EV713C蛋白酶与ZMYM2相互作用及功能初探

Interaction and Function of EV713C Protease and ZMYM2

肠道病毒71型(Enterovirus 71,EV71)为小RNA病毒科肠道病毒属A组病毒的代表株,感染可引发手足口病(Hand-foot and mouth disease,HFMD),严重危害儿童健康。EV713C蛋白酶(3C)是其编码的主要蛋白酶之一,在病毒多蛋白加工过程中发挥关键作用,同时切割细胞蛋白以利于病毒复制。为进一步了解EV71与宿主间博弈关系,本课题组前期以3C为诱饵进行酵母双杂交实验,筛选与3C发生相互作用的可能底物,钓取到锌指MYM型蛋白2(Zinc finger MYM-type protein 2,ZMYM2)。ZMYM2是一种具有锌指结构的转录因子,与细胞中重要的抗病毒小体PML核体(PML nuclear bodies,PML-NBs)的形成及稳定性相关。本文选择3C与ZMYM2关系展开研究,确证二者相互作用并初探生物学功能。首先通过免疫共沉淀实验确证3C与ZMYM2之间存在相互作用;随后分析功能,发现过表达ZMYM2抑制EV71复制;敲减内源ZMYM2有利于EV71的复制;分析3C对ZMYM2影响,发现3C剂量依赖性切割ZMYM2,ZMYM2上至少具有2个3C的识别位点。本研究为解析ZMYM2功能及进一步了解EV71与宿主先天免疫间博弈关系提供了新的实验证据。

Enterovirus 71(EV71)is an emerging pathogen that causes hand,foot,and mouth disease(HFMD)as well as fatal neurologic diseases in infants and young children.Efficacious therapeutic drugs or vaccines for EV71 are lacking due(at least in part)to the molecular basis of the interaction between EV71 and host defense system being poorly understood.ZMYM2 is a cellular transcription factor with a“zinc finger”structure,which is related to the formation and stability of important antiviral PML nuclear bodies(PML⁃NBs)in cells.In order to further understand the interaction between EV71 and the host,we previously conducted a yeast two⁃hybrid experiment with the viral protease 3C as the“bait”to screen possible substrates of 3C,and discovered that ZMYM2 was a candidate.Here,we confirmed that EV713C could interact with ZMYM2,and that overexpression of ZMYM2 inhibited EV71 replication.Depletion of endogenous ZMYM2 by short⁃hairpin RNA promoted EV71 replication.Remarkably,3C targeted ZMYM2 and induced its cleavage,which was dependent upon 3C protease activity.Further analysis revealed that there are at least two cleavage sites of 3C on ZMYM2.These results provide new experimental evidences for analyzing ZMYM2 function,and may reveal a new means for EV71 to evade the host antiviral response.