猪繁殖与呼吸综合征病毒5'非翻译区一顶端茎环结构是病毒复制转录必需的顺式作用元件  

A PROMINENT STEM-LOOP STRUCTURE IN 5' UNTRANSLATED REGION OF PORCINE REPRODUCTIVE AND RESPIRATORY SYNDROME VIRUS IS ESSENTIAL FOR VIRAL REPLICATION AND TRANSCRIPTION

作　　者：高飞[1] 郑浩[1] 姜一峰[1] 李丽薇[1] 郑海红[1] 周艳君[1] 韦祖樟[2] 童光志[1] 

机构地区：[1]中国农业科学院上海兽医研究所,上海200241 [2]广西大学动物科学技术学院,南宁530005

出　　处：《中国动物传染病学报》2014年第4期7-16,共10页Chinese Journal of Animal Infectious Diseases

基　　金：国家自然科学基金(31300140);863计划(2011AA10A208-2);科研院所基本科研业务费(2013JB01;2013JB02)

摘　　要：猪繁殖与呼吸综合征病毒(Porcine reproductive and respiratory syndrome virus,PRRSV)基因组5'非翻译区(untranslated region,UTR)对病毒复制转录等生物合成过程至关重要,但其作用机制尚不清楚。本实验室分别在北美型(pAPRRS)与欧洲型PRRSV(pSHE)感染性克隆骨架的5'UTR与ORF1之间插入碱基"at"形成Nde I位点,构建了两个突变体pTLNd4和pSHEnde。结果显示插入后的碱基未影响病毒感染性。以两株突变体为骨架将两株感染性克隆的5'UTR互换,构建了突变体克隆pTLV8和pSHSP5。结果表明pTLV8仍具有感染性,而pSHSP5株拯救出病毒。对pSHSP5和pTLV8的RNA二级结构预测结果显示,虽然嵌合突变体pTLV8的5'端与亲本pAPRRS的二级结构差异较大,但转录调控序(transcription-regulating sequence,TRS)仍位于顶端茎环结构最突出的位置,并保证了TRS与ORF1之间的起始密码子AUG位于该茎环结构的顶端,而pSHSP5的顶端茎环结构则遭到了破坏。本研究结果表明TRS所在的顶端茎环结构在调控病毒复制转录过程中是关键的顺式作用元件之一,对其结构破坏的突变体将不能拯救出病毒。The 5＇ untranslated region （UTR） of the genomic RNA is believed to be vital for the replication and transcription of Porcine reproductive and respiratory syndrome virus （PRRSV）, yet its functional mechanism remains unclear. Based on infectious full-length cDNA clones pAPRRS and pSHE of type 1 and type 2 PRRSV, exogenous“at”was inserted between 5＇ UTR and ORF1 to form Nde I digestion site, then pTLNd4 and pSHEnde were obtained. After transfection and passage on cell monolayers, the infectiousness of pTLNd4 and pSHEnde was demonstrated. Afterwards, the 5＇ UTR chimeric mutants pTLV8 and pSHSP5 were obtained. Interestingly pTLV8 developed cytopathic effect （CPE） on MARC-145 cells while pSHSP5 was a lethal mutant. Comparison of the secondary structures of 5＇ proximal sequences revealed that a signifi cant change occurred in pSHSP5 but not in pTLV8. The prominent stem-loop structure remained functional in pTLV8 as TRS and AUG of ORF1 were located in the top loop. Taken together, the prominent stem-loop structure where TRS is located is one of the crucial cis- acting elements in regulating viral replication and transcription. Therefore, any changes that alter the stem-loop structure would be lethal for mutant viruses.

关 键 词：猪繁殖与呼吸综合征病毒 5’非翻译区 嵌合突变体 转录调控序列 二级结构预测 

分 类 号：S852.659.6[农业科学—基础兽医学]