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Cytosolic retinoic acid-inducible gene I (RIG-I) is an important innate immune RNA sensor and can induce antiviral cytokines, e.g., interferon-β (IFN-β). Innate immune response to hepatitis B virus (HBV) plays a pivotal role in viral clearance and persistence. However, knowledge of the role that RIG-I plays in HBV infection is limited. The woodchuck is a valuable model for studying HBV infection. To characterize the molecular basis of woodchuck RIG-I (wRIG-I), we analyzed the complete coding sequences (CDSs) of wRIG-I, containing 2778 base pairs that encode 925 amino acids. The deduced wRIG-I protein was 106.847 kD with a theoretical isoelectric point (pI) of 6.07, and contained three important functional structures [caspase activation and recruitment domains (CARDs), DExD/H-box helicases, and a repressor domain (RD)]. In woodchuck fibroblastoma cell line (WH12/6), wRIG-I-targeted small interfering RNA (siRNA) down-regulated RIG-I and its downstrean effector-IFN-β transcripts under RIG-I' ligand, 5'-ppp double stranded RNA (dsRNA) stimulation. We also measured mRNA levels of wRIG-I in different tissues from healthy woodchucks and in the livers from woodchuck hepatitis virus (WHV)-infected woodchucks. The basal expression levels of wRIG-I were abundant in the kidney and liver. Importantly, wRIG-I was significantly up-regulated in acutely infected woodchuck livers, suggesting that RIG-I might be involved in WHV infection. These results may characterize RIG-I in the woodchuck model, providing a strong basis for further study on RIG-I-mediated innate immunity in HBV infection.
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Animais , Linhagem Celular Tumoral , Clonagem Molecular , Proteína DEAD-box 58 , Genética , Alergia e Imunologia , Fibroblastos , Alergia e Imunologia , Patologia , Expressão Gênica , Hepatite B , Genética , Alergia e Imunologia , Patologia , Vírus da Hepatite B da Marmota , Imunidade Inata , Interferon beta , Genética , Alergia e Imunologia , Ponto Isoelétrico , Rim , Alergia e Imunologia , Patologia , Virologia , Fígado , Alergia e Imunologia , Patologia , Virologia , Marmota , Genética , Alergia e Imunologia , Virologia , Fases de Leitura Aberta , Domínios Proteicos , RNA de Cadeia Dupla , RNA Interferente Pequeno , Genética , Metabolismo , Doenças dos Roedores , Genética , Alergia e Imunologia , Patologia , VirologiaRESUMO
The type I interferon and IFNAR play an important role in hepatitis B virus (HBV) infection and anti-HBV therapy. However, its mechanism of action is still poorly understood. To gain more insights into the role of type I interferon and type I interferon receptor (IFNAR) in HBV infection, we established an HBV persistent replication IFNAR knockout (IFNAR(-/-)) mouse model and preliminarily applied this model. At first, the progeny of IFNAR(-/-) mouse was reproduced. Then hydrodynamic injection with pAAV/HBV1.2 plasmid was conducted to establish the persistent HBV replication IFNAR(-/-) mouse model. At last, we applied this model to evaluate the effect of nucleoside analogues entecavir (ETV) on HBV replication. It was found that there was no difference in the serum HBsAg and HBeAg levels and HBcAg expression in the liver tissue between the ETV treated groups and normal saline (NS) treated group, but the serum HBV DNA levels were significantly suppressed 10, 25, 40 and 55 days after the ETV treatment [P=0.035, P=0.00, P=0.149 and P=0.084, IFNAR knockout (KO) control group vs. C57BL/6 ETV groups, respectively; P=0.081, P=0.001, P=0.243 and P=0.147, IFNAR KO control group vs. IFNAR KO ETV groups, respectively]. Interestingly, there was no difference in serum HBV DNA levels between the ETV treated IFNAR(-/-) and C57BL/6 mice. This result suggests that HBV suppression during ETV treatments doesn't depend on type I interferon and IFNAR. Collectively, persistent HBV replication IFNAR(-/-) mouse model that we established is a useful and convenient tool to detect the function of the type I interferon and IFNAR in HBV infection and anti-HBV treatments.
