The Induction of Unfolded Protein Response in Tembusu Virus Infected Ducklings
ZHAO DongMin,, HUANG XinMei, ZHANG LiJiao, LIU QingTao, YANG Jing, HAN KaiKai, LIU YuZhuo, LI Yin,Institute of Veterinary Science, Jiangsu Academy of Agricultural Sciences/Key Laboratory of Veterinary Biologicals Engineering and Technology, Ministry of Agriculture/National Center for Engineering Research of Veterinary Bio-products, Nanjing 210014
Abstract 【Objective】 The aim of this study was to determine the signal pathways (PERK, IRE1 and ATF6) of unfolded protein response induced by tembusu virus in ducklings, so as to provide a theoretical basis for elucidating the pathogenesis of TMUV. [Method] 1-day-old SPF ducklings were infected intraperitoneally with TMUV (JS804). Ducklings inoculated in the same manner with equal volume of RMPI-1640 were used as negative control. Five ducklings from each group were euthanized at 12, 24, 36 h and 48 h post infection, and their brains, livers and hearts were collected. The total RNAs were extracted from collected tissues by using total RNA extraction kit. Then the total RNAs were reverse transcribed into cDNA. Specific target genes representing the three known pathways of unfolded protein response were selected, and the primers were designed based on the published GenBank sequence. The relative expression of target genes was quantified by real time PCR. Real time PCR data were analyzed using the comparative Ct method (2-ΔΔCt). GAPDH was chosen as a reference gene for internal control. 【Result】 In three organs from infected ducklings, it was observed that the viral titers were the highest in the liver, lower in the heart, and the lowest in the brain. The unfolded protein response was characterized by upregulated expression of GRP78. The relative expression of GRP78 in the brain and liver were persistently upregulated and reached a peak at 36 h post infection (4.21 fold and 10.14 fold, respectively). GRP78 expression in the heart was upregulated transiently at 36 h post infection (1.32 fold). ATF4 expression represented the activation of PERK pathway. The ATF4 expression in the liver and brain were persistently upregulated from 24 h and 36 h post infection to 48 h post infection respectively, and peaked at 36 h post infection (2.71 fold and 6.02 fold, respectively). However, the upregulation of ATF4 expression in the heart was observed at 36 h post infection (1.57 fold). The activation of IRE pathway was characterized by XBP1s. In the liver, the expression level of XBP1s increased most significantly (9 fold). In the brain, the expression level of EDEM enhanced most significantly (3.87 fold) and persistently upregulated from 12 to 48 h post infection. Comparing to negative control, the expression of ATF6 pathway marker GRP94 and XBP1u were upregulated in three tissues, which reached a peaked at 36 h post infection, although expression profiles of GRP94 and XBP1u were different at indicated time points. 【Conclusion】 It was the first report that TMUV infection induced three branches of unfolded protein response in ducklings, and these results might be helpful for understanding the interaction between tembusu virus infection and host response. Keywords:Tembusu virus;SPF ducklings;endoplasmic reticulum;unfolded protein response;signaling pathway
