目的探讨丙型肝炎病毒NS5A反式调节蛋白9(hepatitis C virus NS5Atransactivated protein 9,NS5ATP9)在乙型肝炎病毒(hepatitis B virus,HBV)共价闭合环状DNA(covalently closed circular DNA,cccDNA)形成与转录中的作用机制。方法利用...目的探讨丙型肝炎病毒NS5A反式调节蛋白9(hepatitis C virus NS5Atransactivated protein 9,NS5ATP9)在乙型肝炎病毒(hepatitis B virus,HBV)共价闭合环状DNA(covalently closed circular DNA,cccDNA)形成与转录中的作用机制。方法利用1.3拷贝HBV表达质粒转染Huh7和HepG2细胞、整合有4拷贝HBV基因组的HepG2.2.15细胞、在诱导型四环素启动子控制下表达HBV的HepAD38细胞构建NS5ATP9过表达或干扰的HBV细胞模型,收集样品和细胞上清液,提取RNA、HBV核心DNA(coreDNA)、cccDNA和蛋白,利用酶联免疫吸附试验、实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)、Southern blot和Western blot技术检测HBV总RNA、前基因组RNA(pregenomic RNA,pgRNA)、乙型肝炎病毒s抗原(hepatitis B virus s antigene,HBsAg)、乙型肝炎病毒e抗原(hepatitis B virus e antigene,HBeAg)、松弛环状DNA(relax circular DNA,rcDNA)以及cccDNA水平。在HepG2细胞中转染乙型肝炎病毒x蛋白(hepatitis B virus x protein,HBx),通过免疫荧光成像及免疫共沉淀方法检测NS5ATP9与HBx的结合情况。双荧光素酶报告基因实验检测NS5ATP9对HBx启动子活性的影响。利用Huh7细胞转染HBV1.3及HBV稳定表达细胞株HepG2.2.15和HepAD38转染NS5ATP9过表达/干扰质粒,通过Western blot技术检测DDB1和SMC6的蛋白水平。结果在HBV病毒活跃的细胞中,NS5ATP9 mRNA水平[HepG2.2.15细胞:1.891±0.567比1.00±0.034,t=2.87,P=0.0351;HepAD38 tet+细胞:1.978±0.399比1.00±0.034,t=4.131,P=0.0091;HepAD38 tet-细胞:2.642±0.672比1.00±0.034,t=4.127,P=0.0091]和蛋白水平均显著增加。过表达NS5ATP9后可显著增加HBeAg[(5.402±0.327)S/COV比(2.68±0.552)S/COV,t=7.35,P=0.0018]、HBsAg[(2.846±0.185)S/COV比(1.512±0.221)S/COV,t=8.02,P=0.0013]、HBV pgRNA及rcDNA的表达水平,而干扰NS5ATP9后此增加作用消失[HBeAg:(2.029±0.09)S/COV比(3.733±0.445)S/COV,t=6.501,P=0.0029;HBsAg:(1.501±0.105)S/COV比(1.878±0.174)S/COV,t=3.216,P=0.0324)]。机制研究显示,NS5ATP9和HBx蛋白主要位于细胞核核仁内,并具有共定位信号,且NS5ATP9可显著提高HBx启动子(1071.06±79.44比488.47±40.12,t=13.09,P=0.00012)的转录活性。另外,过表达NS5ATP9可显著降低DDB1和SMC6的蛋白水平,而沉默NS5ATP9则可显著提高DDB1和SMC6的蛋白水平。结论HBV上调NS5ATP9的表达,形成HBV-NS5ATP9-HBV cccDNA-HBV的正反馈环路,NS5ATP9通过与HBx相互作用上调肝细胞中HBV cccDNA的形成与转录,进而促进慢性乙型肝炎的发生发展。展开更多
Zika virus (ZIKV), a mosquito-borne flavivirus, has been associated with benign infections for decades. However, it has become a public health concern due to its association with severe fetal and neurological complica...Zika virus (ZIKV), a mosquito-borne flavivirus, has been associated with benign infections for decades. However, it has become a public health concern due to its association with severe fetal and neurological complications. Although many efforts have been made to control ZIKV infection, approved vaccines or antiviral drugs are still lacking. Consequently, the development of new effective anti-ZIKV agents is urgently needed. In this context, we investigated the antiviral potential of pyrazolone derivatives against ZIKV replication using in silico and in vitro methods. The four pyrazolone derivatives evaluated (1a, 1b, 1c, and 1d) inhibited over 50% of ZIKV replication with low cytotoxicity. Among them, compound 1b exhibited the most potent activity (EC50 = 4.3 μM) and the highest selectivity (SI = 342). Mechanism of action studies indicated that these compounds act at early stages of virus replication, and compound 1b can also directly inactivate ZIKV particles. Molecular docking studies suggested that these compounds can bind to and block the activity of ZIKV NS5 methyltransferase. Finally, pharmacokinetic and toxicological predictions have reinforced the safety and drug-like profiles of these derivatives. In conclusion, the pyrazolone scaffold proved to be valuable for anti-ZIKV drug development, and the derivatives studied deserve further investigation.展开更多