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Animais , Feminino , Humanos , Masculino , Camundongos , Doença Crônica , Modelos Animais de Doenças , Hepatite B , Genética , Virologia , Vírus da Hepatite B , Fisiologia , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptor de Interferon alfa e beta , Genética , Metabolismo , Replicação Viral , GenéticaRESUMO
The type I interferon and IFNAR play an important role in hepatitis B virus (HBV) infection and anti-HBV therapy. However, its mechanism of action is still poorly understood. To gain more insights into the role of type I interferon and type I interferon receptor (IFNAR) in HBV infection, we established an HBV persistent replication IFNAR knockout (IFNAR(-/-)) mouse model and preliminarily applied this model. At first, the progeny of IFNAR(-/-) mouse was reproduced. Then hydrodynamic injection with pAAV/HBV1.2 plasmid was conducted to establish the persistent HBV replication IFNAR(-/-) mouse model. At last, we applied this model to evaluate the effect of nucleoside analogues entecavir (ETV) on HBV replication. It was found that there was no difference in the serum HBsAg and HBeAg levels and HBcAg expression in the liver tissue between the ETV treated groups and normal saline (NS) treated group, but the serum HBV DNA levels were significantly suppressed 10, 25, 40 and 55 days after the ETV treatment [P=0.035, P=0.00, P=0.149 and P=0.084, IFNAR knockout (KO) control group vs. C57BL/6 ETV groups, respectively; P=0.081, P=0.001, P=0.243 and P=0.147, IFNAR KO control group vs. IFNAR KO ETV groups, respectively]. Interestingly, there was no difference in serum HBV DNA levels between the ETV treated IFNAR(-/-) and C57BL/6 mice. This result suggests that HBV suppression during ETV treatments doesn't depend on type I interferon and IFNAR. Collectively, persistent HBV replication IFNAR(-/-) mouse model that we established is a useful and convenient tool to detect the function of the type I interferon and IFNAR in HBV infection and anti-HBV treatments.
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<p><b>OBJECTIVE</b>To investigate the possible influence of HBV and its antigens on the expressions of JAK-STAT signal transduction pathway molecules and the antiviral proteins of IFN alpha.</p><p><b>METHODS</b>The HepG2 cells were transfected with pSM2, pHBS2-S and pHBc-EGFP plasmids which express HBV whole particles or S-antigen, Pre-S antigen and core antigens. The infectious supernatant from HepG2.2.15 cells and the pured HBV proteins which contained the S, Pre-S antigens were used to treat the HepG2 cells. Northern blot and RT-PCR were applied to analyse the expressions of the antiviral proteins MxA, 2' -5' OAS, 9-27 and the JAK-STAT signal transduction pathway molecules STAT1 in HepG2 cells responded to the IFN alpha treatment.</p><p><b>RESULTS</b>The HepG2 cells transfected with pSM2, pHBS2-S and pHBc-EGFP plasmids could express whole HBV particles and HBsAg, Pre-S antigen and HBcAg. The quantitation of expressed HBV particles and antigens increased significantly during the course of transfection. Northern blot hybridization analysis indicated that the HepG2 cells expressed IFN alpha antiviral proteins MxA, 2' -5' OAS and 9-27. When transfected with pHBV-dimer, pHBS2-S, pHBc-EGFP plasmids, the IFN/A antiviral proteins MxA, 2' -5' OAS and 9-27 in transfected cells were reduced greatly as compared to the un-transfected HepG2 cells, and the expressed antiviral proteins decreased sharply with the development of transfection time. Furthermore, the expression of IFN alpha JAK-STAT signal transduction pathway molecule STAT1 was also inhibited with the expression of HBV particles and HBV antigens in transfected HepG2 cells.</p><p><b>CONCLUSIONS</b>The HBV and its antigens influence the expressions of IFN alpha JAK-STAT signal transduction pathway molecules and antiviral proteins in the hepatocellular models in vitro. It is indicated that HBV might possess the activity to antagonise or counteract the IFN alpha antiviral action.</p>
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Humanos , 2',5'-Oligoadenilato Sintetase , Metabolismo , Proteínas de Ligação ao GTP , Metabolismo , Células Hep G2 , Antígenos da Hepatite B , Alergia e Imunologia , Vírus da Hepatite B , Alergia e Imunologia , Interferon-alfa , Metabolismo , Proteínas de Resistência a Myxovirus , Proteínas de Ligação a RNA , Metabolismo , Fator de Transcrição STAT1 , Metabolismo , Transdução de Sinais , TransfecçãoRESUMO