PDF (449KB)元数据多维度评价相关文章导出EndNote|Ris|Bibtex收藏本文 本文引用格式 赵冬敏, 黄欣梅, 章丽娇, 刘青涛, 杨婧, 韩凯凯, 刘宇卓, 李银. 坦布苏病毒感染诱导雏鸭体内未折叠蛋白反应[J]. 中国农业科学, 2021, 54(4): 855-863 doi:10.3864/j.issn.0578-1752.2021.04.016 ZHAO DongMin, HUANG XinMei, ZHANG LiJiao, LIU QingTao, YANG Jing, HAN KaiKai, LIU YuZhuo, LI Yin. The Induction of Unfolded Protein Response in Tembusu Virus Infected Ducklings[J]. Scientia Acricultura Sinica, 2021, 54(4): 855-863 doi:10.3864/j.issn.0578-1752.2021.04.016
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0 引言
【研究意义】内质网是病毒蛋白折叠、装配与成熟的场所,病毒蛋白的大量合成常导致内质网应激。发生应激后,为了维持内质网稳态和功能,细胞随即启动未折叠蛋白反应(unfolded protein response,UPR)应对病毒感染导致的内质网应激。UPR不仅可以缓解内质网应激,还可调控细胞自噬与凋亡,调节炎症相关信号通路,抑制干扰素等天然抗病分子的表达,参与病毒致病过程[1]。因此,对UPR进行深入研究有助于阐明病毒的致病机制。坦布苏病毒(tembusu virus,TMUV)是一种危害鸭鹅的黄病毒属病毒,主要引起患病鸭鹅食欲急剧减退、产蛋量骤降、瘫痪及出血性卵巢炎,给鸭鹅养殖业造成了重大经济损失[2]。作为一种新发病原,TMUV的致病机制尚不明确。鉴于UPR在病毒感染及致病机制中的重要作用,本文针对TMUV感染后诱导雏鸭体内UPR进行探讨,不仅加深对TMUV致病机制的认识,也有利于TMUV感染的综合防控。【前人研究进展】坦布苏病毒属于黄病毒科、黄病毒属,病毒基因组为单股正链RNA,大小约为11 kb,可以直接作为mRNA翻译3种结构蛋白和7种非结构蛋白。黄病毒属病毒在细胞中复制的典型结构特征是病毒复制发生在细胞内质网,感染后病毒对宿主细胞内质网进行重塑[3]。内质网是真核细胞中一种重要的细胞器,具有多种生物学功能,如钙离子的储存、细胞内信号转导、跨膜和分泌型蛋白的合成和折叠等,接近三分之一的分泌型蛋白是在内质网中折叠和成熟的[4,5]。为了保证分泌蛋白可以正确折叠,内质网具有一套严谨和复杂的质量控制系统,如果错误折叠的蛋白在内质网腔中大量聚集则会影响其正常生理功能,从而导致内质网应激[6]。为了恢复内质网稳态,应对内质网应激,细胞会启动未折叠蛋白反应(unfolded protein response,UPR)。UPR可通过抑制新生蛋白的合成、降解驻留在内质网的病毒蛋白量或诱导促折叠伴侣分子表达等增强内质网功能[7]。UPR主要包括3条信号通路,分别由3种内质网驻留蛋白起始:双链RNA依赖的蛋白激酶样内质网激酶(PERK)、I型内质网转膜蛋白激酶(IRE1)和活化转录因子6(ATF6)[8]。在PERK信号通路中,真核翻译起始因子2的α亚基(eIF2α)被磷酸化后,抑制eIF2α启动的mRNA翻译,减少蛋白质合成,减轻内质网的负担。同时磷酸化的eIF2α上调激活转录因子4(ATF4)的表达,ATF4可依次上调下游转录因子C/EBPα-同源蛋白(CHOP,也称GADD153)和生长抑制DNA损伤基因34(GADD34)的表达。GADD34可与蛋白磷酸酶1(PP1)结合,水解eIF2αSer51位的磷酸基团,发挥反馈抑制作用,部分恢复蛋白的翻译[9,10]。在IRE1信号通路中,具有转录因子活性的剪切型X盒结合蛋白1(XBP1s)可诱导内质网相关降解蛋白(EDEM)和内质网相关降解途径(ERAD)中分子伴侣的表达,促进错误折叠蛋白的降解[11,12]。在ATF6信号通路中,ATF6被水解后释放出含有b-ZIP的转录激活功能域(ATF6p50),转位入核后,作为转录因子,与内质网应激响应元件(ERSE)结合,编码产生更多的分子伴侣,如GRP78、GRP94和蛋白二硫异构酶(PDI),提高内质网折叠蛋白的能力[13,14]。目前,很多研究报道黄病毒属病毒感染后可诱导UPR。登革病毒(DENV)感染后诱导的UPR途径具有时间依赖性:感染早期主要诱导PERK途径,随后该途径关闭,在感染的中晚期IRE1途径和ATF6途径依次开启[15]。西尼罗病毒(WNV)减毒株感染后可抑制PERK信号通路的开启,而具有神经毒力的强毒株感染诱导的UPR可开启3条信号通路[16,17]。日本乙型脑炎病毒(JEV)感染其易感细胞后可促进分子伴侣的表达[18]。此外,导致新生儿小头畸形和成人神经发育异常的寨卡病毒(ZIKV)可在体内、体外激活IRE1信号通路和ATF6信号通路,缓解ZIKV感染导致的内质网应激[19]。【本研究切入点】目前有关TMUV感染诱导UPR的研究较少,ZHAO等[3]的研究表明TMUV感染BHK-21细胞后,可在不同时期诱导细胞启动不同的UPR信号通路,但是尚无TMUV感染后在体内诱导UPR信号通路的相关报道。【拟解决的关键问题】为了进一步研究TMUV感染后在雏鸭体内诱导的UPR信号通路,本文通过检测UPR不同信号通路中的靶分子表达水平,分析TMUV感染在雏鸭体内诱导的UPR,拓展对TMUV感染诱导UPR的认识,为阐明TMUV致病机理奠定理论基础。
1 材料与方法
1.1 试验材料
坦布苏病毒JS804株由江苏省农业科学院兽医研究所禽病与生物兽药研究室保存;SPF鸭胚购自中国农业科学院哈尔滨兽医研究所,自行孵化至出壳,置于隔离器中饲养。组织总RNA提取试剂盒购自Invitrogen公司。反转录试剂RT SuperMix for qPCR(+gDNA wiper)、2×ChamQ SYBR Master Mix购自南京诺唯赞生物科技有限公司。
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