AIM To investigate the functional role and underlying molecular mechanism of mi R-29 a in hepatitis B virus(HBV) expression and replication.METHODS The levels of mi R-29 a and SMARCE1 in HBV-infected Hep G2.2.15 cells...AIM To investigate the functional role and underlying molecular mechanism of mi R-29 a in hepatitis B virus(HBV) expression and replication.METHODS The levels of mi R-29 a and SMARCE1 in HBV-infected Hep G2.2.15 cells were measured by quantitative real-time PCR and western blot analysis. HBV DNA replication was measured by quantitative PCR and Southern blot analysis. The relative levels of hepatitis B surface antigen and hepatitis B e antigen were detected by enzyme-linked immunosorbent assay. The Cell Counting Kit-8(CCK-8) was used to detect the viability of Hep G2.2.15 cells. The relationship between mi R-29 a and SMARCE1 were identified by target prediction and luciferase reporter analysis.RESULTS mi R-29 a promoted HBV replication and expression, w h i le S MA R C E 1 r e p r e s s e d H B V r e p lic a t io n a n d expression. Cell viability detection indicated that mi R-29 a transfection had no adverse effect on the host cells. Moreover, SMARCE1 was identified and validated to be a functional target of mi R-29 a. Furthermore, restored expression of SMARCE1 could relieve the increased HBV replication and expression caused by mi R-29 a overexpression.CONCLUSION mi R-29 a promotes HBV replication and expression through regulating SMARCE1. As a potential regulator of HBV replication and expression, mi R-29 a could be a promising therapeutic target for patients with HBV infection.展开更多
Chronic infection with the hepatitis B virus(HBV) is the leading risk factor for the development of hepatocellular carcinoma(HCC). With nearly 750000 deaths yearly, hepatocellular carcinoma is the second highest cause...Chronic infection with the hepatitis B virus(HBV) is the leading risk factor for the development of hepatocellular carcinoma(HCC). With nearly 750000 deaths yearly, hepatocellular carcinoma is the second highest cause of cancer-related death in the world. Unfortunately, the molecular mechanisms that contribute to the development of HBV-associated HCC remain incompletely understood. Recently, micro RNAs(mi RNAs), a family of small non-coding RNAs that play a role primarily in post-transcriptional gene regulation, have been recognized as important regulators of cellular homeostasis, and altered regulation of mi RNA expression has been suggested to play a significant role in virus-associated diseases and the development of many cancers. With this in mind, many groups have begun to investigate the relationship between mi RNAs and HBV replication and HBV-associated disease. Multiple findings suggest that some mi RNAs, such as mi R-122, and mi R-125 and mi R-199 family members, are playing a role in HBV replication and HBV-associated disease, including the development of HBV-associated HCC. In this review, we discuss the current state of our understanding of the relationship between HBV and mi RNAs, including how HBV affects cellular mi RNAs, how these mi RNAs impact HBV replication, and the relationship between HBV-mediated mi RNA regulation and HCC development. We also address the impact of challenges in studying HBV, such as the lack of an effective model system for infectivity and a reliance on transformed cell lines, on our understanding of the relationship between HBV and mi RNAs, and proposepotential applications of mi RNA-related techniques that could enhance our understanding of the role mi RNAs play in HBV replication and HBV-associated disease, ultimately leading to new therapeutic options and improved patient outcomes.展开更多
Hepatitis B virus(HBV) infection is one of the major causes of liver diseases, affecting more than 350 million people worldwide. The interferon(IFN)-mediated innate immune responses could restrict HBV replication at t...Hepatitis B virus(HBV) infection is one of the major causes of liver diseases, affecting more than 350 million people worldwide. The interferon(IFN)-mediated innate immune responses could restrict HBV replication at the different steps of viral life cycle. Indeed, IFN-α has been successfully used for treatment of patients with chronic hepatitis B. However, the role of the innate immune response in HBV replication and the mechanism of the anti-HBV effect of IFN-α are not completely explored. In this review, we summarized the currently available knowledge about the IFN-mediated anti-HBV effect in the HBV life cycle and the possible effectors downstream the IFN signaling pathway. The antiviral effect of Toll-like receptors(TLRs) in HBV replication is briefly discussed. The strategies exploited by HBV to evade the IFN- and TLR-mediated antiviral actions are summarized.展开更多
Plus-strand RNA virus replication occurs in tight association with cytoplasmic host cell membranes. Both, viral and cellular factors cooperatively generate distinct organelle-like structures, designated viral replicat...Plus-strand RNA virus replication occurs in tight association with cytoplasmic host cell membranes. Both, viral and cellular factors cooperatively generate distinct organelle-like structures, designated viral replication factories. This compartmentalization allows coordination of the different steps of the viral replication cycle, highly efficient genome replication and protection of the viral RNA from cellular defense mechanisms. Electron tomography studies conducted during the last couple of years revealed the three dimensional structure of numerous plus-strand RNA virus replication compartments and highlight morphological analogies between different virus families. Based on the morphology of virusinduced membrane rearrangements, we propose two separate subclasses: the invaginated vesicle/spherule type and the double membrane vesicle type. This review discusses common themes and distinct differences in the architecture of plus-strand RNA virus-induced membrane alterations and summarizes recent progress that has been made in understanding the complex interplay between viral and co-opted cellular factors in biogenesis and maintenance of plus-strand RNA virus replication factories.展开更多