<p><b>OBJECTIVE</b>To investigate the properties of HBsAb in occult hepatitis B virus infection and its affinity to different serotypes of hepatitis B virus surface antigen (HBsAg).</p><p><b>METHODS</b>Long-term follow-up was conducted in 2 HBsAb positive patients with occult hepatitis B virus infection. HBsAg was detected using multiple diagnostic kits and the HBsAb subtype was determined by performing neutralization experiments with different serotypes of HBsAg. The viral S gene was PCR-amplified and mutation analysis was conducted. Plasmids expressing HBsAgs were constructed by inserting these PCR products into an eukaryotic expression vector and were then transfected into HepG2 cells. The cell culture supernatant and cellular extracts were detected for HBsAg respectively. Neutralization experiments were carried out in the cell culture supernatant from HBsAg plasmids transfected HepG2 cells and serum samples from these patients and others who had been confirmed to be positive for HBsAb.</p><p><b>RESULTS</b>Multiple tests using various diagnostic kits showed that the 2 patients were negative for HBsAg and the three different serotypes of HBsAg (adr, adw, ay) could neutralize 82.1%-100% of HBsAb existed in the 2 patients. Sequence analysis of S gene cloned from these patients revealed that the homology to reference strain were 95.13%-97.79% and 92.04%-95.58% respectively at the nucleotide and amino acid levels. Quantitation of HBsAg showed that the expression levels of HBsAg from the two patients were 41.1% and 22.6% respectively of that of control HBsAg in cell culture supernatant and 48.1% and 59.3% respectively in cellular extract, and the supernatant/cell lysate ratios were 0.85 and 0.38 respectively. In neutralization experiments, HBsAg could be totally absorbed by control serum, whereas could only be partially neutralized by HBsAbs from the two patients (F = 353.6 and 645.2, P is less than 0.01).</p><p><b>CONCLUSION</b>Both the antigenicity and the ability of HBsAg secreted outside of the cells are decreased in these HBsAb-positive patients with occult HBV infection. The HBsAbs are mainly specific for common epitopes among different serotypes of HBsAg and are probably different as compared with those produced by vaccine inoculation.</p>
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Adulto , Humanos , Masculino , Pessoa de Meia-Idade , Hepatite B , Sangue , Virologia , Anticorpos Anti-Hepatite B , Sangue , Antígenos de Superfície da Hepatite B , Sangue , Vírus da Hepatite B , Testes SorológicosRESUMO
<p><b>OBJECTIVE</b>This report aims to investigate the Toll-like receptor (TLR) signaling pathways and induced antiviral activity in hepatocytes.</p><p><b>METHODS</b>We isolated primary hepatocytes from wild-type C57BL/6 mice and examined the expression of TLR by realtime RT-PCR. Hepatocytes were stimulated with TLR 1-9 agonists and the supernatants were harvested. The secretion of cytokines were tested by ELISA. The antiviral effectors in supernatants were assayed via virus protection assay (in EMCV system) and the control of HBV replication were assessed via Southern blotting (in HBV system).</p><p><b>RESULTS</b>We demonstrated that hepatocytes expressed TLR1-9. In accordance with these TLR expression profiles, hepatocytes responded to all TLR ligands by producing inflammatory cytokines (TNF-α or IL-6), to TLR -1,-3,-7 and -9 ligands by producing type I IFN (IFN-α or IFN-β). Only TLR 3 and TLR 7 agonists could stimulate the production of high amounts of antiviral mediators by hepatocytes in virus protection assay. By contrast, supernatants from TLR1, -3 and -4 directly stimulated hepatocytes and TLR 3, -7 and -9 transfected hepatocytes were able to potently suppress HBV replication.</p><p><b>CONCLUSION</b>Primary hepatocytes display a unique TLR signaling pathway and can control HBV replication after stimulation by TLR agonists in mice. It may be helpful for the development of TLR-based therapeutic approaches against hepatotropic virus.</p>
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Animais , Camundongos , Células Cultivadas , Vírus da Hepatite B , Alergia e Imunologia , Fisiologia , Hepatócitos , Alergia e Imunologia , Metabolismo , Imunidade Inata , Camundongos Endogâmicos C57BL , Transdução de Sinais , Receptores Toll-Like , Alergia e Imunologia , Metabolismo , Replicação ViralRESUMO
