【作者】 李杨；

【导师】 汪铭书； 程安春；

【作者基本信息】 四川农业大学 ， 预防兽医学， 2020， 硕士

【摘要】 鸭肠炎病毒(Duck enteritis virus,DEV)皮层蛋白VP16由位于病毒基因组长独特序列(Long unique region,UL)的UL48基因编码,是DEV重要的功能蛋白。本研究通过在鸭胚成纤维细胞(Duck embryo fibroblast,DEF)中表达VP16及其截短突变体,对VP16在抗病毒天然免疫中的功能进行研究,获得以下结果:1.VP16蛋白抑制DEF细胞中IFN-β的转录水平及转录激活将1MOI鸭肠炎病毒接种于鸭胚成纤维细胞,RT-qPCR检测感染后不同时间点鸭IFN-β的mRNA转录水平,结果表明,鸭IFN-β的mRNA转录在感染24h时明显下调,与感染12h时相比降低了85%。在DEF细胞中转染VP16真核表达质粒,瞬时表达VP16蛋白,发现其能剂量依赖性的抑制IFN-β的mRNA转录,表达VP16蛋白的重组表达质粒在DEF细胞中的转染量为10μg/9.6 cm~2时对IFN-βmRNA转录水平的抑制效果较4μg/9.6 cm~2细胞的转染量增加了3倍左右。运用双荧光素酶报告基因检测系统,检测DEF细胞中VP16对IFN-β的启动子活性的影响。结果表明,表达VP16蛋白的实验组中,poly I:C激活的IFN-β启动子的活性被下调了80%,与对照组相比差异显著(p<0.01)。过表达cGAS、STING和IRF7均能明显的诱导DEF中的IFN-β启动子的激活,感染DEV或者瞬时表达VP16都能显著的抑制cGAS、STING和IRF7诱导的IFN-β启动子的激活,抑制效果与对照组相比差异极显著(p<0.001或p<0.005)。2.VP16蛋白抑制IFN-β激活免疫通路的作用位点筛选在DEF细胞中瞬时表达VP16蛋白能同时抑制poly I:C和poly d A:d T激活的抗病毒效应分子Mx蛋白和OASL蛋白的mRNA转录,VP16下调的poly I:C诱导的Mx蛋白mRNA转录水平与对照组相比具有统计学差异(P<0.05);VP16下调的poly I:C诱导的OASL蛋白mRNA转录水平与对照组相比差异显著(P<0.01)。VP16下调poly d A:d T诱导的Mx蛋白和OASL蛋白mRNA转录水平与对照组相比均差异极显著(P<0.001)。表明VP16蛋白作用于DNA病毒识别的天然免疫通路与RNA病毒识别的天然免疫通路的共同节点。在DEF中过表达DNA受体cGAS识别的天然免疫通路中的各节点免疫分子蛋白,过表达cGAS/STING、TBK-1、IRF7或IRF1蛋白均能激活IFN-β的启动子活性,但当与VP16蛋白同时表达时,cGAS/STING、TBK-1和IRF7激活的IFN-β启动子活性均被下调,与对照组相比抑制水平差异显著或极显著(P<0.005或P<0.001)。但VP16蛋白不能抑制由IRF1激活的IFN-β启动子活性,IFN-β启动子活性只降低15%以下。表明VP16蛋白作用于TBK1下游的IRF7。3.VP16蛋白作用于IRF7的分子机制的初步研究利用真核表达细胞系HEK293T共转染表达VP16蛋白和IRF7蛋白,在HEK293T细胞中进行免疫荧光共定位和免疫共沉淀实验,结果VP16能与IRF7共定位于细胞质;免疫共沉淀实验检测到VP16可与IRF7结合。表明VP16与IRF7关联。进一步检测不同的截短VP16蛋白对DEF中IFN-β启动子活性的影响效果,结果表明,截去了大部分C端的VP16(aa1-200)蛋白对poly I:C诱导的IFN-β启动子活性的抑制效果比截去部分N端的VP16(aa110-475)蛋白多70%并且差异显著(p<0.05)。综上,DEV逃逸由cGAS受体识别并激活的天然免疫通路,VP16是其发挥免疫逃逸功能的重要蛋白。VP16通过与IRF7结合,从而抑制IFN-β介导的抗病毒天然免疫,VP16发挥其免疫抑制作用的区域位于其N端。更多还原

