RESUMO
Hepatitis delta virus, the only member of the only species in the genus Deltavirus, is a unique human pathogen. Its ~1.7 kb circular negative-sense RNA genome encodes a protein, hepatitis delta antigen, which occurs in two forms, small and large, both with unique functions. Hepatitis delta virus uses host RNA polymerase II to replicate via double rolling circle RNA synthesis. Newly synthesized linear RNAs are circularized after autocatalytic cleavage and ligation. Hepatitis delta virus requires the envelope of the helper virus, hepatitis B virus (family Hepadnaviridae), to produce infectious particles. This is a summary of the International Committee on Taxonomy of Viruses (ICTV) Report on the taxonomy of Deltavirus which is available at www.ictv.global/report/deltavirus.
Assuntos
Hepatite D/virologia , Vírus Delta da Hepatite/classificação , Vírus Delta da Hepatite/genética , RNA Viral/genética , Genoma Viral , Vírus Auxiliares/fisiologia , Vírus da Hepatite B/fisiologia , Vírus Delta da Hepatite/crescimento & desenvolvimento , Interações Hospedeiro-Patógeno , Humanos , RNA/genética , RNA/metabolismo , RNA Polimerase II/metabolismo , RNA Circular , RNA Viral/metabolismo , Proteínas do Envelope Viral/metabolismo , Replicação ViralRESUMO
Antiviral compounds that increase the resistance of host tissues represent an attractive class of therapeutic. Here, we show that squalamine, a compound previously isolated from the tissues of the dogfish shark (Squalus acanthias) and the sea lamprey (Petromyzon marinus), exhibits broad-spectrum antiviral activity against human pathogens, which were studied in vitro as well as in vivo. Both RNA- and DNA-enveloped viruses are shown to be susceptible. The proposed mechanism involves the capacity of squalamine, a cationic amphipathic sterol, to neutralize the negative electrostatic surface charge of intracellular membranes in a way that renders the cell less effective in supporting viral replication. Because squalamine can be readily synthesized and has a known safety profile in man, we believe its potential as a broad-spectrum human antiviral agent should be explored.
Assuntos
Antivirais/farmacologia , Viroses/tratamento farmacológico , Replicação Viral/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antivirais/química , Linhagem Celular , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Colestanóis/química , Colestanóis/farmacologia , Cricetinae , Feminino , Vírus da Hepatite B/efeitos dos fármacos , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus Delta da Hepatite/efeitos dos fármacos , Vírus Delta da Hepatite/crescimento & desenvolvimento , Hepatócitos/efeitos dos fármacos , Hepatócitos/virologia , Humanos , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Estrutura Molecular , Muromegalovirus/efeitos dos fármacos , Muromegalovirus/crescimento & desenvolvimento , Espalhamento a Baixo Ângulo , Viroses/virologia , Difração de Raios X , Proteínas rac1 de Ligação ao GTP/químicaRESUMO
Hepatitis D virus (HDV) infection involves a distinct subgroup of individuals simultaneously infected with the hepatitis B virus (HBV) and characterized by an often severe chronic liver disease. HDV is a defective RNA agent needing the presence of HBV for its life cycle. HDV is present worldwide, but the distribution pattern is not uniform. Different strains are classified into eight genotypes represented in specific regions and associated with peculiar disease outcome. Two major specific patterns of infection can occur, i.e. co-infection with HDV and HBV or HDV superinfection of a chronic HBV carrier. Co-infection often leads to eradication of both agents, whereas superinfection mostly evolves to HDV chronicity. HDV-associated chronic liver disease (chronic hepatitis D) is characterized by necro-inflammation and relentless deposition of fibrosis, which may, over decades, result in the development of cirrhosis. HDV has a single-stranded, circular RNA genome. The virion is composed of an envelope, provided by the helper HBV and surrounding the RNA genome and the HDV antigen (HDAg). Replication occurs in the hepatocyte nucleus using cellular polymerases and via a rolling circle process, during which the RNA genome is copied into a full-length, complementary RNA. HDV infection can be diagnosed by the presence of antibodies directed against HDAg (anti-HD) and HDV RNA in serum. Treatment involves the administration of pegylated interferon-α and is effective in only about 20% of patients. Liver transplantation is indicated in case of liver failure.
