B19病毒非结构蛋白11 kD对p6启动子和细胞因子启动子的调控作用  

Regulation of viral p6 and cytokine promoter by Parvovirus B19 nonstructural protein 11 kD

作　　者：王媛 柯峰[2] 戈勇 殷焦 黄玉 董衍明 WANG Yuan;KE Feng;GE Yong;YIN Jiao;HUANG Yu;DONG Yan-Ming(Medical College of Hubei University of Arts and Sicence,Xiangyang,Hubei 441053,China;Xiangyang Central Hospital,Xiangyang,Hubei 444102,China;School of Life Sciences,Hubei University,Wuhan,Hubei 430062,China;BGI-Marine,Shenzhen,Guangdong 518083,China;Department of Biological Sciences,George Washington University,Washington DC 20052,USA)

机构地区：[1]湖北文理学院医学院,湖北襄阳441053 [2]襄阳市中心医院,湖北襄阳444102 [3]湖北大学生命科学学院,湖北武汉430062 [4]华大海洋研究院,广东深圳518083 [5]Department of Biological Sciences,George Washington University,Washington DC 20052,USA

出　　处：《微生物学通报》2020年第5期1460-1467,共8页Microbiology China

基　　金：襄阳市科技研究与开发项目(襄科计)[(2017)32号];湖北省自然科学基金面上项目(2017CFB241);中国博士后面上项目(2019M650176)。

摘　　要：【背景】人细小病毒B19是可以感染并引起人类疾病的两种细小病毒科成员之一。B19病毒的唯一启动子p6负责病毒RNA的转录。研究表明p6启动子活性与其转录因子结合位点以及B19病毒NS1蛋白的调节有关。但是,关于其他重要位点和B19病毒蛋白是否也具有影响p6启动子活性的作用还未有报道。【目的】探究影响p6启动子活性的重要因素,并明确B19病毒11 kD蛋白与p6启动子及细胞因子启动子的调控关系。【方法】通过生物信息方法预测分析p6启动子转录结合位点及其CpG位点,利用体外甲基化分析CpG位点对p6启动子活性的影响。同时,利用增强绿色荧光蛋白(enhanced green fluorescent protein,EGFP)和荧光素酶报告系统分析影响p6启动子活性的转录因子结合区域以及11 kD蛋白对p6启动子和细胞因子启动子的影响。【结果】p6启动子在不同非敏感细胞系中均有较高活性,并且其302-479区段的转录结合位点CCCTC结合因子(CCCTC-binding factor,CTCF)、阴阳因子(Yin Yang 1,YY1)、信号传导与转录激活因子(signal transducer and activator of transcription 3,STAT3)、Sp1/3和E2F转录调节因子7(E2F transcription factor 7,E2F7)对维持启动子活性很重要。同时,p6启动子活性可被其CpG位点的甲基化修饰所抑制,但不被11kD蛋白所调控。然而11 kD蛋白却能显著上调肿瘤坏死因子(tumor necrosis factor alpha,TNF-α)、白介素6(interleukin 6,IL6)、STAT3启动子的活性。【结论】本研究明晰了B19病毒p6启动子的潜在转录因子结合位点和CpG位点对维持其启动子活性的重要性,并进一步揭示了非结构蛋白11kD与p6启动子以及细胞内因子启动子的关系,推测11 kD蛋白可能通过影响细胞内相关因子的表达,进而在B19病毒与细胞相互作用过程中扮演重要角色。[Background]Human Parvovirus B19(B19 virus)is a member of the two Parvoviriade families that cause human diseases.The unique viral p6 promoter in B19 virus controls all the viral m RNA transcription.Previous research shows that some transcription factor binding sites(TFBSs)within p6 and nonstructural protein NS1 of B19 virus are both involved in regulation of p6 activity.However,effects of other TFBSs or viral proteins on p6 activtiy have not been reported.[Objective]Here,we investigate the roles of 11 kD protein in regulation of the activities of B19 p6 promoter as well as some cytokine promoters.[Methods]Based on bioinformatics methods,we predicted some new TFBSs and identified the CpG island in p6 region.In vitro methylation assay was carried out to study the effect of CpG on p6 activity.Meanwhile,EGFP and luciferase reporter system were employed to detect the activities of p6 and other cytokine promoters.[Results]Consistent with previous research,p6 shows similar high activity in different non-permissive cells.The truncated p6 mutation assay indicated that CTCF,YY1,STAT3,Sp1/3 and E2 F7 binding sites at nt.302-479 played significant roles in maintaining p6 promoter activity.In addition,in vitro methylation assay demonstrated that CpG methylation on p6 inhibited its promoter activity.Interestingly,though 11 kD protein did not regulate p6 activity,it increased the promoter activity of TNF-α,IL6,STAT3 significantly.[Conclusion]Our findings imply that the newly discovered TFBSs and CpG island in p6 are essential for the maintenance of p6 promoter activity.Meanwhile,the association between nonstructural protein 11 kD and cytokine promoter suggests that 11 kD may participate in B19 virus host interaction by regulating the cytokine factor expression.

关 键 词：B19病毒 11 kD蛋白 p6启动子 细胞因子启动子 

分 类 号：R373[医药卫生—病原生物学]