鼠冠状病毒核衣壳蛋白电荷非均一性与其磷酸化的相关性

The charge heterogeneity of murine coronavirus nucleocapsid protein is related to phosphorylation

核衣壳(nucleocapsid,N)蛋白是病毒必不可少的结构蛋白,具有保护病毒基因组和调控病毒复制进程的重要作用。作为冠状病毒的经典模型,鼠肝炎病毒(mouse hepatitis virus,MHV)N蛋白的磷酸化调控病毒RNA复制转录和装配已有报道。本研究发现MHV-A59感染鼠neuro-2a细胞后期表达的N蛋白呈现明显的电荷不均一性,其中只有小部分发生磷酸化并被装配至病毒颗粒中;等电聚焦电泳显示N蛋白的电荷呈连续分布。用蛋白脂酰化抑制剂2-溴棕榈酸(2-BP)和蛋白酶体抑制剂MG-132处理后显示病毒复制水平不但与N蛋白电负性相关,而且与细胞的PKCαS657磷酸化和内质网蛋白水平相关。结果提示磷酸化修饰与鼠冠状病毒N蛋白电荷不均一性及稳定性密切相关。

The nucleocapsid(N)protein is an essential structural protein of the virus and plays an important role in protecting the viral genome and regulating the process of viral replication.The phosphorylation of N protein of mouse hepatitis virus(MHV),has been proved to be associated with viral transcription,replication and assembly.We found that N protein expressed at late stage rather than early stage in neuro-2a cells infected with MHV-A59 showed a significant charge heterogeneity.Isoelectric focusing electrophoresis showed that the charge of N protein was continuously distributed.Treatment with the protein S-palmitoylation inhibitor 2-BP and the proteasome inhibitor MG-132 showed that viral replication levels were not only associated with N protein electronegativity,but also with the PKCαphosphorylation at S657 and the maintenance of some endoplasmic reticulum components involved in protein modification and degradation.These results suggest that phosphorylation is a factor associated to the heterogeneity and stability of the murine coronavirus N protein.