狂犬病病毒G蛋白的过表达及对病毒的抑制  

Over-expression of rabies virus G protein and its inhibitory effect on the virus in neuroblastoma cells

作　　者：阳佑天[1] 张琼[1] 张博越 刘文俊[1] 罗永文[1] 赵静[1] 梅明珠 张莹[1] 罗均[1] 郭霄峰[1] YANG Youtian;ZHANG Qiong;ZHANG Boyue;LIU Wenjun;LUO Yongwen;ZHAO Jing;MEI Mingzhu;ZHANG Ying;LUO Jun;GUO Xiaofeng(College of Veterinary Medicine,South China Agricultural University,Guangzhou 510642,China)

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《华南农业大学学报》2018年第6期10-17,共8页Journal of South China Agricultural University

基　　金："十三五"国家重点专项(2016YFD0500400);国家自然科学基金(31172322);广东省自然科学基金重点项目(2015A03031103)

摘　　要：【目的】探究G蛋白在狂犬病病毒(Rabies virus,RABV)复制中的作用,以揭示携带双G基因的重组RABV的Hep-dG与亲代毒株rHep-Flury在神经母细胞瘤(NA)细胞中滴度差异的原因,为RABV致病机制的研究奠定基础。【方法】通过病毒吸附、入侵、荧光定量PCR、Western-blot以及中和抗体阻断等试验,检测G蛋白过表达对IFN-β以及相关因子转录的影响。【结果】Hep-dG感染能显著上调NA细胞中IFN-βmRNA的表达,激活了下游因子STAT1的表达与磷酸化,在较低的感染复数(MOI=0.01)下,Hep-dG感染后24 h即可显著促进IFN-β基因的表达,36 h达到最高水平(P<0.001)。该病毒进入细胞后,产生了更多的病毒Leader RNA和RIG-I mRNA,且与IFN-βmRNA的表达高度一致。抗体阻断IFN-β后,Hep-dG在NA细胞中的病毒滴度显著上升(P<0.01),约为阻断前的7.9倍,且与亲代毒株rHep-Flury无显著差异。与阴性对照比较,5μg的pH-G质粒转染能刺激IFN-β的转录(P<0.05),表明真核表达RABV G蛋白能在一定程度上刺激IFN-β的转录。【结论】本研究初步揭示了G蛋白激活先天性免疫应答的原因和作用。RABV G蛋白的过表达,通过促进病毒Leader RNA的转录,激活了RIG-I介导的IFN-β通路,进而抑制了Hep-dG在NA细胞的繁殖。G蛋白的过表达也对干扰素通路起到一定的作用。【Objective】To explore the role of G protein in rabies virus(RABV)replication,reveal the reason for the difference of virus titer in neuroblastoma(NA)cells between the recombinant RABV Hep-dG with dual copy of G gene and the parental strain rHep-Flury,and lay a foundation for the study of RABV pathogenesis.【Method】The effects of G protein over-expression on transcriptions of IFN-βand related factors were examined by the virus binding assay,virus entry assay,fluorescence quantitative PCR,Western-blot and neutralizing antibody blocking assay.【Result】Hep-dG infection significantly increased the expression of IFN-βmRNA and activated the expression of the downstream factor STAT1 in NA cells.Under the low multiplicity of infection(MOI=0.01),the expression of IFN-βgene significantly increased at 24 h after Hep-dG infection and reached the highest level at 36 h.After the virus entered the cells,there were more viral Leader RNA and RIG-I mRNA,which were highly consistent with the expression of IFN-βmRNA.The block of IFN-βexpression by neutralizing antibody in NA cells significantly increased the virus titer of Hep-dG in cell culture supernatant(P<0.01),which was 7.9 times before blocking.Meanwhile the virus titer of Hep-dG had no significant difference with the parental strain rHep-Flury.Compared with the negative control,transfection of 5μg pH-G plasmid could stimulate the transcription of IFN-β(P<0.05),which showed that eukaryotic expression of RABV G protein could stimulate IFN-βtranscription to a certain extent.【Conclusion】This study preliminarily reveals the cause and role of G protein in activating innate immune response.Over-expression of RABV G protein activates the RIG-I-mediated IFN-βpathway by promoting transcription of the viral Leader RNA,which in turn inhibits Hep-dG replication in NA cells and finally results in the lower virus titer in NA cells.

关 键 词：狂犬病病毒 G蛋白 IFN-β通路 Β干扰素 病毒滴度 

分 类 号：S855.3[农业科学—临床兽医学]