APOBEC3A通过脱氨基作用抑制HBV复制

doi:10.13523/j.cb.20151202

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中国生物工程杂志 ›› 2015, Vol. 35 ›› Issue (12) : 8-14. DOI: 10.13523/j.cb.20151202

研究报告

APOBEC3A通过脱氨基作用抑制HBV复制

陈彦猛, 黄瑶, 胡杰, 胡源

作者信息 +

APOBEC3A Inhibited HBV Replication Dependent Its Deamination

CHEN Yan-meng, HUANG Yao, HU Jie, HU Yuan

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文章历史 +

摘要

目的:研究APOBEC3A抑制HBV复制的分子机制。方法:首先在肝癌细胞HuH7中过表达APOBEC3A,通过MTT法检测了APOBEC3A对细胞毒性的影响;通过免疫荧光检测了APOBEC3A在细胞中的定位,通过IP试验进一步证实了APOBEC3A与病毒颗粒的相互作用;并通过ELISA,特异性荧光定量PCR检测了HBV复制的参数包括上清中HBsAg,病毒核心颗粒中HBV DNA以及核内的cccDNA的表达水平;最后通过3D PCR方法分析了核心颗粒中HBV DNA脱氨基作用。结果:过表达APOBEC3A对HuH7细胞毒性没有显著性差异;APOBEC3A主要位于细胞核,但APOBEC3A可以与病毒颗粒结合,在逆转录环节对HBV复制发生抑制作用;共转染HBV复制质粒和APOBEC3A表达质粒后,细胞上清中HBsAg,核心颗粒中的HBV DNA以及核内的cccDNA均显著下降;最后通过3D PCR和克隆测序表明核心颗粒中的HBV DNA负链发生了大量的G-A突变,同时正链也发生了较多的C-T突变。结论:APOBEC3A可与病毒颗粒结合,在HBV复制的逆转录环节可作用于HBV单链,发生脱氨基作用,从而抑制HBV的复制。

Abstract

Purpose:To investigate the mechanism of APOBEC3A on HBV replication. Methods:(1) The cytotoxicity effect of exogenous APOBEC3A in HuH7 cells was evaluated by MTT assay. (2) The location of APOBEC3A in the HuH7 cells was measured by immunofluorescence, and the interaction of APOBEC3A with virus core particles was further confirmed by IP. (3) The relative levels of HBV replication parameters:including HBsAg in the supernatant, HBV DNA in virus core particles and intranuclear cccDNA were measured by ELISA and specific real-time PCR in the presence of exogenous APOBEC3A. (4) The deamination of HBV DNA in core particles was analyzed by 3D PCR. Results:(1) The expression of exogenous APOBEC3A had no effect on cell viability of HuH7 cells. (2)APOBEC3A was mainly located in the nucleus, but APOBEC3A combined with virus core particles to deaminate DNA during the process of HBV reverse transcription. (3) After co-transfection of HBV replication plasmid and APOBEC3A expression plasmid, the levels of HBsAg in the supernatant, HBV DNA in particles, and cccDNA were significantly decreased by 80%, 64% and 82%, respectively.(4) Hypermutations of G-A accumulated in the HBV minus strand DNA and C-T in the plus strand DNA of core particles in the presence of exogenous APOBEC3A were confirmed by 3D PCR and sequencing. Conclusion:APOBEC3A can interact with virus core particles, target HBV single strand DNA during the process of HBV reverse transcription, and inhibit HBV replication dependent its deamination.

导出引用

陈彦猛, 黄瑶, 胡杰, 胡源. APOBEC3A通过脱氨基作用抑制HBV复制[J]. 中国生物工程杂志, 2015, 35(12): 8-14 https://doi.org/10.13523/j.cb.20151202

CHEN Yan-meng, HUANG Yao, HU Jie, HU Yuan. APOBEC3A Inhibited HBV Replication Dependent Its Deamination[J]. China Biotechnology, 2015, 35(12): 8-14 https://doi.org/10.13523/j.cb.20151202

中图分类号： Q789

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