【Abstract】 Duck enteritis virus(DEV)tegument protein VP16 encoded by the UL48 gene locatingin the long unique region(UL)of the viral genome and is an important and highly functionally protein of DEV.In this study,VP16 and its truncated mutants are expressed in duck embryo fibroblast(DEF)cells,the abilityof VP16 in antiviral innate immunity was studied,following results obtained:1.VP16 inhibited the mRNA transcriptional level and activation of IFN-βin DEFs Duck embryo fibroblast cells were inoculated with 1MOI duck enteritis virus.RT-qPCR were used to detectthe transcription level of duck IFN-βmRNA at different time points after infection.Results showed that duck IFN-βmRNA transcription level was significantly down-regulated at 24h after infection,it is 85%lower than that at 12h.Transfecting VP16expressing plasmids in DEF cells,DEV VP16 protein was functionally expressed,we found that it can dose-dependently inhibit IFN-βmRNA transcription.The recombinant expression plasmids which express VP16 protein was transfected in DEF cells at amount of10μg/9.6 cm~2,the inhibitory effect of IFN-βmRNA transcription level was increased by about 3 times compared with the group that transfected 4μg/9.6 cm~2cells.Using dual-luciferase reporter system to detect the effect of VP16 on the promoter activity of IFN-βin DEF cells.Results showed that in the experimental group expressing VP16 protein,the IFN-βpromoter activity which up-regulated by poly I:C was down-regulated by 80%,which was significantly different from the control group(p<0.01).Overexpression of cGAS,STING and IRF7 can obviously induce the IFN-βpromoter activity in DEF.Infection with DEV or transient expression of VP16 can significantly inhibit the activationof IFN-βpromoter that is induced by cGAS,STING and IRF7,meanwhile p CAGGS did not exhibit inhibitory ability by comparing with the control group,the difference was extremely significant(p<0.001 or p<0.005).2.Screening of the key site where VP16 protein utilized to dampen IFN-βactivation.VP16 protein can not only inhibit poly I:C induced Mx and OASL mRNA transcription,but also exerted the same function to poly d A:d T induced ISGs transcription.The transcription level of Mx protein mRNA induced by poly I:C down-regulated by VP16 was statistically different,P value<0.05.The transcription level of OASL protein mRNA induced by poly I:C down-regulated by VP16 had significantlystatistical difference,P value<0.01.VP16 down-regulated poly d A:d T-induced Mx protein and OASL protein mRNA transcription levels,both of which had extremely significant statistical differences,P value<0.001.The results being that VP16 protein acted on the common node of innateimmune pathway recognized by DNA virus andRNA virus.The over-expression of heterogeneous DNA sensor-cGAS stimulated the down-stream immunologic pathway.Overexpression of cGAS/STING,TBK-1,IRF7 or IRF1 protein can activate the promoter activity of IFN-β,but when VP16 protein was expressed as well,the activity of IFN-βpromoter activated by cGAS/STING,TBK-1 and IRF7 were all down-regulated,and the level of inhibition was significantly or significantly different compared with the control group(P<0.005 or P<0.001).However,VP16 protein cannot inhibit the activity of IFN-βpromoter activated by IRF1,and the activity of IFN-βpromoter is only reduced by less than 15%.This indicated that the VP16 protein could act at IRF7 level which is downstream of TBK1.3.Preliminary study on the mechanism of VP16 protein acting on IRF7 site.Using the eukaryotic expression cell line HEK293T to co-transfect and express VP16protein and IRF7 protein.Immunofluorescence colocalization and immunoprecipitation experiments were carried out in HEK293T cells.As a result,VP16 and IRF7 could co-colocalized with each other in the cytoplasm,whatsmore,VP16 combined with IRF7 using co-IP method,indicating that VP16 associated with IRF7 in vitro.Further study focused on the different truncated VP16 proteins affecting IFN-βpromoter activity in DEF,the results showed that VP16(aa1-200)lacking most of C-terminal down-regulated 70%more poly I:C-induced IFN-βpromoter activity than that caused by VP16(aa110-475)which lacks most of the N-terminal.the difference between these two groups was significant(p<0.05).In summary,DEV owns the immune-evasion ability to escape host innate immunity,especially exerts their function on the cGAS recognized pathway.VP16 is an important immune-modulatory protein who helps DEV to be free of elimination from immune system.VP16affected interferon regulatory factor 7 by binding with it and utilizing N-terminal region to inhibit interferon beta-mediated antiviral immunity.更多还原