BACKGROUND: Hepatitis B related end-stage liver disease is recently acknowledged as one of the main indications for orthotopic liver transplantation (OLT). However, the high recurrence rate of hepatitis B virus infect...BACKGROUND: Hepatitis B related end-stage liver disease is recently acknowledged as one of the main indications for orthotopic liver transplantation (OLT). However, the high recurrence rate of hepatitis B virus infection following transplantation is regarded as a major factor affecting the long-term survival of transplant recipients especially in Chi- na. Cyclosporine A (CsA), which is routinely used to pre- vent the allograft rejection, is reported to have the inhibito- ry activity on hepatitis B virus (HBV) replication in vitro. In this paper, we review the inhibitory effect and its possi- ble mechanisms of CsA on HBV replication in vitro. DATA RESOURCES: An English-language literature search was conducted using MEDLINE (1990-2004) on cyclospo- rine A, hepatitis B virus, mitochondria, calcium and other related reports and review articles. RESULTS: Hepatitis B x protein (HBx) is essential to HBV replication. The cytosolic calcium signaling mediated by mitochondria and the Src kinase pathway were involved during HBx activation of HBV replication. CsA inhibits the HBV replication in vitro by its binding to mitochondrial cy- clophilin D, then blocking the mitochondria-mediated cy- tosolic calcium signaling. The derivates of CsA also have the HBV replication inhibitory effect in vitro. CONCLUSIONS; By interacting with mitochondria, pre- venting the release of intramitochondrial calcium, and then blocking the cytosolic calcium signaling, CsA inhibits the HBV replication in vitro. The derivates of CsA also have this activity.展开更多
AIM: To examine the effect of hepatitis C virus (HCV) structural mimics of regulatory regions of the genome on HCV replication.METHODS: HCV RNA structural mimics were constructed and tested in a HCV genotype 1b aBB7 r...AIM: To examine the effect of hepatitis C virus (HCV) structural mimics of regulatory regions of the genome on HCV replication.METHODS: HCV RNA structural mimics were constructed and tested in a HCV genotype 1b aBB7 replicon,and a Japanese fulminant hepatitis-1 (JFH-1) HCV genotype 2a infection model.All sequences were computer-predicted to adopt stem-loop structures identical to the corresponding elements in full-length viral RNA.Huh7.5 cells bearing the BB7 replicon or infected with JFH-1 virus were transfected with expression vectors generating HCV mimics and controls.Cellular HCV RNA and protein levels were quantified by real-time polymerase chain reaction and Western blotting,respectively.To evaluate possible antisense effects,complementary RNAs spanning a mimic were prepared.RESULTS: In the BB7 genotype 1b replicon system,mimics of the polymerase (NS-5B),X and BA regions inhibited replication by more than 90%,50%,and 60%,respectively.In the JFH-1 genotype 2 infection system,mimics that were only 74% and 46% identical in sequence relative to the corresponding region in JFH-1 inhibited HCV replication by 91.5% and 91.2%,respectively,as effectively as a mimic with complete identity to HCV genotype 2a.The inhibitory effects were confirmed by NS3 protein levels.Antisense RNA molecules spanning the 74% identical mimic had no significant effects.CONCLUSION: HCV RNA structural mimics can inhibit HCV RNA replication in replicon and infectious HCV systems and do so independent of close sequence identity with the target.展开更多
Hepatitis C virus (HCV) is one of the main etiological factors responsible for liver disease worldwide. It has been estimated that there are over 170 million people infected with HCV worldwide. Of these infected indiv...Hepatitis C virus (HCV) is one of the main etiological factors responsible for liver disease worldwide. It has been estimated that there are over 170 million people infected with HCV worldwide. Of these infected individuals, approximately 75% will go on to develop a life long necroinflammatory liver disease, which over decades, can result in serious complications, such as cirrhosis and hepatocellular carcinoma. Currently there is no effective vaccine and whilst antiviral therapies have been improved, they are still only effective in approximately 50% of individuals. HCV infection stands as a major cause of global morbidity and suffering, and places a signifi cant burden on health systems. The second highest cause of liver disease in the western world is alcoholic liver disease. Frequently, HCV infected individuals consume alcohol, and the combined effect of HCV and alcohol consumption is deleterious for both liver disease and response to treatment. This review discusses the impact of alcohol metabolism on HCV replication and the negative impact on interferon (IFN)-α treatment, with a particular focus on how alcohol and HCV act synergistically to increase oxidative stress, ultimately leading to exacerbated liver disease and a reduction in the effi cacy of IFN-α treatment. A better understanding of the complicated mechanisms at play in hepatocytes infected with HCV and metabo- lizing alcohol will hopefully provide better treatment options for chronic hepatitis C individuals that consume alcohol.展开更多
Objectives: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression...Objectives: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. Methods: (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. Results: Our data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The ex- pression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. Conclusion: Our results demonstrated that siRNAs exerted robust and specific inhibi- tion on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.展开更多
AIM: To investigate the effects of suberoylanilide hydroxamic acid(SAHA) on proliferation and apoptosis of a human hepatocellular carcinoma cell line(HepG2.2.15) and hepatitis B virus(HBV) replication.METHODS: HepG2.2...AIM: To investigate the effects of suberoylanilide hydroxamic acid(SAHA) on proliferation and apoptosis of a human hepatocellular carcinoma cell line(HepG2.2.15) and hepatitis B virus(HBV) replication.METHODS: HepG2.2.15 cells were treated with different concentrations of SAHA.Cell morphology was examined by confocal laser scanning microscopy,and cell proliferation was determined using a MTT colorimetric assay.Flow cytometry was used to detect apoptosis and determine cell cycle phase,while hepatitis B surface antigen and hepatitis B e antigen content were measured using chemiluminescence.Reverse transcription polymerase chain reaction was performed to measure HBV DNA in cell lysate.RESULTS: Cell proliferation rates were significantly reduced by the addition of SAHA.The inhibitory effect of SAHA on cell proliferation was both time-and dosedependent.After 24 h of treatment with SAHA,the early cell apoptotic rate increased from 3.25% to 21.02%(P = 0.041).The proportion of G0 /G1 phase cells increased from 50.3% to 65.3%(P = 0.039),while that of S phase cells decreased from 34.9% to 20.6%(P = 0.049).After 48 h of treatment,hepatitis B surface antigen and hepatitis B e antigen content increased from 12.33 ± 0.62 to 25.42 ± 2.67(P = 0.020) and 28.92 ± 1.24 to 50.48 ± 1.85(P = 0.026),respectively.Furthermore,HBV DNA content increased from 4.54 ± 0.46 to 8.34 ± 0.59(P = 0.029).CONCLUSION: SAHA inhibits HepG2.2.15 cell proliferation,promotes apoptosis,and stimulates HBV replication.In combination with anti-HBV drugs,SAHA may potentially be used cautiously for treatment of hepatocellular carcinoma.展开更多