Assuntos
Hepatite D Crônica , Vírus Delta da Hepatite , Antivirais/uso terapêutico , Progressão da Doença , Genótipo , Anticorpos Anti-Hepatite/sangue , Vírus da Hepatite B/patogenicidade , Hepatite B Crônica/complicações , Hepatite D Crônica/complicações , Hepatite D Crônica/diagnóstico , Hepatite D Crônica/epidemiologia , Hepatite D Crônica/terapia , Vírus Delta da Hepatite/genética , Vírus Delta da Hepatite/crescimento & desenvolvimento , Vírus Delta da Hepatite/imunologia , Vírus Delta da Hepatite/patogenicidade , Humanos , Transplante de Fígado , RNA Viral/sangue , Superinfecção , Resultado do Tratamento , Carga Viral , Replicação ViralRESUMO
Co-infection and superinfection of hepatitis B virus (HBV) with hepatitis delta virus (HDV) leads to suppression of HBV replication both in patients and in animal and cellular models. The mechanisms behind this inhibition have not previously been explored fully. HBV replication is governed by four promoters and two enhancers, Enh1 and Enh2. Repression of these enhancers has been reported to be one of the main mechanisms of HBV inhibition. Moreover, in a previous study, it has been demonstrated that alpha interferon (IFN-alpha)-inducible MxA protein inhibits HBV replication. HDV encodes two proteins, p24 and p27. p27 was shown to activate several heterologous promoters, including HBV promoters. In an attempt to analyse the mechanisms of HBV inhibition by HDV, the question was raised whether HDV proteins could act directly by repressing HBV enhancers, and/or indirectly by activating the MxA gene. This issue was addressed in a co-transfection model in Huh-7 cells, using p24- or p27-expressing plasmids along with Enh1, Enh2, HBV and MxA promoter-luciferase constructs. Enh1 and Enh2 were strongly repressed, by 60 and 80 % and 40 and 60 %, by p24 and p27, respectively. In addition, p27 was responsible for threefold activation of the MxA promoter and potentiation of IFN-alpha on this promoter. MxA mRNA quantification and a virus yield reduction assay confirmed these results. In conclusion, this study shows that HDV proteins inhibit HBV replication by trans-repressing its enhancers and by trans-activating the IFN-alpha-inducible MxA gene.
Assuntos
Elementos Facilitadores Genéticos , Proteínas de Ligação ao GTP/biossíntese , Regulação Viral da Expressão Gênica , Vírus da Hepatite B/fisiologia , Vírus Delta da Hepatite/fisiologia , Proteínas Virais/metabolismo , Fusão Gênica Artificial , Linhagem Celular , Genes Reporter , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus Delta da Hepatite/crescimento & desenvolvimento , Humanos , Luciferases/genética , Luciferases/metabolismo , Proteínas de Resistência a Myxovirus , Ligação ProteicaRESUMO
Hepatitis B virus (HBV) and hepatitis delta virus (HDV) share the HBV envelope proteins. When woodchucks chronically infected with woodchuck hepatitis virus (WHV) are superinfected with HDV, they produce HDV with a WHV envelope, wHDV. Several lines of evidence are provided that wHDV infects not only cultured primary woodchuck hepatocytes (PWH) but also primary human hepatocytes (PHH). Surprisingly, HBV-enveloped HDV (hHDV) and wHDV infected PHH with comparable efficiencies; however, hHDV did not infect PWH. The basis for these host range specificities was investigated using as inhibitors peptides bearing species-specific pre-S (where S is the small envelope protein) sequences. It was found that pre-S1 contributed to the ability of wHDV to infect both PHH and PWH. In addition, the inability of hHDV to infect PWH was not overcome using a chimeric form of hHDV containing WHV S protein, again supporting the essential role of pre-S1 in infection of target cells. One interpretation of these data is that host range specificity of HDV is determined entirely by pre-S1 and that the WHV and HBV pre-S1 proteins recognize different receptors on PHH.
