KNK437抑制乙型肝炎病毒复制及转录  

KNK437 Inhibits Replication and Transcription of the Hepatitis B Virus

作　　者：胡康洪[1] 黄亚运[1] 穆敬芳[2] 程智逵[2] 朱祥[1] HU Kanghong HUANG Yayun MU Jingfang CHENG Zhikui ZHU Xiang(Hubei University of Technology, Institute of Biomedical and Pharmaceutical Sciences, Sino-Germary Biomedical Center, Wuhan 430068, China State Key Laboratory of Virology, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan 430071, China)

机构地区：[1]湖北工业大学生物医药研究院中德生物医学中心,武汉430068 [2]中国科学院武汉病毒研究所病毒学国家重点实验室,武汉430071

出　　处：《病毒学报》2017年第1期24-35,共12页Chinese Journal of Virology

基　　金：湖北省自然科学基金重点项目(项目号:2014CFA075);题目:RNA诱饵分子抗HBV功效的体内研究;武汉市科技局应用基础研究计划(项目号:2015060101010033);题目:肝癌早期诊断与靶向药物设计

摘　　要：乙型肝炎病毒(Hepatitis B virus,HBV)在肝细胞内复制过程中,病毒反转录酶(P蛋白)与前基因组RNA(Pregenomic RNA,pgRNA)上的RNA包装信号ε形成P-ε复合物,启动反转录合成和核衣壳装配。封闭P-ε相互作用是一种极具吸引力的抗HBV策略。已知P-ε形成RNP复合物正常发挥活性时,需要热休克蛋白(Heat-shock proteins,Hsps)参与。本研究探索Hsps抑制剂KNK437对HBV复制及转录的影响。主要运用了三种工作模型:HepG2.2.15细胞系、瞬时转染1.05×HBV(pCH9-3091)质粒的Huh7细胞系和瞬时转染1.3×HBV(pGEM-1.3×HBV)质粒的Huh7细胞系。采用CCK-8方法检测KNK437的细胞毒性,ELISA测定细胞培养液上清中HBsAg和HBeAg的分泌水平;q-PCR和qRT-PCR分别检测细胞内HBV核衣壳中DNA和细胞内HBV RNA水平;Western blotting检测细胞内衣壳亚单位(core)表达水平;qRT-PCR检测细胞内Hsps本身的转录水平。结果表明:20μM KNK437对细胞没有毒性,且在该浓度下KNK437除HepG2.2.15模型外,均下调HBV HBsAg和HBeAg的胞外分泌,抑制细胞内HBV核衣壳中DNA合成,降低细胞内HBV RNA转录水平,最低可使DNA水平降至1.5%左右,RNA水平降至30%左右,从而表明KNK437抑制HBV复制和转录。在HepG2.2.15中的Western blotting显示,KNK437明显减少细胞内衣壳亚单位(core)表达水平。KNK437也抑制Hsp70,Hsp90b,Hsp40等三种热休克蛋白RNA的转录,从而验证了它的确是一种泛Hsps抑制剂。本研究结果显示KNK437具有潜在抗HBV应用前景。During replication of the hepatitis B virus （HBV） in liver cells, the reverse transcription of pregenomic RNA （pgRNA） is initiated by protein priming at an RNA packaging signal ε located near the 5＇ end of pgRNA. Heat-shock proteins （Hsps） such as Hsc70, Hsp40, and Hsp90 have been reported to he involved in the reconstitution of HBV polymerase （P protein） and ε The P - ε complex initiates the reverse transcription and assembly of nucleocapsids. Hence, blockade of P - ε interactions is an attractive target for drug intervention. We explored the influence of the Hsp inhibitor KNK437 on replication and transcription of the HBV. Three working models were applied： HepG2. 2. 15 cell line; Huh7 cells transfected transiently with the 1. 05 × HBV （pCH9-3091） plasmid; Huh7 cells transfeeted transiently with the 1.3 × HBV （pGEM-1. 3 × HBV） plasmid. Cytotoxic effects of KNK437 were detected by the CCK-8 method. Levels of hepatitis B surface antigen （HBsAg） and hepatitis B viral protein （HBeAg） in the media secreted from cells were measured using an ELISA. Intracellular HBV DNAs within nucleocapsids were measured by quantitative polymerase chain reaction （qPCR）, and intracellular HBV RNAs by quantitative reverse transcription-polymerase chain reaction （qRT-PCR）. Transcription of Hsps in cells was determined by qRT-PCR. Data suggested that KNK437 reduced the extracellular secretion of HBsAg and HBeAg in most cases; it downregulated expression of intracellular HBV DNAs within nucleocapsids and RNA transcripts. The lowest rate of viral DNAs in KNK437-treated hepatocytes for all experimental groups was ≈1.5% （control, 100%）, whereas that for RNAs was≈30 %. Western blotting revealed KNK437 to inhibit intracellular core expression in HepG2. 2.15. As a general inhibitor, KNK437 suppressed transcription of hsp70, hsp90b, and hsp40. These data suggest that KNK437 may be a potent anti-HBV inhibitor for future therapy against chronic hepatitis.

关 键 词：乙型肝炎病毒(HBV) 热休克蛋白(Hsps) KNK437 抑制剂 

分 类 号：R373.2[医药卫生—病原生物学]