AIM:To evaluate the value of the hepatitis B virus(HBV) replication mouse model with regard to several aspects of the study of HBV biology.METHODS:To evaluate the HBV replication mouse model in detecting the efficacy ...AIM:To evaluate the value of the hepatitis B virus(HBV) replication mouse model with regard to several aspects of the study of HBV biology.METHODS:To evaluate the HBV replication mouse model in detecting the efficacy of anti-HBV agents,the interferon inducer polyinosinic-polytidylin acid(polyIC) and nucleotide analogues adefovir and entecavir were administered to mice injected with wild type pHBV4.1,and the inhibiting effect of these agents on HBV DNA replication was evaluated.To identify the model's value in a replication ability study of HBV drug-resistant mutants and a HBx-minus mutant,telbivudine resistance mutants(rtM204I,ayw subtype),adefovir resistance mutants(rtA181V + rtN236T,ayw subtype) and HBxminus mutants were injected respectively,and their corresponding HBV DNA replication intermediates in mouse liver were assessed.RESULTS:Compared with the wild type HBV replication mouse model without antiviral agent treatment,the HBV DNA replication intermediates of the polyICtreated group were decreased 1-fold;while in the entecavir-and adefovir-treated groups,the levels of HBV DNA replication intermediates were inhibited 13.6-fold and 1.4-fold,respectively.For the mouse models injected with telbivudine resistance mutant,adefovir resistance mutant and HBx-minus mutant,HBV DNA replication intermediates could still be detected,but the levels of HBV DNA replication intermediates of these mutants decreased 4.5-fold,5.6-fold and 2.9-fold respectively,compared with the mouse model with wild type HBV plasmid.CONCLUSION:The HBV replication mouse model we established was a useful and convenient tool to detect the efficacy of antiviral agents and to study the replication ability of HBV mutants in vivo.展开更多
Isatis indigotica Fort.(Ban-Lan-Gen)is an herbal medicine prescribed for influenza treatment.However,its active components and mode of action remain mostly unknown.In the present study,erucic acid was isolated from Is...Isatis indigotica Fort.(Ban-Lan-Gen)is an herbal medicine prescribed for influenza treatment.However,its active components and mode of action remain mostly unknown.In the present study,erucic acid was isolated from Isatis indigotica Fort.,and subsequently its underlying mechanism against influenza A virus(IAV)infection was investigated in vitro and in vivo.Our results demonstrated that erucic acid exhibited broad-spectrum antiviral activity against IAV resulting from reduction of viral polymerase transcription activity.Erucic acid was found to exert inhibitory effects on IAV or viral(v)RNA-induced pro-inflam-matory mediators as well as interferons(IFNs).The molecular mechanism by which erucic acid with antiviral and anti-inflammatory properties was attributed to inactivation of NF-kB and p38 MAPK signaling.Furthermore,the NF-kB and p38 MAPK inhibitory effect of erucic acid led to diminishing the transcriptional activity of interferon-stimulated gene factor 3(ISGF-3),and thereby reducing IAV-triggered pro-inflammatory response amplification in IFN-β-sensitized cells.Additionally,IAV-or vRNA-triggered apoptosis of alveolar epithelial A549 cells was prevented by erucic acid.In vivo,erucic acid administration consistently displayed decreased lung viral load and viral antigens expression.Meanwhile,erucic acid markedly reduced CD8+cytotoxic T lymphocyte(CTL)recruitment,pro-apoptotic signaling,hyperactivity of multiple signaling pathways,and exacerbated immune inflammation in the lung,which resulted in decreased lung injury and mortality in mice with a mouse-adapted A/FM/1/47-MA(H1N1)strain infection.Our findings provided a mechanistic basis for the action of erucic acid against IAV-mediated inflammation and injury,suggesting that erucic acid may have a therapeutic potential in the treatment of influenza.展开更多
AIM To investigate the effect of(-)-epigallocatechin-3-gallate(EGCG) on polyinosinic-polycytidylic acid(poly I:C)-triggered intracellular innate immunity against hepatitis C virus(HCV) in hepatocytes. METHODS A cell c...AIM To investigate the effect of(-)-epigallocatechin-3-gallate(EGCG) on polyinosinic-polycytidylic acid(poly I:C)-triggered intracellular innate immunity against hepatitis C virus(HCV) in hepatocytes. METHODS A cell culture model of HCV infection was generated by infecting a hepatoma cell line, Huh7, with HCV JFH-1 strain(JFH-1-Huh7). Poly I:C with a high molecular weight and EGCG were used to stimulate the JFH-1-Huh7 cells. Real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of intracellular m RNAs and of intracellular and extracellular HCV RNA. Enzyme-linked immunosorbent assay was used to evaluate the interferon(IFN)-λ1 protein level in the cell culture supernatant. Immunostaining was used to examine HCV core protein expression in Huh7 cells.RESULTS Our recent study showed that HCV replication could impair poly I:C-triggered intracellular innate immune responses in hepatocytes. In the current study, we showed that EGCG treatment significantly increased the poly I:C-induced expression of Toll-like receptor 3(TLR3), retinoic acid-inducible gene I, and IFN-λ1 in JFH-1-Huh7 cells. In addition, supplementation with EGCG increased the poly I:C-mediated antiviral activity in JFH-1-Huh7 cells at the intracellular and extracellular HCV RNA and protein levels. Further investigation of the mechanisms showed that EGCG treatment significantly enhanced the poly I:C-induced expression of IFN-regulatory factor 9 and several antiviral IFNstimulated genes, including ISG15, ISG56, myxovirus resistance A, and 2'-5'-oligoadenylate synthetase 1, which encode the key antiviral elements in the IFN signaling pathway. CONCLUSION Our observations provide experimental evidence that EGCG has the ability to enhance poly I:C-induced intracellular antiviral innate immunity against HCV replication in hepatocytes.展开更多
Objective To develop an effective and specific medicine targeting hepatitis B virus(HBV) pregenome. Based on the identified accessible target sites for hammerhead ribozyme in our previous researches, a recombinant hep...Objective To develop an effective and specific medicine targeting hepatitis B virus(HBV) pregenome. Based on the identified accessible target sites for hammerhead ribozyme in our previous researches, a recombinant hepatitis delta virus(HDV) ribozyme was chosen and used to demonstrate the effective cleavage in vitro and in vivo. Methods Three hammerhead ribozymes for potential target sites(S, X and C genes) and co-expression plasmid(pTr-dB, pTdδ-dB, pTrX-dB and pTrC-dB) as well as four HDV-ribozyme chimera constructs with HBV(pTdXX, pTdXC, pTdSX and pTdSC) were severally chosen to validate the inhibition of the replication and expression of HBV. The co-expression plasmids(pTdδ and pTr-Db) in physiological saline were hydrodynamically injected to mice by tail vein. Results Compared with the group injected with pTr-dB in Huh-7 