Assuntos
Vírus da Hepatite B da Marmota/genética , Vírus Delta da Hepatite/crescimento & desenvolvimento , Hepatócitos/virologia , Proteínas do Envelope Viral/metabolismo , Montagem de Vírus , Sequência de Aminoácidos , Animais , Células Cultivadas , Vírus Delta da Hepatite/genética , Humanos , Marmota , Modelos Moleculares , Alinhamento de Sequência , Proteínas do Envelope Viral/genéticaRESUMO
Hepatitis D virus (HDV) is a defective RNA virus that requires the presence of hepatitis B virus (HBV) for its life cycle. The in vitro HDV infection system is widely used as a surrogate model to study cellular infection with both viruses owing to its practical feasibility. However, previous methods for running this system were less efficient for high-throughput screening and large-scale studies. Here, we developed a novel method for the production of infectious HDV by adenoviral vector (AdV)-mediated transduction. We demonstrated that the AdV-based method yields 10-fold higher viral titers than the transient-transfection approach. The HDV-containing supernatant derived from AdV-infected Huh7 cells can be used as the inoculum in infectivity assays without requiring further concentration prior to use. Furthermore, we devloped a chemiluminescent immunoassay (HDV-CLEIA) to quantitatively determine intracellular HDAg with a dynamic range of 5-11,000 pg/mL. HDV-CLEIA can be used as an alternative approach to assess HDV infection. The advantages of our updated methodology were demonstrated through in vitro HDV infection of HepaRG cells and by evaluating the neutralization activity using antibodies that target various regions of the HBV/HDV envelope proteins. Together, the methods presented here comprise a novel toolbox of in vitro assays for studying HDV infection.
Assuntos
Técnicas Citológicas/métodos , Hepatite D/patologia , Vírus Delta da Hepatite/crescimento & desenvolvimento , Modelos Biológicos , Adenoviridae/genética , Linhagem Celular , Vetores Genéticos , Hepatócitos/virologia , Humanos , Transdução GenéticaRESUMO
AIM: To study the hepatitis B virus (HBV) and hepatitis D virus (HDV) replication interferences in patients with chronic hepatitis delta infected with different HBV genotypes. METHODS: We conducted a transversal study including 68 chronic hepatitis delta (CHD) (37 HIV-positive) patients and a control group of 49 chronic hepatitis B (CHB) (22 HIV-positive) patients. In addition, a dynamic follow-up was performed in 16 CHD patients. In all the samples, the surface antigen of hepatitis B (HBsAg) serum titers were analyzed with the Monolisa HBsAg Ultra system (Bio-Rad), using as quantification standard a serial dilution curve of an international HBsAg standard. Serum HBV-DNA titers were analyzed using the Roche Cobas TaqMan (Roche, Barcelona, Spain), and the serum HDV-RNA using an in-house real-time qRT-PCR method, with TaqMan probes. HBV genotype was determined with the line immunoassay LiPA HBV genotyping system (Innogenetics, Ghent, Belgium). In those patients negative for LiPA assay, a nested PCR method of complete HBsAg coding region, followed by sequence analysis was applied. RESULTS: No differences in the HBV-DNA levels were found in CHB patients infected with different HBV genotypes. However, in CHD patients the HBV-DNA levels were lower in those infected with HBV-A than in those with HBV-D, both in HIV negative [median (IQR): 1.25 (1.00-1.35) vs 2.95 (2.07-3.93) log10 (copies/mL), P = 0.013] and HIV positive patients [2.63 (1.24-2.69) vs 7.25 (4.61-7.55) log10 (copies/mL), P < 0.001]. This was confirmed in the dynamic study of the HBV/HDV patients. These differences induce an under-estimation of HBV-A incidence in patients with CHD analyzed with LiPA assay. Finally, the HBsAg titers reflected no significant differences in CHD patients infected with HBV-A or D. CONCLUSION: Viral replication interference between HBV and HDV is HBV-genotype dependent, and more evident in patients infected with HBV-genotype A, than with HBV-D or E.
