新城疫P蛋白磷酸化位点的质谱分析和功能鉴定  

IDENTIFICATION AND FUNCTIONAL ANALYSIS OF PHOSPHORYLATION SITES IN NEWCASTLE DISEASE VIRUS PHOSPHOPROTEIN

作　　者：李继红[1] 徐腾飞 张耀丹[1] 汪伟[1] 孟春春[1] 孙英杰[1] 谭磊[1] 宋翠萍[1] 廖瑛[1] 仇旭升[1] 丁铲[1] LI Ji-hong;XU Teng-fei;ZHANG Yao-dan;WANG Wei;MENG Chun-chun;SUN Ying-jie;TAN Lei;SONG Cui-ping;LIAO Ying;QIU Xu-sheng;DING Chan(Shanghai Veterinary Research Institute,CAAS,Shanghai 200241,China)

机构地区：[1]中国农业科学院上海兽医研究所

出　　处：《中国动物传染病学报》2019年第5期1-6,共6页Chinese Journal of Animal Infectious Diseases

基　　金：国家重点研发计划重点专项(2016YFD0501603);国家自然科学基金(31101822,31530074)

摘　　要：副黏病毒磷蛋白,即P蛋白(phosphoprotein),富含丝氨酸和苏氨酸,在自然条件下磷酸化水平较高。副黏病毒P蛋白的磷酸化对病毒的转录和复制具有重要的作用,但新城疫病毒(Newcastle disease virus,NDV)P蛋白磷酸化位点及其功能依然尚不明确。本研究通过LC-MS/MS质谱对NDV La Sota病毒P蛋白的磷酸化位点进行了检测,鉴定了Ser56、Ser77、Thr78、Thr82、Ser164和Ser141六个磷酸化位点。通过在线分析,预测这些磷酸化位点的磷酸化激酶分别是PKC、CKII和GSK3。为了鉴定6个磷酸化位点的生物学功能,在已建立的表达GFP基因的NDV微型基因组质粒pTVT-LGT基础上,用荧光素酶报告基因(luciferase)替换GFP报告基因,建立了表达荧光素酶的NDV微型基因组系统。利用“丙氨酸扫描”对6个磷酸化位点进行突变后,在NDV微型基因组平台中进行功能鉴定。结果显示,T78、T82和S164位磷酸化位点的缺失显著影响了病毒RNA依赖性RNA聚合酶复合体(RNA dependent RNA polymerase,RdRp)的复制,其生物学意义有待进一步的研究。The P protein(phosphoprotein)of paramyxoviruses is characterized by incorporating highly phosphorylated serine and threonine.The phosphorylation of paramyxovirus P protein plays an important role in viral transcription and duplication.However,the position and function of P protein phosphor-sites of Newcastle disease virus(NDV)are still not clear.In this study,the LaSota P protein was analyzed by LC-MS/MS and 6 phosphorylation sites were found at Ser56,Ser77,Thr78,Thr82,Ser164 and Ser141,which were caused by kinases PKC,CKII and GSK3.In order to determine the function of these phosphor-sites,the NDV minigenome system was established for expression of the reporter gene Luciferase.Plasmids expressing P mutants lacking Ser56,Ser77,Thr78,Thr82,Ser164 and Ser141 were constructed and detected via NDV minigenome system.As a result,the mutations at T78,T82 and S164 significantly influenced the expression of report gene,suggesting these phosphor-sites might play a role in the process of RNA dependent RNA polymerase(RdRp)functioning and need to be further investigated.

关 键 词：新城疫病毒 质谱 微型基因组 磷酸化位点 

分 类 号：S852.659.5[农业科学—基础兽医学]