摘要
本研究旨在探究日本脑炎病毒(JEV)感染细胞后产生非编码亚基因组RNA(sfRNA)的生物学功能。应用Northern blot检测JEV感染细胞产生sfRNA的动态特征;通过Western blot和qPCR检测JEV感染细胞中PKR、IFITM2等干扰素刺激基因的表达;通过体外转录制备JEV sfRNA,转染细胞研究其功能。实验结果表明:JEV感染Huh7细胞后期可下调PKR、IFITM2的表达,与sfRNA产生的时间规律一致;将体外转录制备的JEV sfRNA转染细胞后可下调IFN-α刺激细胞中IFITM2、PKR蛋白的含量,但其mRNA水平与对照组差异不显著,推测JEV sfRNA抑制其翻译过程;SL-Ⅱ区域缺失的JEV sfRNA不能有效下调干扰素诱导细胞中IFITM2和PKR蛋白的表达。因此,本研究发现JEV sfRNA在翻译阶段抑制PKR和IFITM2蛋白的表达,为病毒增殖营造有利环境。
The aim of this research was to investigate the biological function of subgenomic flaviviral RNA(sfRNA)produced in Japanese encephalitis virus(JEV)infected cells.The dynamic characteristics of sfRNA production in JEV infected cells was determined by Northern blot.The expression of interferon-stimulated genes such as PKR and IFITM2 in JEV infected cells were identified by Western blot and qPCR.JEV sfRNA was developed by transcription in vitro and its function was investigated through transfection experiments.The results show that the expression of PKR and IFITM2 were down regulated at late infection stage in JEV infected cells,which was consistent with the time when sfRNA was generated.In cells transfected with JEV sfRNA synthesis in vitro,the protein expression of IFITM2 and PKR stimulated by IFN-αwas decreased,while their mRNA levels show no significant difference compared with the control group,which suggested the translation process was inhibited.Deletion of the SL-Ⅱregion of JEV sfRNA did not down regulate the expression of IFITM2 and PKR proteins effectively in interferon treated cells.In conclusion,JEV sfRNA inhibited the expression of PKR and IFITM2 proteins during translation,creating a favorable environment for JEV proliferation.
作者
杨兴淼
徐昊
曹瑞兵
YANG Xingmiao;XU Hao;CAO Ruibing(College of Veterinary Medicine,Nanjing Agricultural University,Nanjing 210095,China)
出处
《中国动物传染病学报》
CAS
北大核心
2022年第3期29-39,共11页
Chinese Journal of Animal Infectious Diseases
基金
国家重点研发计划(2016YFD0500402)
国家自然科学基金面上项目(31772756)。