Assuntos
Coinfecção , Vírus da Hepatite B/crescimento & desenvolvimento , Hepatite B/virologia , Hepatite D Crônica/virologia , Vírus Delta da Hepatite/crescimento & desenvolvimento , Replicação Viral , Adulto , Biomarcadores/sangue , Estudos Transversais , DNA Viral/sangue , DNA Viral/genética , Feminino , Genótipo , Infecções por HIV/complicações , Infecções por HIV/virologia , Hepatite B/complicações , Hepatite B/diagnóstico , Antígenos de Superfície da Hepatite B/sangue , Vírus da Hepatite B/genética , Vírus da Hepatite B/imunologia , Hepatite D Crônica/complicações , Hepatite D Crônica/diagnóstico , Vírus Delta da Hepatite/genética , Humanos , Estudos Longitudinais , Masculino , Pessoa de Meia-Idade , RNA Viral/sangue , RNA Viral/genética , Estudos Retrospectivos , Fatores de Tempo , Carga ViralRESUMO
The use of HBV and HDV experimental models has significantly contributed to understand the viral life cycle and to systematically test antiviral effects of various drugs on a pre-clinical level. Similar replication strategies of related hepadna viruses permit the use of chimpanzees (Pan troglodytes), woodchucks (Marmota monax), ground and tree squirrels (Spermophilus beecheyi) or Pekin ducks (Anas domesticus) as appropriate animal models. Cell culture systems for in vitro infection or transfection using both primary cultures of human and non-human hepatocytes and non-hepatocytes and cell lines have recently been identified. The advantages and restrictions of these experimental models with respect to evaluation of interferon effects on viral and hepatocellular gene expression are discussed.
Assuntos
Vírus da Hepatite B/crescimento & desenvolvimento , Vírus Delta da Hepatite/crescimento & desenvolvimento , Interferons/farmacologia , Animais , Células Cultivadas , Modelos Animais de Doenças , Humanos , Replicação Viral/efeitos dos fármacosRESUMO
The development of tissue culture technology has led to invaluable information in many fields of modern virology. Until recently, the lack of an in vitro culture system for the hepatitis B virus (HBV) was a considerable impediment to the study of its life cycle at the cellular and molecular levels. However, it did not prevent its isolation and molecular cloning. Such has been the case also for the hepatitis delta virus (HDV), the genome of which was cloned and sequenced before its replication could be observed in cultured cells. In recent years, tissue culture systems for HBV and HDV have been developed progressively by the identification of permissive, established cell lines for production of virions and susceptible primary hepatocyte cultures for infection assays. I will briefly review here the recent experiments that have contributed to replicate HBV and HDV in cell culture systems.
Assuntos
Vírus da Hepatite B/crescimento & desenvolvimento , Vírus Delta da Hepatite/crescimento & desenvolvimento , Cultura de Vírus , Animais , HumanosRESUMO
Viral hepatitis remains a worldwide public health problem. The hepatitis D virus (HDV) must either coinfect or superinfect with the hepatitis B virus (HBV). HDV contains a small RNA genome (approximately 1.7 kb) with a single open reading frame (ORF) and requires HBV supplying surface antigens (HBsAgs) to assemble a new HDV virion. During HDV replication, two isoforms of a delta antigen, a small delta antigen (SDAg) and a large delta antigen (LDAg), are produced from the same ORF of the HDV genome. The SDAg is required for HDV replication, whereas the interaction of LDAg with HBsAgs is crucial for packaging of HDV RNA. Various clinical outcomes of HBV/HDV dual infection have been reported, but the molecular interaction between HBV and HDV is poorly understood, especially regarding how HBV and HDV compete with HBsAgs for assembling virions. In this paper, we review the role of endoplasmic reticulum stress induced by HBsAgs and the molecular pathway involved in their promotion of LDAg nuclear export. Because the nuclear sublocalization and export of LDAg is regulated by posttranslational modifications (PTMs), including acetylation, phosphorylation, and isoprenylation, we also summarize the relationship among HBsAg-induced endoplasmic reticulum stress signaling, LDAg PTMs, and nuclear export mechanisms in this review.
