为了探索猪繁殖与呼吸综合征病毒(PRRSV)N蛋白磷酸化对于病毒复制和转录的影响,本研究利用Phos-tag和点突变技术实现了N蛋白磷酸化形式的分离并鉴定出磷酸化位点为第120位丝氨酸(Ser120).通过反向遗传操作系统将N蛋白的磷酸化位点突变并拯救出病毒.空斑实验、IFA检测、病毒生长曲线结果显示,磷酸化位点突变对于病毒的感染性和N蛋白的核定位没有影响,但是病毒的复制能力减弱.进一步利用荧光定量RT-PCR方法检测突变病毒与亲本病毒在不同感染时间的基因组RNA和亚基因组RNA的转录水平,结果表明突变病毒的gRNA和长链sgRNAs转录水平明显低于亲本病毒,表明N蛋白Ser120磷酸化可能参与了病毒的转录调控过程.%In order to explore the functional significance of nucleocapsid protein phosphorylation of porcine reproductive and respiratory syndrome virus (PRRSV) during its infection,The N protein phosphorylation site was identified as serine 120 by using site-directed mutagenesis and Phos-tag SDS-PAGE based western blot.Furthermore,the phosphorylation site was mutated by reverse genetics manipulation and the mutant virus was rescued.Plaque assay,IFA detection and viral growth curve results showed that abrogation of N protein phosphorylation had no impact on virus viability,infectivity,and N protein subcellular localization.However,the growth ability of the mutant virus was impaired.Real-time RT-PCR results showed that the abundance of genome and longer sgRNAs of mutant virus were significantly lower than that of parental virus.Thus,we conclude that N protein Serl20 phosphorylation is not essential for virus viability,but may play an important role in regulating viral RNA transcription during PRRSV infection.
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