<p><b>OBJECTIVE</b>To establish the reference sequences of genotype B and C of hepatitis B virus in China.</p><p><b>METHODS</b>Genome sequences of Hepatitis B virus isolated from different area in China were retrieved from GenBank. These genome sequences were alignmented and the most common sequences were regarded as reference sequences. The amino acid sequences were also alignmented.</p><p><b>RESULTS</b>The homology was 99.32% between Bc genome and Ba genome, and it was 95.52% between Bc genome and Bj genome. The S gene sequence homology was 99.71% between Bc and Ba, and it was 98.68% between Bc and Bj. The homology was 98.44% between Cc genome and C genome, and it was 93.97% between Cc genome and Caus genome. The S gene sequence homology was 99.27% between Cc and C, and it was 95.01% between Cc and Caus. There was significant difference at the sites of 1762, 1764, 1858 between genotype B and genotype C (P < 0.05). The amino acid sequences were also different between genotype B and genotype C.</p><p><b>CONCLUSION</b>Bc and Cc sequences of Hepatitis B virus can be regarded as the reference sequences of genotype B and C.</p>
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Humanos , Sequência de Aminoácidos , Sequência de Bases , China , Primers do DNA , Variação Genética , Genoma Viral , Genótipo , Vírus da Hepatite B , Classificação , Genética , Hepatite B Crônica , Virologia , Dados de Sequência Molecular , Padrões de Referência , Alinhamento de Sequência , Homologia de SequênciaRESUMO
<p><b>OBJECTIVE</b>To establish a new method for rapidly selecting anti-hepatitis B virus drugs in clinical therapy.</p><p><b>METHODS</b>The full-length hepatitis B virus (HBV) genomes from 8 patients with chronic hepatitis B (CHB) were generated by polymerase chain reaction (PCR). All patients were resistant to lamivudine therapy. Their HBV DNA fragments were inserted into Sap I site of pHY106 eukaryotic expression vector separately. The recombinant plasmids containing 1.1 copies of HBV genome were transfected into Huh7 cell line; the levels of HBsAg, HBeAg and HBV DNA in supernatants of Huh7 cells were measured by ELISA and real-time quantitative PCR, and intracellular HBV replicative intermediates were detected by Southern blot. Antiviral effects of lamivudine and adefovir were evaluated in this vitro system.</p><p><b>RESULTS</b>The 8 recombinant plasmids containing a full-length genome of clinical HBV isolates could replicate and be expressed in Huh 7 cells. There were 6 isolates with polymerase YVDD mutations and 2 isolates with polymerase YIDD mutations. Adefovir, but not lamivudine, inhibited the HBV replication and gene expression in vitro. Furthermore, adefovir inhibited HBV replication in these CHB patients.</p><p><b>CONCLUSION</b>The method described here enables a rapid selection of anti-HBV drugs in clinical therapy and is very useful in antiviral therapy for CHB patients.</p>
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Adulto , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Adulto Jovem , Antivirais , Farmacologia , Avaliação Pré-Clínica de Medicamentos , Farmacorresistência Viral , Hepatite B , Virologia , Vírus da Hepatite B , Genética , VirossomosRESUMO
To study the effect of HCV core protein on the interferon-induced antiviral genes expression and its mechanisms. Methods HepG2 cells were transiently transfected with HCV core protein expression plasmid and the blank plasmid respectively. RT-PCR was used to analyze the effect of HCV core protein on PKR and 2'-5'OAS expression. The effect of HCV core protein on ISRE-medicated gene expression was detected by luciferase activity assay. Western-blot assay was performed to observe the change of mRNA and protein levels of SOCS3, STAT1 and p-STAT1 following HCV core expression. In the presence of HCV core protein, the transcription of PKR and 2'-5' OAS are down-regulated. ISRE-medicated reporter gene expression and STAT1 phosphorylation were inhibited. The transcription and expression of SOCS3 were induced compared with blank plasmid-transfected group. In HepG2 cells, HCV core protein can down-regulate the expression of some interferon-induced antiviral genes, which involves the induction of SOCS3 and the inhibition of STAT1 phosphorylation.
