自噬相关基因Atg3在埃博拉病毒组装过程中的作用研究

The Role of Autophagy-related Gene 3 in the Assembly Process of Ebola Virus

自噬是真核细胞清除胞内冗余物质的降解机制,也是细胞针对病原体入侵的一种天然免疫机制。自噬相关基因3(Autophagy-related gene 3,Atg3)在细胞自噬发生过程中起决定性作用。为研究Atg3对埃博拉病毒(Ebola virus,EBOV)组装的影响,本研究首先根据CRISPR/Cas9技术构建了携带有Atg3基因靶向向导RNA(guide RNA,gRNA)、表达Cas9的重组质粒pSpCas-Atg3,将此质粒转染至人胚胎肾细胞293T中,之后采用流式分选结合有限稀释法筛选单细胞克隆。细胞基因测序及Western Blot试验表明成功获得了Atg3基因敲除的细胞系。在Agt3基因敲除细胞系和野生型细胞中分别共同转染表达EBOV囊膜蛋白(Glycoprotein,GP)和基质蛋白的重组质粒,包装病毒样颗粒(Virus-like particles,VLP);同时在两种细胞中组装整合有EBOV的重组HIV假病毒,获得的假病毒感染非洲绿猴肾细胞(Vero细胞),通过测定报告基因的活性判定病毒对细胞的侵入能力。结果显示Atg3基因缺失后,VLP所整合的GP蛋白显著增多,假病毒对细胞的侵染能力也明显增强(P<0.001),表明Agt3能够负调控EBOV的组装和入侵。本研究为开发新抗病毒药物提供了新思路。

Autophagy is a cellular process in which redundant proteins are subjected for degradation.It also represents an innate immune mechanism against pathogens.Autophagy-related gene 3(Atg3)is important in the regulation of autophagy.In order to study the effect of Atg3 for assembly of Ebola virus(EBOV),we first constructed a Atg3-specific guide RNA(gRNA),as well as Cas9-expressing recombinant plasmid(namely pSpCas-Atg3)to screen Atg3 knockout(KO)cell lines by using CRISPR/Cas9 technology.After transfection with pSpCas-Atg3,the green fluorescent protein(GFP)-positive 293 T cells were sorted and diluted to obtain monoclonal cells.Through cellular genetic sequencing and Western Blot analysis,it was found that the Atg3 was successfully knocked out.The Atg3 KO cells were co-transfected with two plasmids expressing EBOV glycoprotein(GP)and VP40 respectively to package EBOV-like particles(VLPs).In addition,the GP and human immunodeficiency virus(HIV)backbone plasmids were also co-transfected into the genetic KO cells to produce HIV-pseudotyped viruses.Relative infectivity was determined according to the expression of reporter gene(luciferase)after Vero cells were transducted with the pseudotyped virus.Results indicated that,in Atg3 KO cells,the incorporation of EBOV GP onto VLP was much higher than that in wild type(WT)cells.Accordingly,the pseudotyped virus produced in KO cells showed higher infectivity than that produced in WT cells.Results of current study clearly demonstrate that Atg3 can regulate EBOV assembly negatively,and may provide a new strategy for the design of anti-viral drugs.