Assuntos
Coinfecção , Vírus da Hepatite B/patogenicidade , Hepatite B/virologia , Hepatite D/virologia , Vírus Delta da Hepatite/patogenicidade , Superinfecção , Replicação Viral , Transporte Ativo do Núcleo Celular , Animais , DNA Viral/biossíntese , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/virologia , Estresse do Retículo Endoplasmático , Hepatite B/diagnóstico , Hepatite B/metabolismo , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/metabolismo , Hepatite D/diagnóstico , Hepatite D/metabolismo , Vírus Delta da Hepatite/crescimento & desenvolvimento , Vírus Delta da Hepatite/metabolismo , Antígenos da Hepatite delta/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Processamento de Proteína Pós-Traducional , RNA Viral/biossíntese , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
The hepatitis delta virus (HDV) is a small (~1700 nucleotides) RNA pathogen which encodes only one open reading frame. Consequently, HDV is dependent on host proteins to replicate its RNA genome. Recently, we reported that ASF/SF2 binds directly and specifically to an HDV-derived RNA fragment which has RNA polymerase II promoter activity. Here, we localized the binding site of ASF/SF2 on the HDV RNA fragment by performing binding experiments using purified recombinant ASF/SF2 combined with deletion analysis and site-directed mutagenesis. In addition, we investigated the requirement of ASF/SF2 for HDV RNA replication using RNAi-mediated knock-down of ASF/SF2 in 293 cells replicating HDV RNA. Overall, our results indicate that ASF/SF2 binds to a purine-rich region distant from both the previously published initiation site of HDV mRNA transcription and binding site of RNAP II, and suggest that this protein is not involved in HDV replication in the cellular system used.
Assuntos
Vírus Delta da Hepatite/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , RNA/genética , Sítios de Ligação , Genoma Viral , Células HEK293 , Vírus Delta da Hepatite/crescimento & desenvolvimento , Vírus Delta da Hepatite/patogenicidade , Humanos , Regiões Promotoras Genéticas , Ligação Proteica , RNA Polimerase II/genética , Fatores de Processamento de Serina-Arginina , Replicação Viral/genéticaRESUMO
Hepatitis delta virus (HDV) consists of a circular single-stranded RNA genome which assembles two viral proteins and acquires a lipid envelope in which the hepatitis B surface antigens (HBsAg) are embedded. HDV does not encode its own polymerase, but exploits a cellular enzyme for its replication. A better understanding of the mechanisms of HDV replication mechanism would provide new insights for antiviral strategies. Based on genomic variability, eight major genotypes of HDV have been identified, which differ as much as 40% in the nucleotide sequence. The cloning of HDV-RNA has provided genetic probes for the measurement of HDV-RNA in serum and liver; the sensitivity of HDV-RNA detection improved significantly when the reverse transcriptase-polymerase chain reaction (PCR) technique was introduced. As no commercial test is standardized for viral load detection, home-made assays have been developed in the different referral centers, which may not be comparable. Quantification of HDV in serum by real-time PCR has been recently proposed in the management of chronically infected patients. No specific inhibitors of HDV are available at present and, in spite of the crucial relationship between HDV and HBV, drugs that block HBV have only a theoretical but no sound effect on HDV replication.
Assuntos
Hepatite D/genética , Vírus Delta da Hepatite/genética , RNA Viral/sangue , Hepatite B/complicações , Hepatite D/sangue , Hepatite D/tratamento farmacológico , Hepatite D/virologia , Vírus Delta da Hepatite/crescimento & desenvolvimento , Vírus Delta da Hepatite/patogenicidade , HumanosRESUMO
A myristate linked to the N-terminus of the large hepatitis B virus (HBV) envelope protein was found to be required for infectivity of the hepatitis delta virus (HDV). Myristoylation of the large HBV envelope protein being known as indispensable for HBV infectivity, this result further demonstrates the similarities between the HBV and HDV entry pathways. In addition, the transfer of the N-myristoylation signal from the large to the middle or the small HBV envelope protein led in both cases to a loss of HDV infectivity. Hence, it is suggested that viral entry could depend on a physical link, or a spatial association, between the N-terminal receptor-binding polypeptide of the large protein and the myristoyl anchor linked to glycine-2.