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Humanos , 2',5'-Oligoadenilato Sintetase , Genética , Metabolismo , Carcinoma Hepatocelular , Patologia , Regulação para Baixo , Hepacivirus , Genética , Metabolismo , Fator Gênico 3 Estimulado por Interferon , Genética , Metabolismo , Interferon-alfa , Genética , Alergia e Imunologia , Neoplasias Hepáticas , Patologia , Proteínas Quinases , Genética , Metabolismo , Fator de Transcrição STAT1 , Genética , Metabolismo , Fator de Transcrição STAT2 , Genética , Metabolismo , Transcrição Gênica , Transfecção , Células Tumorais Cultivadas , Proteínas do Core Viral , Genética , Metabolismo , FisiologiaRESUMO
<p><b>OBJECTIVE</b>To search for and verify some common B cell epitopes in the core proteins of woodchuck hepatitis virus and human hepatitis B virus.</p><p><b>METHODS</b>Monoclonal antibodies against both core proteins of woodchuck hepatitis virus (WHV) and human hepatitis B virus (HBV) were prepared by inoculating Balb/c mice with denatured recombination WHV and HBV core proteins. ELISA and immunoblotting assays for WHcAg and HBcAg were carried out by using these antibodies. Immunohistochemistry was carried out with liver tissue sections of both WHV-infected woodchucks and chronic HBV-infected patients. The epitopes were mapped with the mouse mAbs (6D1 and 1H4) by using a panel of 24 16mer overlapping peptides covering the entire WHcAg. The amino acid sequences of WHcAg and HBcAg were compared.</p><p><b>RESULTS</b>Cross-reactions were observed between mAbs (6D1 and 1H4) and WHcAg and between Mabs and HBcAg/HBcAg in ELISA and immunoblotting assay. Liver tissue sections of both WHV-infected woodchucks and chronic HBV-infected patients could be stained specifically by mAbs. The epitopes were mapped at aa1-8 (6D1) and aa125-140 (1H4) of the core proteins of both WHV and HBV by using ELISA assay. WHcAg and HBcAg share similar amino acids sequences at aa1-8 and aa125-140 respectively.</p><p><b>CONCLUSION</b>The core proteins of woodchuck hepatitis virus and human hepatitis B virus share common linear B cell epitopes which span aa1-8 and aa125-140 respectively.</p>
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Animais , Humanos , Camundongos , Anticorpos Monoclonais , Linfócitos B , Alergia e Imunologia , Linhagem Celular Tumoral , Reações Cruzadas , Epitopos de Linfócito B , Alergia e Imunologia , Antígenos do Núcleo do Vírus da Hepatite B , Alergia e Imunologia , Vírus da Hepatite B da Marmota , Genética , Alergia e Imunologia , Vírus da Hepatite B , Genética , Alergia e Imunologia , Marmota , Proteínas do Core Viral , Alergia e ImunologiaRESUMO
<p><b>OBJECTIVE</b>To study whether the substitutions at the major hydrophilic region II (MHRII) of hepatitis B surface antigen (HBsAg) will impair the antigenicity of HBsAg.</p><p><b>METHODS</b>Four recombinant plasmids expressing mutant HBsAg (mtHBsAg) P1120T, C121S, K122I and T123N were constructed. HepG2 cells were transfected with the four plasmids and a plasmid expressing G145R HBsAg. The immunoreactivity of the cells expressing mtHBsAg with P1120T, C121S, K122I, T123N and G145R were detected by immunofluorescence (IF) staining and ELISA with 4 antibodies and 7 HBsAg diagnostic kits respectively.</p><p><b>RESULTS</b>mtHBsAg with P120T was recognized by mAb1 and mAb2. mtHBsAg with C121S and K122I was not recognized by any mAbs. mtHBsAg with T123N in lysates was recognized by mAb2, but not recognized in the supernatants.</p><p><b>CONCLUSION</b>Substitutions at amino acid positions 120-123 of HBsAg strongly impaired the antigenicity of HBsAg, a fact that was not appreciated previously.</p>