cell, hepatitis B surface antigen(HBsAg) was reduced by 31% in the group injected with pTdδ-dB, by 54%, 26%, 72% and 97% in the group injected with recombinant-ribozymes pTdSX, pTdSC, pTdXC and pTdXX, respectively. The inhibiting effects of endogenous ribozymes RzX and RzC on the HBsAg expression were 66% and 57%, respectively. Compared with the positive control, the amount of HBsAg was decreased in mice injected with pTdXX through tail vein by 88% and 96% on the second day and the third day, respectively. HBsAg was undetectable on the 6th day and could not primitively be detected on the 9th day in the sera from all mice. HBV DNA was not detected in the sera of BALB/c mice injected with pTdXX-dB, pTrX-dB or replicating-defective plasmid pHBV, while HBV DNA replication in control group could be detected on the 6th day. While HBcAg could not be detected in liver tissues of mice injected with plasmid pTdXX-dB on the 3rd day. Conclusions Encoding regions of HBV S, C and X gene were the effective cleavage sites for hammerhead ribozyme in vitro and in vivo, which provides basis for further construction of therapeutic recombinant HDV and the development of targeting antiviral gene therapy.展开更多
BACKGROUND Previously,we have successfully constructed replication-competent hepatitis B virus(HBV)vectors by uncoupling the P open reading frame(ORF)from the preC/C ORF to carefully design the transgene insertion sit...BACKGROUND Previously,we have successfully constructed replication-competent hepatitis B virus(HBV)vectors by uncoupling the P open reading frame(ORF)from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence.Consequently,the replication-competent HBV vectors carrying foreign genes,including pCH-BsdR,carrying blasticidin resistance gene(399 bp),and pCH-hrGFP,carrying humanized renilla green fluorescent protein gene(720 bp),were successfully obtained.However,the replication efficiency of the former is higher but it is tedious to use,while that of the latter is poor and cannot be quantified.Hence,we need to search for a new reporter gene that is convenient and quantifiable for further research.AIM To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies.METHODS We utilized the replication-competent HBV viral vectors constructed by our laboratory,combined with the secreted luciferase reporter gene,to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase(SecNluc).HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene.RESULTS The replication-competent HBV vector carrying the SecNluc reporter gene pCHsNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression.HBV replication intermediates could be produced from this vector.Via transfection with pTRE-sNLuc and selection by hygromycin,we obtained isolated cell clones,named HBV-NLuc-35 cells,which could secrete secNLuc recombinant viruses,and were sensitive to existing anti-HBV drugs.Using differentiated HepaRG cells,it was verified that recombinant HBV possessed infectivity.CONCLUSION Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability,and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene.More importantly,the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.展开更多
AIM: To address the effect of heat-shock protein 90(HSP90) inhibitors on the release of the hepatitis C virus(HCV), a cell culture-derived HCV(JFH1/HCVcc) from Huh-7 cells was examined.METHODS: We quantified both the ...AIM: To address the effect of heat-shock protein 90(HSP90) inhibitors on the release of the hepatitis C virus(HCV), a cell culture-derived HCV(JFH1/HCVcc) from Huh-7 cells was examined.METHODS: We quantified both the intracellular and extracellular(culture medium) levels of the components(RNA and core) of JFH-1/HCVcc. The intracellular HCV RNA and core levels were determined after the JFH1/HCVcc-infected Huh-7 cells were treated with radicicol for 36 h. The extracellular HCV RNA and core protein levels were determined from the medium of the last 24 h of radicicol treatment. To determine the possible role of the HSP90 inhibitor in HCV release, we examined the effect of a combined application of low doses of the HSP90 inhibitor radicicol and the RNA replication inhibitors cyclosporin A(Cs A) or interferon. Finally, we statistically examined the combined effect of radicicoland Cs A using the combination index(CI) and graphical representation proposed by Chou and Talalay.RESULTS: We found that the HSP90 inhibitors had greater inhibitory effects on the HCV RNA and core protein levels measured in the medium than inside the cells. This inhibitory effect was observed in the presence of a low level of a known RNA replication inhibitor(Cs A or interferon-α). Treating the cells with a combination of radicicol and cyclosporin A for 24 h resulted in significant synergy(CI < 1) that affected the release of both the viral RNA and the core protein. CONCLUSION: In addition to having an inhibitory effect on RNA replication, HSP90 inhibitors may interfere with an HCV replication step that occurs after the synthesis of viral RNA, such as assembly and release.展开更多
To the Editor:I have previously reported a type II diabetic patient complicated with chronic hepatitis C virus(HCV)infection.1]Recently,Riva et al[2]reported an association of truncated CXCL10 with failure to achieve ...To the Editor:I have previously reported a type II diabetic patient complicated with chronic hepatitis C virus(HCV)infection.1]Recently,Riva et al[2]reported an association of truncated CXCL10 with failure to achieve spontaneous clearance of acute HCV infection.They showed展开更多
文摘AIM To investigate the functional role and underlying molecular mechanism of mi R-29 a in hepatitis B virus(HBV) expression and replication.METHODS The levels of mi R-29 a and SMARCE1 in HBV-infected Hep G2.2.15 cells were measured by quantitative real-time PCR and western blot analysis. HBV DNA replication was measured by quantitative PCR and Southern blot analysis. The relative levels of hepatitis B surface antigen and hepatitis B e antigen were detected by enzyme-linked immunosorbent assay. The Cell Counting Kit-8(CCK-8) was used to detect the viability of Hep G2.2.15 cells. The relationship between mi R-29 a and SMARCE1 were identified by target prediction and luciferase reporter analysis.RESULTS mi R-29 a promoted HBV replication and expression, w h i le S MA R C E 1 r e p r e s s e d H B V r e p lic a t io n a n d expression. Cell viability detection indicated that mi R-29 a transfection had no adverse effect on the host cells. Moreover, SMARCE1 was identified and validated to be a functional target of mi R-29 a. Furthermore, restored expression of SMARCE1 could relieve the increased HBV replication and expression caused by mi R-29 a overexpression.CONCLUSION mi R-29 a promotes HBV replication and expression through regulating SMARCE1. As a potential regulator of HBV replication and expression, mi R-29 a could be a promising therapeutic target for patients with HBV infection.
