KNK437抑制乙型肝炎病毒复制及转录

发布时间：2019-02-22 19:21

【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。
[Abstract]:During the replication of hepatitis B virus (Hepatitis B virus,HBV) in hepatocytes, the retrovirus reverse transcriptase (P protein) formed P- 蔚 complex with the RNA packaging signal 蔚 on the pregenomic RNA (Pregenomic RNA,pgRNA. Initiating reverse transcription synthesis and core-capping assembly. Closed P- 蔚 interaction is an attractive anti-HBV strategy. It is known that heat shock protein (Heat-shock proteins,Hsps) is involved in P- 蔚 forming RNP complex. The aim of this study was to investigate the effects of Hsps inhibitor KNK437 on HBV replication and transcription. Three working models were used: HepG2.2.15 cell line, Huh7 cell line with 1. 05 脳 HBV (pCH9-3091) plasmid and Huh7 cell line with 1. 3 脳 HBV (pGEM-1.3 脳 HBV) plasmid. The cytotoxicity of KNK437 was detected by CCK-8, the levels of HBsAg and HBeAg in the supernatant of cell culture medium were measured by ELISA, DNA and HBV RNA in the core-shell of HBV were detected by q-PCR and qRT-PCR, respectively. Western blotting was used to detect the expression of (core) and qRT-PCR was used to detect the transcriptional level of Hsps itself. The results showed that 20 渭 M KNK437 was not toxic to cells, and at this concentration, KNK437 could down-regulate the extracellular secretion of HBV HBsAg and HBeAg, inhibit the synthesis of DNA in HBV nucleocapsid and decrease the level of HBV RNA transcription. The lowest level of KNK437 can reduce the DNA level to about 1.5% and the RNA level to about 30%, which indicates that KNK437 inhibits HBV replication and transcription. Western blotting in HepG2.2.15 showed that KNK437 significantly reduced the (core) expression level of cell underwear shell subunit. KNK437 also inhibited the transcription of three heat shock proteins (Hsp70,Hsp90b,Hsp40) RNA, which proved that it was indeed a pan-Hsps inhibitor. The results of this study indicate that KNK437 has a potential application prospect of anti-HBV.
【作者单位】：湖北工业大学生物医药研究院中德生物医学中心;中国科学院武汉病毒研究所病毒学国家重点实验室;
【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
【分类号】：R373.21

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