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Humanos , Substituição de Aminoácidos , Variação Antigênica , Células Hep G2 , Antígenos de Superfície da Hepatite B , Genética , Alergia e Imunologia , Vírus da Hepatite B , Genética , Alergia e Imunologia , Mutação , Plasmídeos , TransfecçãoRESUMO
Guanylate binding protein-1(GBP-1) is an interferon-induced protein. To observe its antiviral effect against Hepatitis B virus (HBV) and Coxsackie virus B3 (CVB3), we constructed an eukaryotic expression vector of human GBP-1(hGBP-1). Full-length encoding sequence of hGBP-1 was amplified by long chain RT-PCR and inserted into a pCR2.1 vector, then subcloned into a pCDNA3.1(-) vector. Recombinant hGBP-1 plasmids and pHBV1.3 carrying 1.3-fold genome of HBV were contransfected into HepG2 cells, and inhibition effect of hGBP-1 against HBV replication was observed. Hela cells transfected with recombinant hGBP-1 plasmids were challenged with CVB3, and viral yield in cultures were detected. The results indicated that recombinant eukaryotic expression plasmid of hGBP-1 was constructed successfully and the hGBP-1 gene carried in this plasmid could be efficiently expressed in HepG2 cells and Hela cells. hGBP-1 inhibit CVB3 but not HBV replication in vitro. These results demonstrate that hGBP-1 mediates an antiviral effect against CVB3 but not HBV and perhaps plays an important role in the interferon-mediated antiviral response against CVB3.
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To establish a replication cellular model of hepatitis B virus (HBV) and determine its application in antiviral drug evaluation,we constructed an expression plasmid which contained 1.3 copies of the HBV genome,and measured the level of viral replication after transient transfection in Huh7 cells.We then observed the effect of antiviral drug administration.1.3 fold of the HBV(ayw) gene fragment was cloned into pCR2.1 by PCR and restriction endonuclease digestion.The recombinant plasmid was trans ient transfected into Huh7 cells,HBsAg,HBeAg and HBV DNA in supernatant of Huh7 cells were measured by ELISA and real-time PCR respectively; intracellular HBV replicative intermediates and intracellular HBV transcripts were detected by Southern blot and Northern blot respectively.The antiviral effect of adefovir,a novel anti-HBV nucleotide analogue,was evaluated in this cellular model system.The results indicated that a recombinant plasmid of HBV replicon was constructed successfully; the HBV genome carried in plasmid pHBV1.3 could efficiently replicate and be expressed in Huh 7 cells,adefovir could inhibit HBV replication in this cellular model,and the inhibition was dosage-dependent.The conclusion is HBV replicon,which can initiate viral replication efficiently in hepatoma cells,may be a useful tool in the study of HBV replication and antiviral drug.