基金Supported by Pennsylvania state CURE grant,No.4100057658,[to Steel LF and Bouchard MJ(partially)]a Ruth L Kirschstein(F31)Predoctoral Fellowship,No.5F31CA171712-03,[to Lamontagne J(partially)]
文摘Chronic infection with the hepatitis B virus(HBV) is the leading risk factor for the development of hepatocellular carcinoma(HCC). With nearly 750000 deaths yearly, hepatocellular carcinoma is the second highest cause of cancer-related death in the world. Unfortunately, the molecular mechanisms that contribute to the development of HBV-associated HCC remain incompletely understood. Recently, micro RNAs(mi RNAs), a family of small non-coding RNAs that play a role primarily in post-transcriptional gene regulation, have been recognized as important regulators of cellular homeostasis, and altered regulation of mi RNA expression has been suggested to play a significant role in virus-associated diseases and the development of many cancers. With this in mind, many groups have begun to investigate the relationship between mi RNAs and HBV replication and HBV-associated disease. Multiple findings suggest that some mi RNAs, such as mi R-122, and mi R-125 and mi R-199 family members, are playing a role in HBV replication and HBV-associated disease, including the development of HBV-associated HCC. In this review, we discuss the current state of our understanding of the relationship between HBV and mi RNAs, including how HBV affects cellular mi RNAs, how these mi RNAs impact HBV replication, and the relationship between HBV-mediated mi RNA regulation and HCC development. We also address the impact of challenges in studying HBV, such as the lack of an effective model system for infectivity and a reliance on transformed cell lines, on our understanding of the relationship between HBV and mi RNAs, and proposepotential applications of mi RNA-related techniques that could enhance our understanding of the role mi RNAs play in HBV replication and HBV-associated disease, ultimately leading to new therapeutic options and improved patient outcomes.
基金Supported by National Natural Science Foundation of China to Pei RJ and Chen XC,Nos.31200135 and 31200699German Research Foundation to Lu MG,Nos.TRR60,GK1045/2 and GK1949
文摘Hepatitis B virus(HBV) infection is one of the major causes of liver diseases, affecting more than 350 million people worldwide. The interferon(IFN)-mediated innate immune responses could restrict HBV replication at the different steps of viral life cycle. Indeed, IFN-α has been successfully used for treatment of patients with chronic hepatitis B. However, the role of the innate immune response in HBV replication and the mechanism of the anti-HBV effect of IFN-α are not completely explored. In this review, we summarized the currently available knowledge about the IFN-mediated anti-HBV effect in the HBV life cycle and the possible effectors downstream the IFN signaling pathway. The antiviral effect of Toll-like receptors(TLRs) in HBV replication is briefly discussed. The strategies exploited by HBV to evade the IFN- and TLR-mediated antiviral actions are summarized.
基金Supported by The DFG,SFB638,TP A5 and SFB/TRR83,TP 13
文摘Plus-strand RNA virus replication occurs in tight association with cytoplasmic host cell membranes. Both, viral and cellular factors cooperatively generate distinct organelle-like structures, designated viral replication factories. This compartmentalization allows coordination of the different steps of the viral replication cycle, highly efficient genome replication and protection of the viral RNA from cellular defense mechanisms. Electron tomography studies conducted during the last couple of years revealed the three dimensional structure of numerous plus-strand RNA virus replication compartments and highlight morphological analogies between different virus families. Based on the morphology of virusinduced membrane rearrangements, we propose two separate subclasses: the invaginated vesicle/spherule type and the double membrane vesicle type. This review discusses common themes and distinct differences in the architecture of plus-strand RNA virus-induced membrane alterations and summarizes recent progress that has been made in understanding the complex interplay between viral and co-opted cellular factors in biogenesis and maintenance of plus-strand RNA virus replication factories.
基金This study was supported by a grant from the National Key Basic ResearchProgram of China (No. 2003CB515501).
文摘BACKGROUND: Hepatitis B related end-stage liver disease is recently acknowledged as one of the main indications for orthotopic liver transplantation (OLT). However, the high recurrence rate of hepatitis B virus infection following transplantation is regarded as a major factor affecting the long-term survival of transplant recipients especially in Chi- na. Cyclosporine A (CsA), which is routinely used to pre- vent the allograft rejection, is reported to have the inhibito- ry activity on hepatitis B virus (HBV) replication in vitro. In this paper, we review the inhibitory effect and its possi- ble mechanisms of CsA on HBV replication in vitro. DATA RESOURCES: An English-language literature search was conducted using MEDLINE (1990-2004) on cyclospo- rine A, hepatitis B virus, mitochondria, calcium and other related reports and review articles. RESULTS: Hepatitis B x protein (HBx) is essential to HBV replication. The cytosolic calcium signaling mediated by mitochondria and the Src kinase pathway were involved during HBx activation of HBV replication. CsA inhibits the HBV replication in vitro by its binding to mitochondrial cy- clophilin D, then blocking the mitochondria-mediated cy- tosolic calcium signaling. The derivates of CsA also have the HBV replication inhibitory effect in vitro. CONCLUSIONS; By interacting with mitochondria, pre- venting the release of intramitochondrial calcium, and then blocking the cytosolic calcium signaling, CsA inhibits the HBV replication in vitro. The derivates of CsA also have this activity.
