猪繁殖与呼吸综合征病毒5′非翻译区一茎环结构的功能解析  

Function Analysis of a Conserved Stem-Loop Structure in the 5′ Untranslated Region of the Porcine Reproductive and Respiratory Syndrome Virus Genome

作　　者：陆嘉琦[1] 高飞[1] 刘萍[1] 袁世山[1] 

机构地区：[1]中国农业科学院上海兽医研究所/中国动物卫生与流行病学中心上海分中心猪传染病研究室农业部动物寄生虫病重点实验室,上海200241

出　　处：《畜牧兽医学报》2010年第11期1428-1434,共7页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：国家自然科学基金面上项目(30972204);国家重点基础研究发展计划"973计划"(2005CB523202);上海市自然科学基金项目(08ZR1422800)

摘　　要：以猪繁殖与呼吸综合征病毒(PRRSV)北美株感染性克隆为平台进行反向遗传操作,利用突变PCR将5′非翻译区(5′UTR)中部分一级序列进行系列突变,以改变5′UTR二级结构中的一保守茎部结构,从而解析PRRSV5′UTR中结构与功能的关系。通过DNA转染MARC-145细胞观察突变体克隆的感染性,并通过免疫荧光及Northern blot来研究拯救后突变病毒的复制、转录特性,以及通过空斑形态学分析突变病毒的生长特性。结果表明,PRRSV5′UTR中保守茎部结构的二级结构与病毒的拯救无直接关系,但其一级核苷酸序列却是PRRSV病毒复制所必需的,作者还找到直接调控病毒复制的核心位点。由此证实了5′UTR中调控PRRSV复制过程的必需序列,为进一步解析PRRSV复制过程的调控元件奠定了基础。The objective of this study was to analyze the secondary structure and function of Porcine Reproductive and Respiratory Syndrome Virus（PRRSV） 5′ UTR.Based on the PRRSV type 2 full-length infectious cDNA clone pAPRRS,a series of mutants which can modify a conserved stem structure were obtained through PCR-based mutagenesis.The mutants are transfected into MARC-145 cells for cytopathic effect（CPE） observation.Meanwhile,the indirect immunofluorescence analysis（IFA） and Northern blot analysis show the genomic replication and subgenomic transcription for the rescued viruses.In addition,the growth features are also detected by plaque morphology compared with the parental virus.It was found that the secondary structure of the conserved stem loop in PRRSV 5′ UTR wasn’t essential for virus rescue.Novertheless,the primary sequence of the stem structure is functional for viral replication.In addition,it is demonstrated that a key nucleotide in PRRSV 5′ UTR directly regulate the viral replication.All results demonstrate that the essential sequences in PRRSV 5′ UTR are functional in viral genomic replication.

关 键 词：猪繁殖与呼吸综合征病毒 5′UTR 高级结构与功能 病毒复制 基因表达调控 

分 类 号：S852.659.6[农业科学—基础兽医学]