Assuntos
Vírus da Hepatite B/química , Vírus Delta da Hepatite/patogenicidade , Proteínas do Envelope Viral/química , DNA Viral/genética , Vírus da Hepatite B/genética , Vírus Delta da Hepatite/crescimento & desenvolvimento , Ácido Mirístico/metabolismo , Proteínas do Envelope Viral/imunologia , Proteínas do Envelope Viral/fisiologia , Virulência/efeitos dos fármacosRESUMO
High dose interferon treatment for 1 year is the only established treatment for chronic hepatitis D, but it is associated with a high relapse rate after treatment discontinuation. In this study, patients were treated with 10 MU interferon alpha 2b, thrice weekly for 2 years. Twenty-three patients were recruited and 15 completed the 2-year treatment and 6 months follow-up periods. Treatment response was assessed biochemically [normal alanine aminotransferase (ALT)], virologically (undetectable hepatitis D virus RNA) and histologically (at least 2 point decrease in the Knodell score) at the end of treatment (EOT) and at the end of follow-up. Out of 15 patients who finished the 2-year treatment period, seven patients (47%) had a biochemical response but only two (13%) had a normal ALT after follow-up. ALT decreased from the baseline value of 143.1 +/- 121.7 (mean +/- SD) to 39.7 +/- 20.6 (P < 0.01) at EOT. Virological response was observed in six patients at EOT and in two patients at follow-up. Two patients lost hepatitis B surface antigen. Of the 12 patients with paired liver biopsies, a histological improvement was observed in eight patients. Interferon treatment leads to a complete or partial response in a substantial number of patients but 2 years of treatment does not appear to increase sustained response rates over 1 year treatment.
Assuntos
Antivirais/uso terapêutico , Hepatite D/tratamento farmacológico , Vírus Delta da Hepatite/crescimento & desenvolvimento , Interferon-alfa/uso terapêutico , Adulto , Alanina Transaminase/sangue , Biópsia , Feminino , Hepatite D/enzimologia , Hepatite D/patologia , Hepatite D/virologia , Histocitoquímica , Humanos , Interferon alfa-2 , Masculino , Projetos Piloto , RNA Viral/sangue , RNA Viral/química , RNA Viral/genética , Proteínas Recombinantes , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Hepatitis B virus (HBV) replication produces three envelope proteins (L, M, and S) that have a common C terminus. L, the largest, contains a domain, pre-S1, not present on M. Similarly M contains a domain, pre-S2, not present on S. The pre-S1 region has important functions in the HBV life cycle. Thus, as an approach to studying these roles, the pre-S1 and/or pre-S2 sequences of HBV (serotype adw2, genotype A) were expressed as N-terminal fusions to the Fc domain of a rabbit immunoglobulin G chain. Such proteins, known as immunoadhesins (IA), were highly expressed following transfection of cultured cells and, when the pre-S1 region was present, >80% were secreted. The IA were myristoylated at a glycine penultimate to the N terminus, although mutation studies showed that this modification was not needed for secretion. As few as 30 amino acids from the N terminus of pre-S1 were both necessary and sufficient to drive secretion of IA. Even expression of pre-S1 plus pre-S2, in the absence of an immunoglobulin chain, led to efficient secretion. Overall, these studies demonstrate an unexpected ability of the N terminus of pre-S1 to promote protein secretion. In addition, some of these secreted IA, at nanomolar concentrations, inhibited infection of primary human hepatocytes either by hepatitis delta virus (HDV), a subviral agent that uses HBV envelope proteins, or HBV. These IA have potential to be part of antiviral therapies against chronic HDV and HBV, and may help understand the attachment and entry mechanisms used by these important human pathogens.