基金Supported by the National Science Fund for Distinguished Young Scholars from the National Natural Science Foundation of China,No.30325036a grant from the National Natural Science Foundation of China,No.30571640
文摘瞄准：与肝炎 B (HBV ) 复制建立一个快速、方便的动物模型。方法：HBV-replication-competent 原生质标志的一个裸体 DNA 解决方案经由尾巴静脉被转移到 BALB/C 鼠标，用一个水动力学在活体内 transfection 过程。在注射以后，这些老鼠在 d 上被牺牲 1， 3， 4， 5， 7 和 10。在肝的 HBV DNA 复制中介被南部的污点杂交分析。肝炎 B 核心抗原(HBcAg ) 和在肝的肝炎 B 表面抗原(HBsAg ) 的表示被免疫组织化学检查。浆液 HBsAg 和肝炎 B e 抗原(HBeAg ) 被连接酶的免疫吸着剂试金(ELISA ) 检测。HBV 复制的抑制在与 polyinosinic-polytidylin 酸(polyIC ) intraperitoneally 对待的 HBV 复制模型老鼠被比较或缓冲磷酸盐 saline (PBS ) 。结果：在水动力学在活体内 transfection 以后，在老鼠肝的 HBV DNA 复制中介在 d 上是可检测的 1 并且在 d 上丰富 3 和 4，层次稍微被减少并且在 d 之间仍然保持相对稳定 5 和 7，并且在 d 上是几乎无法发现的 10。HBcAg 和 HBsAg 的表示模式类似于 HBV 复制中介 DNA 的，除了他们在 d 上到达了一座山峰之外 1 在注射以后。在 HBV DNA 复制中介的明显的差别都没在肝的左、正确、中间的脑叶被观察。有 polyIC 的术后疗法，在肝的 HBV 中间的 DNA 的水平在与 PBS 注射的控制老鼠是比那低的。结论：有 HBV 复制的高水平的一个快速、方便的老鼠模型被开发并且过去常在 HBV 复制上调查 polyIC 的禁止的效果，它为未来提供一个有用工具 HBV 染色体的功能的研究。
基金Supported by In part Grants from NIDDK DK042182 and the Herman Lopata Chair for Hepatitis Research (Wu GY)
文摘AIM: To examine the effect of hepatitis C virus (HCV) structural mimics of regulatory regions of the genome on HCV replication.METHODS: HCV RNA structural mimics were constructed and tested in a HCV genotype 1b aBB7 replicon,and a Japanese fulminant hepatitis-1 (JFH-1) HCV genotype 2a infection model.All sequences were computer-predicted to adopt stem-loop structures identical to the corresponding elements in full-length viral RNA.Huh7.5 cells bearing the BB7 replicon or infected with JFH-1 virus were transfected with expression vectors generating HCV mimics and controls.Cellular HCV RNA and protein levels were quantified by real-time polymerase chain reaction and Western blotting,respectively.To evaluate possible antisense effects,complementary RNAs spanning a mimic were prepared.RESULTS: In the BB7 genotype 1b replicon system,mimics of the polymerase (NS-5B),X and BA regions inhibited replication by more than 90%,50%,and 60%,respectively.In the JFH-1 genotype 2 infection system,mimics that were only 74% and 46% identical in sequence relative to the corresponding region in JFH-1 inhibited HCV replication by 91.5% and 91.2%,respectively,as effectively as a mimic with complete identity to HCV genotype 2a.The inhibitory effects were confirmed by NS3 protein levels.Antisense RNA molecules spanning the 74% identical mimic had no significant effects.CONCLUSION: HCV RNA structural mimics can inhibit HCV RNA replication in replicon and infectious HCV systems and do so independent of close sequence identity with the target.
文摘Hepatitis C virus (HCV) is one of the main etiological factors responsible for liver disease worldwide. It has been estimated that there are over 170 million people infected with HCV worldwide. Of these infected individuals, approximately 75% will go on to develop a life long necroinflammatory liver disease, which over decades, can result in serious complications, such as cirrhosis and hepatocellular carcinoma. Currently there is no effective vaccine and whilst antiviral therapies have been improved, they are still only effective in approximately 50% of individuals. HCV infection stands as a major cause of global morbidity and suffering, and places a signifi cant burden on health systems. The second highest cause of liver disease in the western world is alcoholic liver disease. Frequently, HCV infected individuals consume alcohol, and the combined effect of HCV and alcohol consumption is deleterious for both liver disease and response to treatment. This review discusses the impact of alcohol metabolism on HCV replication and the negative impact on interferon (IFN)-α treatment, with a particular focus on how alcohol and HCV act synergistically to increase oxidative stress, ultimately leading to exacerbated liver disease and a reduction in the effi cacy of IFN-α treatment. A better understanding of the complicated mechanisms at play in hepatocytes infected with HCV and metabo- lizing alcohol will hopefully provide better treatment options for chronic hepatitis C individuals that consume alcohol.
基金Project (No. 30471943) supported partly by the National Natural Science Foundation of China
文摘Objectives: To evaluate the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of hepatitis B virus (HBV) transcripts on the viral replication and antigen expression in vitro. Methods: (1) Seven siRNAs targeting surface (S), polymerase (P) or precore (PreC) region of HBV genome were designed and chemically synthesized. (2) HBV-producing HepG2.2.15 cells were treated with or without siRNAs for 72 h. (3) HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. (4) Intracellular viral DNA was quantified by real-time PCR (Polymerase Chain Reaction). (5) HBV viral mRNA was reverse transcribed and quantified by real-time PCR. (6) The change of cell cycle and apoptosis was determined by flow cytometry. Results: Our data demonstrated that synthetic small interfering RNAs (siRNAs) targeting S and PreC gene could efficiently and specifically inhibit HBV replication and antigen expression. The ex- pression of HBsAg and HBeAg and the replication of HBV could be specifically inhibited in a dose-dependent manner by siRNAs. Furthermore, our results showed that the combination of siRNAs targeting various regions could inhibit HBV replication and antigen expression in a more efficient way than the use of single siRNA at the same final concentration. No apoptotic change was observed in the cell after siRNA treatment. Conclusion: Our results demonstrated that siRNAs exerted robust and specific inhibi- tion on HBV replication and antigen expression in a cell culture system and combination of siRNAs targeting different regions exhibited more potency.
文摘AIM: To investigate the effects of suberoylanilide hydroxamic acid(SAHA) on proliferation and apoptosis of a human hepatocellular carcinoma cell line(HepG2.2.15) and hepatitis B virus(HBV) replication.METHODS: HepG2.2.15 cells were treated with different concentrations of SAHA.Cell morphology was examined by confocal laser scanning microscopy,and cell proliferation was determined using a MTT colorimetric assay.Flow cytometry was used to detect apoptosis and determine cell cycle phase,while hepatitis B surface antigen and hepatitis B e antigen content were measured using chemiluminescence.Reverse transcription polymerase chain reaction was performed to measure HBV DNA in cell lysate.RESULTS: Cell proliferation rates were significantly reduced by the addition of SAHA.The inhibitory effect of SAHA on cell proliferation was both time-and dosedependent.After 24 h of treatment with SAHA,the early cell apoptotic rate increased from 3.25% to 21.02%(P = 0.041).The proportion of G0 /G1 phase cells increased from 50.3% to 65.3%(P = 0.039),while that of S phase cells decreased from 34.9% to 20.6%(P = 0.049).After 48 h of treatment,hepatitis B surface antigen and hepatitis B e antigen content increased from 12.33 ± 0.62 to 25.42 ± 2.67(P = 0.020) and 28.92 ± 1.24 to 50.48 ± 1.85(P = 0.026),respectively.Furthermore,HBV DNA content increased from 4.54 ± 0.46 to 8.34 ± 0.59(P = 0.029).CONCLUSION: SAHA inhibits HepG2.2.15 cell proliferation,promotes apoptosis,and stimulates HBV replication.In combination with anti-HBV drugs,SAHA may potentially be used cautiously for treatment of hepatocellular carcinoma.
