/epidemiología , Niño , Chile/epidemiología , Femenino , Genotipo , Virus de la Hepatitis B/aislamiento & purificación , Hepatitis B Crónica/epidemiología , Humanos , Masculino , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Polimorfismo de Longitud del Fragmento de Restricción , Prevalencia , Adulto Joven

RESUMEN

Infectious pancreatic necrosis virus (IPNV) is a bi-segmented, dsRNA virus of the Birnaviridae family. The structural protein VP1 has been postulated as the RNA-dependent RNA polymerase (RdRp), but its transcriptional activity has not been unequivocally identified from viral particles. Here, we assayed partially purified IPNV in an in vitro RNA synthesis system. To test the RdRp, dialdehyde-nucleotide analogs were used to covalently inhibit the polymerase-associated activity. Our results showed that dialdehyde-nucleotide analogs completely abrogated IPNV in vitro RNA synthesis. The protein involved in this process was identified as viral VP1, since: (a) after incubation of IPNV with [alpha-(32)P]2',3'-dialdehyde-UTP, labeled VP1 protein was identified and (b) VP1 was unable to bind [alpha-(32)P]GTP when particles were preincubated with 2',3'-dialdehyde-ATP. Thus, within viral particles, inhibition of the transcriptional activity is a result of the binding of 2',3'-dialdehyde-nucleotide analogs to the RdRp, VP1.

Asunto(s)

Proteínas de la Cápside/antagonistas & inhibidores , ARN Polimerasas Dirigidas por ADN/antagonistas & inhibidores , Virus de la Necrosis Pancreática Infecciosa/metabolismo , Nucleótidos/metabolismo , Virión/enzimología , Proteínas de la Cápside/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Nucleótidos/química , Radioisótopos de Fósforo/metabolismo , ARN Viral/biosíntesis

RESUMEN

In this study, we have analyzed the morphogenesis of the birnavirus infectious pancreatic necrosis virus throughout the infective cycle in CHSE-214 cells by using a native agarose electrophoresis system. Two types of viral particles (designated A and B) were identified, isolated, and characterized both molecularly and biologically. Together, our results are consistent with a model of morphogenesis in which the genomic double-stranded RNA is immediately assembled, after synthesis, into a large (66-nm diameter) and uninfectious particle A, where the capsid is composed of both mature and immature viral polypeptides. Upon maturation, particles A yield particles B through the proteolytic cleavage of most of the remaining viral precursors within the capsid, the compaction of the particle (60-nm diameter), and the acquisition of infectivity. These studies will provide the foundation for further analyses of birnavirus particle assembly and RNA replication.

Asunto(s)

Genoma Viral , Virus de la Necrosis Pancreática Infecciosa/crecimiento & desarrollo , Virus de la Necrosis Pancreática Infecciosa/patogenicidad , ARN Viral/metabolismo , Virión/metabolismo , Ensamble de Virus , Animales , Células Cultivadas , Virus de la Necrosis Pancreática Infecciosa/metabolismo , Morfogénesis , ARN Bicatenario/metabolismo , Salmón/virología , Replicación Viral