猪繁殖与呼吸障碍综合征病毒(PRRSV)3′末端非翻译区中调控序列的研究

Identification of porcine reproductive and respiratory syndrome virus regulation sequence in 3′-Untranslated region

摘要:

以北美株PRRSV感染性克隆pCBC2为平台进行反向遗传操作,将3′UTR中的一级结构进行了系列缺失或插入突变,并改变二级结构中的一个保守的茎环结构,构建全长PRRSV突变体克隆,解析3′UTR突变对病毒感染性的影响,旨在界定调控PRRSV3′UTR的启动子序列及二级结构,即复制过程中的最小调控元件。以空斑和Northern blot来研究拯救后重组病毒的复制、转录和生长特性,发现重组病毒感染动力学与亲本病毒无可见差别。结果表明PRRSV3′UTR的5′端可耐受一定数目的核苷酸的缺失(41nt)与插入(23nt)突变,但进一步9nt缺失造成保守的环结构突变后就使病毒失去了感染性。证明了这是3′UTR中控制PRRSV复制过程的的必需序列及二级结构,为进一步解析PRRSV复制过程的调控元件奠定了基础。

Abstract:

Based on our established infectious clone of PRRSV，designated as pCBC2，a series of mutagenesis of 3′-untranslated region (3′-UTR) at primary structure and secondary structure level were constructed. Then the full length mutant clones were transfected into MARC-145 cells，from which the influences of the discrete 3′-UTR mutation on PRRSV replication and transcription were analyzed. The properties of the rescued mutant viruses were then further characterized by Northern Blot and plaque morphology analysis. Our results demonstrated that PRRSV could tolerate more than 41 nucleotides deletion and 23nt insertion in the 3′-UTR，however，minor changes in the conserved stem loop region destroyed virus infectivity. To sum up，the stem-loop structure was essential for virus viability，but 5′ end of the 3′-UTR tolerates certain level of nucleotide deletion or insertion. This is the first report to define the essential sequence and secondary structure for PRRSV genome replication and it is useful for future research about the regulation element.