基金Supported by The National Basic Research Program of China,No. 2007CB512902The National Basic Research Program of China, No. 2006CB504302Key Technologies Research of Sichuan Province,No. 2008SZ0039
文摘AIM:To evaluate the value of the hepatitis B virus(HBV) replication mouse model with regard to several aspects of the study of HBV biology.METHODS:To evaluate the HBV replication mouse model in detecting the efficacy of anti-HBV agents,the interferon inducer polyinosinic-polytidylin acid(polyIC) and nucleotide analogues adefovir and entecavir were administered to mice injected with wild type pHBV4.1,and the inhibiting effect of these agents on HBV DNA replication was evaluated.To identify the model's value in a replication ability study of HBV drug-resistant mutants and a HBx-minus mutant,telbivudine resistance mutants(rtM204I,ayw subtype),adefovir resistance mutants(rtA181V + rtN236T,ayw subtype) and HBxminus mutants were injected respectively,and their corresponding HBV DNA replication intermediates in mouse liver were assessed.RESULTS:Compared with the wild type HBV replication mouse model without antiviral agent treatment,the HBV DNA replication intermediates of the polyICtreated group were decreased 1-fold;while in the entecavir-and adefovir-treated groups,the levels of HBV DNA replication intermediates were inhibited 13.6-fold and 1.4-fold,respectively.For the mouse models injected with telbivudine resistance mutant,adefovir resistance mutant and HBx-minus mutant,HBV DNA replication intermediates could still be detected,but the levels of HBV DNA replication intermediates of these mutants decreased 4.5-fold,5.6-fold and 2.9-fold respectively,compared with the mouse model with wild type HBV plasmid.CONCLUSION:The HBV replication mouse model we established was a useful and convenient tool to detect the efficacy of antiviral agents and to study the replication ability of HBV mutants in vivo.
基金funded by the National Natural Science Foundation of China(Grantno.81873065)the Secondary Development Projects of Guangdong Famous and Excellent TraditionalChinese Patent Medicines(Grant no.20174005)+1 种基金the Natural Science Foundation of Guangdong Province(Grant no.2018A030310172)the China Postdoctoral Science Foundation(Grant no.2017M622652,2019M652987)。
文摘Isatis indigotica Fort.(Ban-Lan-Gen)is an herbal medicine prescribed for influenza treatment.However,its active components and mode of action remain mostly unknown.In the present study,erucic acid was isolated from Isatis indigotica Fort.,and subsequently its underlying mechanism against influenza A virus(IAV)infection was investigated in vitro and in vivo.Our results demonstrated that erucic acid exhibited broad-spectrum antiviral activity against IAV resulting from reduction of viral polymerase transcription activity.Erucic acid was found to exert inhibitory effects on IAV or viral(v)RNA-induced pro-inflam-matory mediators as well as interferons(IFNs).The molecular mechanism by which erucic acid with antiviral and anti-inflammatory properties was attributed to inactivation of NF-kB and p38 MAPK signaling.Furthermore,the NF-kB and p38 MAPK inhibitory effect of erucic acid led to diminishing the transcriptional activity of interferon-stimulated gene factor 3(ISGF-3),and thereby reducing IAV-triggered pro-inflammatory response amplification in IFN-β-sensitized cells.Additionally,IAV-or vRNA-triggered apoptosis of alveolar epithelial A549 cells was prevented by erucic acid.In vivo,erucic acid administration consistently displayed decreased lung viral load and viral antigens expression.Meanwhile,erucic acid markedly reduced CD8+cytotoxic T lymphocyte(CTL)recruitment,pro-apoptotic signaling,hyperactivity of multiple signaling pathways,and exacerbated immune inflammation in the lung,which resulted in decreased lung injury and mortality in mice with a mouse-adapted A/FM/1/47-MA(H1N1)strain infection.Our findings provided a mechanistic basis for the action of erucic acid against IAV-mediated inflammation and injury,suggesting that erucic acid may have a therapeutic potential in the treatment of influenza.
基金Supported by the National Natural Science Foundation of China,No.81500449the Natural Science Foundation of Shanghai,No.14ZR1434200+2 种基金Shanghai Municipal Commission of Health and Family Planning,No.20144Y0175the Scientific Research Foundation for the Returned Overseas Chinese Scholarsthe State Education Ministry of China,No.20150909-6
文摘AIM To investigate the effect of(-)-epigallocatechin-3-gallate(EGCG) on polyinosinic-polycytidylic acid(poly I:C)-triggered intracellular innate immunity against hepatitis C virus(HCV) in hepatocytes. METHODS A cell culture model of HCV infection was generated by infecting a hepatoma cell line, Huh7, with HCV JFH-1 strain(JFH-1-Huh7). Poly I:C with a high molecular weight and EGCG were used to stimulate the JFH-1-Huh7 cells. Real-time reverse transcription-polymerase chain reaction was used to detect the expression levels of intracellular m RNAs and of intracellular and extracellular HCV RNA. Enzyme-linked immunosorbent assay was used to evaluate the interferon(IFN)-λ1 protein level in the cell culture supernatant. Immunostaining was used to examine HCV core protein expression in Huh7 cells.RESULTS Our recent study showed that HCV replication could impair poly I:C-triggered intracellular innate immune responses in hepatocytes. In the current study, we showed that EGCG treatment significantly increased the poly I:C-induced expression of Toll-like receptor 3(TLR3), retinoic acid-inducible gene I, and IFN-λ1 in JFH-1-Huh7 cells. In addition, supplementation with EGCG increased the poly I:C-mediated antiviral activity in JFH-1-Huh7 cells at the intracellular and extracellular HCV RNA and protein levels. Further investigation of the mechanisms showed that EGCG treatment significantly enhanced the poly I:C-induced expression of IFN-regulatory factor 9 and several antiviral IFNstimulated genes, including ISG15, ISG56, myxovirus resistance A, and 2'-5'-oligoadenylate synthetase 1, which encode the key antiviral elements in the IFN signaling pathway. CONCLUSION Our observations provide experimental evidence that EGCG has the ability to enhance poly I:C-induced intracellular antiviral innate immunity against HCV replication in hepatocytes.
