基于粗提物的水稻条纹病毒体外“抓帽”体系  

An in vitro Cap-Snatching System of Rice Stripe Tenuivirus Based on Crude Virion Preparations

作　　者：林文忠 吴然 金晶[1] 丘萍 张洁[1] 吴祖建[1] 杜振国[1] LIN WenZhong;WU Ran;JIN Jing;QIU Ping;ZHANG Jie;WU ZuJian;DU ZhenGuo(Plant Virus Research Institute,Fujian Agriculture and Forestry University/State Key Laboratory of Ecological Pest Control for Fujian and Taiwan Crops,Fuzhou 350002)

机构地区：[1]福建农林大学植物病毒研究所/闽台作物有害生物生态防控国家重点实验室,福州350002

出　　处：《中国农业科学》2020年第21期4440-4448,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31672005,31870150)、福建省自然科学基金(2019J01656,2016J05071)。

摘　　要：【背景】布尼亚病毒目(Bunyavirales)和正黏病毒科(Orthomyxoviridae)病毒从帽子结构下游10—20个碱基处切割寄主mRNA,以5′端切割产物(帽子序列)作为引物起始自身基因组的转录,生成含一段异源帽子序列的病毒mRNA,这一过程称为“抓帽”。水稻条纹病毒(rice stripe tenuivirus,RSV)是一种布尼亚病毒,其“抓帽”机制还不甚清楚。【目的】研究RSV粗提物能否从溶液中的外源mRNA“抓帽”,以建立一种便捷的体外体系,用于解析RSV“抓帽”和转录机制。【方法】以PEG沉淀和超速离心法粗提RSV,利用聚丙烯酰胺凝胶电泳(SDS-PAGE)和质谱技术分析粗提物成分。然后取少量粗提液,加入富含珠蛋白(globin)mRNA的兔网织红细胞裂解液(rabbit reticulocyte lysate,RCL)和合适浓度的金属离子配制体外“抓帽”体系。待体外反应结束,提取体系中总RNA,以巢式RT-PCR扩增含珠蛋白-αmRNA帽子序列的RSV mRNA,并对其进行克隆,通过序列分析比较RSV体外“抓帽”与体内“抓帽”的异同。【结果】从100 g感染RSV的水稻叶片获得RSV粗提液2 mL。除RSV病毒粒子外,粗提液中还含核酮糖-1,5-二磷酸羧化酶等多种叶绿体蛋白。取2μL粗提液,加入4 mmol·L-1 MgCl2、2 mmol·L-1 NTP、0.8 U·μL-1 RNA酶抑制剂和8μL RCL,配成20μL反应体系,30℃孵育1.5 h后,巢式RT-PCR扩增到了目的条带,表明RSV粗提物能切割溶液中的珠蛋白-αmRNA,并利用其帽子序列合成自身mRNA。在反应体系中加入帽子结构类似物m7G(5′)ppp(5′)G后进行相同反应,目的条带变淡,且变淡程度与所加入m7G(5′)ppp(5′)G的浓度成正比,表明识别帽子结构是RSV从溶液中切割利用珠蛋白-αmRNA的前提。对巢式RT-PCR产物克隆和测序后分析发现,与在体内的情况类似,RSV从帽子结构下游的A或C处切割珠蛋白-αmRNA,得到的帽子序列与病毒模板链3′端的U或G配对后引发转录。在利用珠蛋白-αmRNA帽子序列�【Background】Viruses of the order Bunyavirales and the family Orthomyxoviridae cleave host cellular mRNAs 10-20 nucleotides downstream of the cap structure and use the 5′terminal cleavage product as a primer to initiate the transcription of their template RNAs.This process is called cap-snatching,the result of which is that all viral mRNAs contain a host-derived capped-RNA leader(CRL).Rice stripe tenuivirus(RSV)is a plant-infecting bunyavirus for which the mechanism of cap-snatching remains poorly characterized.【Objective】The objective of this study is to establish a convenient in vitro system to dissect the cap-snatching mechanisms of RSV,this study is designed to test whether crude preparations of RSV snatch CRLs from exogenously supplied mRNAs.【Method】Crude preparations of RSV were obtained by PEG precipitation combined with differential centrifugation.The protein components of the preparations were analyzed by SDS-PAGE and mass spectrometry.After adding rabbit reticulocyte lysate(RCL)rich in globin mRNAs,the preparations were used to establish an in vitro cap-snatching reaction mix.Total RNA was extracted from the reaction mix at the end of the reaction.RSV mRNAs with CRLs obtained from globin-αmRNAs were detected using nested RT-PCR.The products of the nested RT-PCR were cloned,and the sequences were analyzed.【Result】Two mL of crude RSV preparations was obtained from 100 g of virus-infected rice.The preparations contain chloroplast proteins including ribulose-1,5-bisphosphate carboxylase/oxygenase in addition to RSV virions.A 20μL reaction system containing 2μL RSV crude preparations,8μL RCL,4 mmol·L-1 MgCl2,2 mmol·L-1 of each NTP and 0.8 U·μL-1 RNase inhibitor was prepared and incubated at 30℃for 1.5 h.Clearly discernable bands with expected sizes were obtained in the nested RT-PCR,indicating that RSV crude preparations can cleave globin-αmRNAs and use their CRLs.After adding the cap analogue m7G(5′)ppp(5′)G,the brightness of the bands was decreased and the extent of the decr

关 键 词：水稻条纹病毒 抓帽机制 体外转录 

分 类 号：S435.111.4[农业科学—农业昆虫与害虫防治]