文摘目的探讨丙型肝炎病毒NS5A反式调节蛋白9(hepatitis C virus NS5Atransactivated protein 9,NS5ATP9)在乙型肝炎病毒(hepatitis B virus,HBV)共价闭合环状DNA(covalently closed circular DNA,cccDNA)形成与转录中的作用机制。方法利用1.3拷贝HBV表达质粒转染Huh7和HepG2细胞、整合有4拷贝HBV基因组的HepG2.2.15细胞、在诱导型四环素启动子控制下表达HBV的HepAD38细胞构建NS5ATP9过表达或干扰的HBV细胞模型,收集样品和细胞上清液,提取RNA、HBV核心DNA(coreDNA)、cccDNA和蛋白,利用酶联免疫吸附试验、实时荧光定量聚合酶链反应(polymerase chain reaction,PCR)、Southern blot和Western blot技术检测HBV总RNA、前基因组RNA(pregenomic RNA,pgRNA)、乙型肝炎病毒s抗原(hepatitis B virus s antigene,HBsAg)、乙型肝炎病毒e抗原(hepatitis B virus e antigene,HBeAg)、松弛环状DNA(relax circular DNA,rcDNA)以及cccDNA水平。在HepG2细胞中转染乙型肝炎病毒x蛋白(hepatitis B virus x protein,HBx),通过免疫荧光成像及免疫共沉淀方法检测NS5ATP9与HBx的结合情况。双荧光素酶报告基因实验检测NS5ATP9对HBx启动子活性的影响。利用Huh7细胞转染HBV1.3及HBV稳定表达细胞株HepG2.2.15和HepAD38转染NS5ATP9过表达/干扰质粒,通过Western blot技术检测DDB1和SMC6的蛋白水平。结果在HBV病毒活跃的细胞中,NS5ATP9 mRNA水平[HepG2.2.15细胞:1.891±0.567比1.00±0.034,t=2.87,P=0.0351;HepAD38 tet+细胞:1.978±0.399比1.00±0.034,t=4.131,P=0.0091;HepAD38 tet-细胞:2.642±0.672比1.00±0.034,t=4.127,P=0.0091]和蛋白水平均显著增加。过表达NS5ATP9后可显著增加HBeAg[(5.402±0.327)S/COV比(2.68±0.552)S/COV,t=7.35,P=0.0018]、HBsAg[(2.846±0.185)S/COV比(1.512±0.221)S/COV,t=8.02,P=0.0013]、HBV pgRNA及rcDNA的表达水平,而干扰NS5ATP9后此增加作用消失[HBeAg:(2.029±0.09)S/COV比(3.733±0.445)S/COV,t=6.501,P=0.0029;HBsAg:(1.501±0.105)S/COV比(1.878±0.174)S/COV,t=3.216,P=0.0324)]。机制研究显示,NS5ATP9和HBx蛋白主要位于细胞核核仁内,并具有共定位信号,且NS5ATP9可显著提高HBx启动子(1071.06±79.44比488.47±40.12,t=13.09,P=0.00012)的转录活性。另外,过表达NS5ATP9可显著降低DDB1和SMC6的蛋白水平,而沉默NS5ATP9则可显著提高DDB1和SMC6的蛋白水平。结论HBV上调NS5ATP9的表达,形成HBV-NS5ATP9-HBV cccDNA-HBV的正反馈环路,NS5ATP9通过与HBx相互作用上调肝细胞中HBV cccDNA的形成与转录,进而促进慢性乙型肝炎的发生发展。
文摘Zika virus (ZIKV), a mosquito-borne flavivirus, has been associated with benign infections for decades. However, it has become a public health concern due to its association with severe fetal and neurological complications. Although many efforts have been made to control ZIKV infection, approved vaccines or antiviral drugs are still lacking. Consequently, the development of new effective anti-ZIKV agents is urgently needed. In this context, we investigated the antiviral potential of pyrazolone derivatives against ZIKV replication using in silico and in vitro methods. The four pyrazolone derivatives evaluated (1a, 1b, 1c, and 1d) inhibited over 50% of ZIKV replication with low cytotoxicity. Among them, compound 1b exhibited the most potent activity (EC50 = 4.3 μM) and the highest selectivity (SI = 342). Mechanism of action studies indicated that these compounds act at early stages of virus replication, and compound 1b can also directly inactivate ZIKV particles. Molecular docking studies suggested that these compounds can bind to and block the activity of ZIKV NS5 methyltransferase. Finally, pharmacokinetic and toxicological predictions have reinforced the safety and drug-like profiles of these derivatives. In conclusion, the pyrazolone scaffold proved to be valuable for anti-ZIKV drug development, and the derivatives studied deserve further investigation.