文摘Objective To develop an effective and specific medicine targeting hepatitis B virus(HBV) pregenome. Based on the identified accessible target sites for hammerhead ribozyme in our previous researches, a recombinant hepatitis delta virus(HDV) ribozyme was chosen and used to demonstrate the effective cleavage in vitro and in vivo. Methods Three hammerhead ribozymes for potential target sites(S, X and C genes) and co-expression plasmid(pTr-dB, pTdδ-dB, pTrX-dB and pTrC-dB) as well as four HDV-ribozyme chimera constructs with HBV(pTdXX, pTdXC, pTdSX and pTdSC) were severally chosen to validate the inhibition of the replication and expression of HBV. The co-expression plasmids(pTdδ and pTr-Db) in physiological saline were hydrodynamically injected to mice by tail vein. Results Compared with the group injected with pTr-dB in Huh-7 cell, hepatitis B surface antigen(HBsAg) was reduced by 31% in the group injected with pTdδ-dB, by 54%, 26%, 72% and 97% in the group injected with recombinant-ribozymes pTdSX, pTdSC, pTdXC and pTdXX, respectively. The inhibiting effects of endogenous ribozymes RzX and RzC on the HBsAg expression were 66% and 57%, respectively. Compared with the positive control, the amount of HBsAg was decreased in mice injected with pTdXX through tail vein by 88% and 96% on the second day and the third day, respectively. HBsAg was undetectable on the 6th day and could not primitively be detected on the 9th day in the sera from all mice. HBV DNA was not detected in the sera of BALB/c mice injected with pTdXX-dB, pTrX-dB or replicating-defective plasmid pHBV, while HBV DNA replication in control group could be detected on the 6th day. While HBcAg could not be detected in liver tissues of mice injected with plasmid pTdXX-dB on the 3rd day. Conclusions Encoding regions of HBV S, C and X gene were the effective cleavage sites for hammerhead ribozyme in vitro and in vivo, which provides basis for further construction of therapeutic recombinant HDV and the development of targeting antiviral gene therapy.
基金Supported by the National Natural Science Foundation of China,No.81672041the National Major Science and Technology Special Project for Infectious Diseases of China,No.2012ZX10004503-012
文摘BACKGROUND Previously,we have successfully constructed replication-competent hepatitis B virus(HBV)vectors by uncoupling the P open reading frame(ORF)from the preC/C ORF to carefully design the transgene insertion site to overcome the compact organization of the HBV genome and maintain HBV replication competence.Consequently,the replication-competent HBV vectors carrying foreign genes,including pCH-BsdR,carrying blasticidin resistance gene(399 bp),and pCH-hrGFP,carrying humanized renilla green fluorescent protein gene(720 bp),were successfully obtained.However,the replication efficiency of the former is higher but it is tedious to use,while that of the latter is poor and cannot be quantified.Hence,we need to search for a new reporter gene that is convenient and quantifiable for further research.AIM To establish a helpful tool for intracellular HBV replication and anti-viral drugs screening studies.METHODS We utilized the replication-competent HBV viral vectors constructed by our laboratory,combined with the secreted luciferase reporter gene,to construct replication-competent HBV vectors expressing the reporter gene secretory Nanoluc Luciferase(SecNluc).HepG2.TA2-7 cells were transfected with this vector to obtain cell lines with stably secreted HBV particles carrying secNluc reporter gene.RESULTS The replication-competent HBV vector carrying the SecNluc reporter gene pCHsNLuc could produce all major viral RNAs and a full set of envelope proteins and achieve high-level secreted luciferase expression.HBV replication intermediates could be produced from this vector.Via transfection with pTRE-sNLuc and selection by hygromycin,we obtained isolated cell clones,named HBV-NLuc-35 cells,which could secrete secNLuc recombinant viruses,and were sensitive to existing anti-HBV drugs.Using differentiated HepaRG cells,it was verified that recombinant HBV possessed infectivity.CONCLUSION Our research demonstrated that a replication-competent HBV vector carrying a secreted luciferase transgene possesses replication and expression ability,and the established HBV replication and expression cell lines could stably secrete viral particles carrying secNluc reporter gene.More importantly,the cell line and the secreted recombinant viral particles could be used to trace HBV replication or infection.
文摘AIM: To address the effect of heat-shock protein 90(HSP90) inhibitors on the release of the hepatitis C virus(HCV), a cell culture-derived HCV(JFH1/HCVcc) from Huh-7 cells was examined.METHODS: We quantified both the intracellular and extracellular(culture medium) levels of the components(RNA and core) of JFH-1/HCVcc. The intracellular HCV RNA and core levels were determined after the JFH1/HCVcc-infected Huh-7 cells were treated with radicicol for 36 h. The extracellular HCV RNA and core protein levels were determined from the medium of the last 24 h of radicicol treatment. To determine the possible role of the HSP90 inhibitor in HCV release, we examined the effect of a combined application of low doses of the HSP90 inhibitor radicicol and the RNA replication inhibitors cyclosporin A(Cs A) or interferon. Finally, we statistically examined the combined effect of radicicoland Cs A using the combination index(CI) and graphical representation proposed by Chou and Talalay.RESULTS: We found that the HSP90 inhibitors had greater inhibitory effects on the HCV RNA and core protein levels measured in the medium than inside the cells. This inhibitory effect was observed in the presence of a low level of a known RNA replication inhibitor(Cs A or interferon-α). Treating the cells with a combination of radicicol and cyclosporin A for 24 h resulted in significant synergy(CI < 1) that affected the release of both the viral RNA and the core protein. CONCLUSION: In addition to having an inhibitory effect on RNA replication, HSP90 inhibitors may interfere with an HCV replication step that occurs after the synthesis of viral RNA, such as assembly and release.
基金Supported by the National Natural Science Foundation of China, No. 30271170 and 30571646 the National Key Basic Research Program of China, No. 20014CB510008
文摘To the Editor:I have previously reported a type II diabetic patient complicated with chronic hepatitis C virus(HCV)infection.1]Recently,Riva et al[2]reported an association of truncated CXCL10 with failure to achieve spontaneous clearance of acute HCV infection.They showed