Assuntos
Antivirais/farmacologia , Antígenos de Superfície da Hepatite B/farmacologia , Vírus da Hepatite B/crescimento & desenvolvimento , Vírus Delta da Hepatite/crescimento & desenvolvimento , Fragmentos Fc das Imunoglobulinas/farmacologia , Fatores Imunológicos/farmacologia , Precursores de Proteínas/farmacologia , Linhagem Celular , Antígenos de Superfície da Hepatite B/genética , Antígenos de Superfície da Hepatite B/metabolismo , Vírus da Hepatite B/genética , Humanos , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/metabolismo , Fatores Imunológicos/genética , Fatores Imunológicos/metabolismo , Precursores de Proteínas/genética , Precursores de Proteínas/metabolismo , Transporte Proteico , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologiaRESUMO
Beginning with three partial cDNA clones of the RNA genome of human hepatitis delta virus (HDV), we assembled the complete 1,679-base sequence on a single molecule and then inserted a trimer of this into plasmid pSLV, a simian virus 40-based eucaryotic expression vector. This construct was used to transfect both monkey kidney (COS7) and human hepatocellular carcinoma (HuH7) cell lines. In this way we obtained replication of the HDV RNA genome and the appearance, in the nucleoli, of the delta antigen, the only known virus-coded protein. This proved both that the HDV genome could replicate in nonliver as well as liver cells and that there was no requirement for the presence of hepatitis B virus sequences or proteins. When the pSVL construct was made with a dimer of an HDV sequence with a 2-base-pair deletion in the open reading frame, genome replication was reduced at least 40-fold. However, when we cotransfected with a plasmid that expressed the correct delta antigen, the mutated dimer achieved a level of genome replication comparable to that of the nonmutated sequence. We thus conclude that the delta antigen can act in trans and is essential for replication of the HDV genome.
Assuntos
Antígenos Virais/fisiologia , Vírus Delta da Hepatite/crescimento & desenvolvimento , RNA Viral/biossíntese , Replicação Viral , Western Blotting , Linhagem Celular , Clonagem Molecular , Imunofluorescência , Genes Virais , Vírus Delta da Hepatite/genética , Antígenos da Hepatite delta , Humanos , Técnicas In Vitro , RNA Viral/genética , TransfecçãoRESUMO
Monolayer cell cultures of primary woodchuck hepatocytes, prepared by perfusing the liver in situ with collagenase type I, yielded hepatocytes with a viability of greater than 90% which could be held in culture for up to 3 months. Cultures of primary woodchuck hepatocytes were infected one day after plating with hepatitis delta virus (HDV) which had been passaged five times in woodchucks and was therefore identified as woodchuck hepatitis delta virus (WHDV). Replication of WHDV was demonstrated by the appearance of genomic WHDV RNA of ca. 1.7 kb beginning 7 days after infection, with an increase of copy numbers up to 2 weeks after inoculation. Synthesis of hepatitis delta virus-associated antigen (HDAg) in hepatocytes was detected by immunofluorescence staining of hepatocytes. Preincubation of the inoculum with rabbit sera containing antibodies against woodchuck hepatitis virus surface antigen (anti-WHs) reduced the infectivity of WHDV to an undetectable level compared with inocula which were treated with anti-WHs negative sera.
Assuntos
Hepatite D/fisiopatologia , Vírus Delta da Hepatite/crescimento & desenvolvimento , Marmota/microbiologia , RNA Viral/biossíntese , Sciuridae/microbiologia , Replicação Viral , Animais , Antígenos Virais/análise , Northern Blotting , Southern Blotting , Células Cultivadas , Regulação da Expressão Gênica , Fígado/microbiologia , Proteínas Virais/biossínteseRESUMO
Hepatitis delta virus (HDV) is a subviral agent of humans which is dependent upon hepatitis B virus as a helper for transmission. HDV can be experimentally transmitted to woodchucks by using woodchuck hepatitis virus (WHV) as the helper. We used this model system to study two types of HDV infections: those of animals already chronically infected with WHV and those of animals without any evidence of prior exposure to WHV. At 5 to 10 days after infection with HDV, liver biopsies of these two groups of animals indicated that around 1% of the hepatocytes were infected (HDV antigen positive). Moreover, similar amounts of replicative forms of HDV RNA were detected. In contrast, by 20 days postinfection, the two groups of animals were quite different in the extent of the HDV infection. The animals chronically infected with WHV showed spread of the infection within the liver and the release of high titers of HDV into the serum. In contrast, the animals not previously exposed to WHV showed a progressive reduction in liver involvement, and at no time up to 165 days postinfection could we detect HDV particles in the serum. However, if these animals were inoculated with a relatively high titer of WHV at either 7 or even 33 days after the HDV infection, HDV viremia was observed. Our data support the interpretation that in these animals, hepatocytes were initially infected in the absence of helper virus, HDV genome replication took place, and ultimately these replicating genomes were rescued by the secondary WHV infection. The observation that HDV can survive in the liver for at least 33 days in the absence of coinfecting helper virus may be relevant to the reemergence of HDV infection following liver transplantation.