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Emerging data indicated that HCV subverts the antiviral activity of interferon (IF); however,whether HCV core protein contributes to the process remains controversial. In the present study, we examined the effect of HCV core protein on interferon-induced antiviral gene expression and whether the effect is involved in the activation and negative regulation of the Jak/STAT signaling pathway. Our results showed that, following treatment with IFN-α, the transcription of PKR, MxA and 2'-5'OAS were down-regulated in HepG2 cells expressing the core protein. In the presence of HCV core protein,ISRE-dependent luciferase activity also decreased. Further study indicated that the core protein could inhibit the tyrosine phosphorylation of STAT1, whereas the level of STAT1 expression was unchanged.Accordingly, SOCS3, the negative regulator of the Jak/STAT pathway, was induced by HCV core protein. These results suggests that HCV core protein may interfere with the expression of some interferon-induced antiviral genes by inhibiting STAT1 phosphorylation and induction of SOCS3.
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<p><b>OBJECTIVE</b>To investigate the function of interferon alpha (IFNalpha) in a Chinese marmot model of hepatitis B, we expressed the Chinese marmot (Marmota himalayana) IFNalpha family gene (IFNA) in eukaryotic cells and prokaryotic cells.</p><p><b>METHODS</b>Eukaryotic and prokaryotic expression plasmids harboring Chinese marmot interferon alpha gene with different genotypes were generated using molecular cloning technology. We detected the biological activity of all expression products by viral protection assay, and analyzed their differences and species restriction of the biological activity.</p><p><b>RESULTS</b>The Chinese marmot functional genotype IFNalpha was expressed in the baby hamster kidney (BHK) cell line, and these products protected WH12/6 cells challenged by encephalomyocarditis virus (EMCV). The Chinese marmot IFN-alpha5 also expressed in E. Coli induced by IPTG, and purified fusion protein had antiviral biological activity. The biologic activity displayed differences among different subtype IFNalpha, and it had strict species restriction.</p><p><b>CONCLUSION</b>The IFNalpha family gene of the Chinese marmot can be expressed in both eukaryotic and prokaryotic cells, and the expression products show antiviral activity in a protection assay. This study provides, for the first time, evidence that IFNalpha from the Chinese marmot has an antiviral function in vitro and can be used to improve the efficacy of current therapies for HBV infection in our Chinese marmot model.</p>
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Animais , Células Eucarióticas , Metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Hepatite B , Metabolismo , Interferon-alfa , Genética , Fisiologia , Marmota , Metabolismo , Células Procarióticas , Metabolismo , Transdução de Sinais , Fatores de TranscriçãoRESUMO
<p><b>OBJECTIVE</b>To investigate the expression profile of genes which are involved in IFN antiviral activity and IFN signal transduction pathway in Hep G2 and HepG2.2.15 cells.</p><p><b>METHODS</b>Genes of interest were selected from the UniGene database (http://www.ncbi.nlm.gov/UniGene/Hs.Home.html). The 5'IMAGE clones with 0.5-0.8 kb length were chosen and ordered from RZPD company. The cDNA inserts were amplified by PCR and then were spotted onto the Hybond-N+ membranes. The membranes were denatured and neutralized for Macroarray analysis. HepG2.2.15 and Hep G2 cells were treated without or with IFN-alpha for 6 h, and the total cellular RNA was isolated using Trizol Reagent. Radio-labelled cDNA was generated from 20 microgram of RNA by reverse transcription using 360 units of reverse transcriptase in the presence of 30 microCi of alpha-32P dCTP. Hybridization was performed between 32P-labelled cDNA and membrane arrays. The membranes were then scanned, and the intensity of autoradiographic spots was quantitated by Cyclone Storage Phosphor System. The images were subsequently analysed by the OptiQuant Imager Analysis Software and converted into digital data.</p><p><b>RESULTS</b>The authors found that just partially IFN-inducible genes were expressed in Hep G2 and HepG2.2.15 cells, and the majority of IFN-inducible genes was lowly responsive or non-responsive to IFN-a treatment. Some interferon-stimulated genes (ISGs) were inhibited or blocked, especially in HepG2.2.15 cells. Interestingly, the authors found that the IFN signal transduction pathway (Jak-STAT) was intact and unimpaired in HepG2.2.15 cells.</p><p><b>CONCLUSION</b>Differential gene expression profiles in response to IFN were found between Hep G2 and HepG2.2.15 cells.</p>