Dataset Information

The anti-HBV effect mediated by a novel recombinant eukaryotic expression vector for IFN-?.

ABSTRACT:

Background

Chronic hepatitis B is a primary cause of liver-related death. Interferon alpha (IFN-?) is able to inhibit the replication of hepadnavirus, and the sustained and stable expression of IFN-? at appropriate level may be beneficial to HBV clearance. With the development of molecular cloning technology, gene therapy plays a more and more important role in clinical practice. In light of the findings, an attempt to investigate the anti-HBV effects mediated by a eukaryotic expression plasmid (pSecTagB-IFN-?) in vitro was carried out.

Methods

HBV positive cell line HepG2.2.15 and its parental cell HepG2 were transfected with pSecTagB-IFN-? or empty plasmid by using Lipofectamine™ 2000 reagent. The expression levels of IFN-? were determined by reverse transcriptase polymerase chain reaction (RT-PCR) and ELISA methods. The effects of pSecTagB-IFN-? on HBV mRNA, DNA and antigens were analyzed by real-time fluorescence quantitative PCR (qRT-PCR) and ELISA assays. RT-PCR, qRT-PCR and western blot were employed to investigate the influence of pSecTagB-IFN-? on IFN-?-induced signal pathway. Furthermore, through qRT-PCR and ELISA assays, the suppressive effects of endogenously expressed IFN-? and the combination with lamivudine on HBV were also examined.

Results

pSecTagB-IFN-? could express efficiently in hepatoma cells, and then inhibited HBV replication, characterized by the decrease of HBV S gene (HBs) and HBV C gene (HBc) mRNA, the reduction of HBV DNA load, and the low contents of hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg). Mechanism research showed that the activation of Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signal pathway, the up-regulation of IFN-?-induced antiviral effectors and double-stranded (ds) RNA sensing receptors by delivering pSecTagB-IFN-?, could be responsible for these phenomena. Furthermore, pSecTagB-IFN-? vector revealed effectively anti-HBV effect than exogenously added IFN-?. Moreover, lamivudine combined with endogenously expressed IFN-? exhibited stronger anti-HBV effect than with exogenous IFN-?.

Conclusion

Our results showed that endogenously expressed IFN-? can effectively and persistently inhibit HBV replication in HBV infected cells. These observations opened a promising way to design new antiviral genetic engineering drugs based on IFN-?.

SUBMITTER: Yu H

PROVIDER: S-EPMC3766191 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature

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Publications

The anti-HBV effect mediated by a novel recombinant eukaryotic expression vector for IFN-α.

Yu Haotian H Hou Zhaohua Z Han Qiuju Q Zhang Cai C Zhang Jian J

Virology journal 20130829

<h4>Background</h4>Chronic hepatitis B is a primary cause of liver-related death. Interferon alpha (IFN-α) is able to inhibit the replication of hepadnavirus, and the sustained and stable expression of IFN-α at appropriate level may be beneficial to HBV clearance. With the development of molecular cloning technology, gene therapy plays a more and more important role in clinical practice. In light of the findings, an attempt to investigate the anti-HBV effects mediated by a eukaryotic expression ...[more]

PMID: 23984795

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Project description:BACKGROUND:Hepatitis B virus covalently closed circular DNA (HBV cccDNA) is assembled by histones and non-histones into a chromatin-like cccDNA minichromosome in the nucleus. The cellular histone acetyltransferase GCN5, displaying succinyltransferase activity, is recruited onto cccDNA to modulate HBV transcription in cells. Clinically, IFN-? is able to repress cccDNA. However, the underlying mechanism of IFN-? in the depression of cccDNA mediated by GCN5 is poorly understood. Here, we explored the effect of IFN-? on GCN5-mediated succinylation in the epigenetic regulation of HBV cccDNA minichromosome. RESULTS:Succinylation modification of the cccDNA minichromosome has been observed in HBV-infected human liver-chimeric mice and HBV-expressing cell lines. Moreover, histone H3K79 succinylation by GCN5 was identified in the system. Interestingly, the mutant of histone H3K79 efficiently blocked the replication of HBV, and interference with GCN5 resulted in decreased levels of HBV DNA, HBsAg, and HBeAg in the supernatant from de novo HBV-infected HepaRG cells. Consistently, the levels of histone H3K79 succinylation were significantly elevated in the livers of HBV-infected human liver-chimeric mice. The knockdown or overexpression of GCN5 or the mutant of GCN5 could affect the binding of GCN5 to cccDNA or H3K79 succinylation, leading to a change in cccDNA transcription activity. In addition, Southern blot analysis validated that siGCN5 decreased the levels of cccDNA in the cells, suggesting that GCN5-mediated succinylation of histone H3K79 contributes to the epigenetic regulation of cccDNA minichromosome. Strikingly, IFN-? effectively depressed histone H3K79 succinylation in HBV cccDNA minichromosome in de novo HepG2-NTCP and HBV-infected HepaRG cells. CONCLUSIONS:IFN-? epigenetically regulates the HBV cccDNA minichromosome by modulating GCN5-mediated succinylation of histone H3K79 to clear HBV cccDNA. Our findings provide new insights into the mechanism by which IFN-? modulate the epigenetic regulation of HBV cccDNA minichromosome.

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