Assuntos
Vírus da Hepatite B da Marmota/crescimento & desenvolvimento , Vírus Delta da Hepatite/crescimento & desenvolvimento , Hepatite Viral Animal/microbiologia , Marmota/microbiologia , Animais , Antígenos Virais/metabolismo , Expressão Gênica , Vírus Auxiliares/crescimento & desenvolvimento , Hepatite B/microbiologia , Antígenos da Hepatite delta , Fígado/microbiologia , RNA Mensageiro/genética , RNA Viral/genética , Fatores de TempoRESUMO
Hepatitis delta virus requires a helper function from hepatitis B virus for packaging, release, and infection of hepatocytes. The assembly of large delta antigen (HDAg) is mediated by copackaging with the small surface antigen of hepatitis B virus (HBsAg), and the assembly of small HDAg requires interactions with large HDAg. To examine the molecular mechanisms by which small HBsAg, large HDAg, and small HDAg interact, we have established a virion assembly system in COS7 cells by cotransfecting plasmids encoding the small HBsAg, the small HDAg, and large HDAg mutants. Results indicate that sequences within the C-terminal 19-amino-acid domain flanking the Cxxx isoprenylation motif are important for the assembly of large HDAg. In addition, a large HDAg mutant bearing extra sequences separating the C-terminal 19-amino-acid domain from the common regions of the small and large HDAgs is capable, like the wild-type large HDAg, of copackaging with small HBsAg. The ability of assembly is also demonstrated for a large HDAg mutant from which nuclear localization signals have been removed. Furthermore, a cryptic signal within the N-terminal 50 amino acid residues other than the putative N-terminal coiled-coil structure and a subdomain between amino acid residues 50 and 65 of the large HDAg are important for the assembly of small HDAg as well as the trans-dominant negative regulation of large HDAg in hepatitis delta virus replication.
Assuntos
Antígenos Virais/genética , Vírus Defeituosos/genética , Vírus Delta da Hepatite/crescimento & desenvolvimento , Vírus Delta da Hepatite/genética , Sequência de Aminoácidos , Animais , Antígenos Virais/biossíntese , Transporte Biológico , Compartimento Celular/genética , Células Cultivadas , Análise Mutacional de DNA , Antígenos de Superfície da Hepatite B/biossíntese , Antígenos da Hepatite delta , Fígado/metabolismo , Dados de Sequência Molecular , Especificidade de Órgãos , Prenilação de Proteína/genética , Deleção de Sequência , Vírion/crescimento & desenvolvimento , Replicação ViralRESUMO
The large hepatitis delta antigen (HDAg) has been found to be essential for the assembly of the hepatitis delta virion. Furthermore, in a cotransfection experiment, the large HDAg itself, without the hepatitis delta virus (HDV) genome and small HDAg, could be packaged into hepatitis B surface antigen (HBsAg) particles. By deletion analysis, it was shown that the amino-terminal leucine zipper domain was dispensable for packaging. The large HDAg could also help in copackaging of the small HDAg into HBsAg particles without the need for HDV RNA. This process was probably mediated through direct interaction of the two HDAgs as a mutated large HDAg whose leucine zipper domain was deleted such that it could not help in copackaging of the small HDAg. This mutated large HDAg did not suppress HDV replication, suggesting that this effect is probably also via protein interaction. These results indicated that functional domains of the large HDAg responsible for packaging with HBsAg particles and for the trans-negative effect on HDV replication can be separated.