【前沿速递】南京大学医学院公共健康研究中心吴稚伟团队发现早幼粒细胞白血病蛋白（P

　　导 读肠道病毒71型（Enterovirus 71, EV71）是导致手足口病（Hand Foot and Mouth disease, HFMD）的主要病原体之一， EV71能够有效抑制宿主的天然免疫，在某些病儿中引起脑炎。阐明EV71-宿主相互作用，如何抑制和逃逸宿主免疫防御是免疫学的重大科学问题。EV71感染可以诱导自噬水平的明显升高，同时EV71病毒在复制的过程也充分利用自噬来帮助自己在宿主细胞中实现病毒增殖。2018年6月，著名国际学术期刊Frontiers in Immunology 在线发表了吴稚伟教授团队的最新研究成果《PML restricts enterovirus 71 replication by inhibiting autophagy》。研究揭示了早幼粒细胞白血病蛋白（PML）在EV71复制过程中起到限制性因子作用的分子机制，以及病毒如何通过自编码蛋白降解PML逃逸宿主的免疫抑制。

　　研究背景EV71是引起手足口病重症病例和死亡病例的主要病原之一。手足口病表现在皮肤和口腔上，但病毒会侵犯心、脑、肾等重要器官。早幼粒细胞白血病蛋白核体（PML-NBs）是动态细胞结构，由许多瞬时和永久定位的蛋白质组成。早幼粒细胞白血病（PML）也称为TRIM19，MYL，PP8675或RNF71，是PML-NBs的主要成分，在基因组稳定性，程序性细胞死亡和抗病毒活性中起着重要作用。 PML最早被发现是肿瘤的负调控蛋白。PML在细胞核内主要由6种亚型组成，来源于单个基因的选择性剪接。所有异构体都有一个由外显子1-3编码的类似N-末端区域，并含有RBCC基序，PML通过该基序多聚形成环状结构，与核基质结合形成PML-NBs。C端区域决定不同亚型的PML具有不同的生物学功能。 PML启动子区包含PML的靶点基因，可被包括I型和II型干扰素（IFN）直接诱导，上调PML亚型的表达和PML-NBs数量及体积。在最新的研究中，我们发现PML可以作为宿主限制性因子抑制EV71复制。 PML通过抑制细胞中的自噬来抑制EV71的复制，反之PML缺陷引发自噬，从而辅助病毒复制。此外，IFN-β介导的抗病毒天然免疫功能需要PML的参与，这表明ISG可能需要PML的存在才能建立抗病毒状态。 但是，EV71能够通过其编码的3Cpro 病毒蛋白酶诱导PML降解，破坏PML核体结构，从而逃逸宿主的抗病毒机制。 这些发现证明了PML在针对EV71的天然免疫防御中的重要作用，并阐述了病毒逃避先天免疫的新机制。

　　结果速览本研究主要通过降低和敲出PML表达后，在HeLa和MEF细胞里发现了自噬水平的改变。与野生型细胞比较，降低和敲出的细胞里，自噬水平升高；同时在EV71分别感然细胞后，降低和敲出PML的细胞里的自噬水平在原有的基础上并未发生明显的升高，我们推测这里自噬水平的增高完全来源于PML的缺失和降低造成（图1）。Fig. 1. PML降低和缺失后在HeLa 和MEF细胞里自噬水平的变化 利用免疫荧光的方法发现EV71感染细胞后可以导致感染细胞核内的PML核体的减少和消失（图2）。并且发现主要由3C蛋白酶切割PML（图3）。Fig. 2. 免疫荧光检测在感染EV71的细胞核内PML核体的减少Fig. 3 .3C蛋白酶切割降解PML

　　结 语该研究的主要发现：在PML敲低或者敲除的细胞中EV71复制增加；在PML的6种亚型中，PML III和IV被鉴定为对抗EV71的抗病毒而PMLIII或PMLIV的过表达抑制EV71病毒生产。 因此，PML是EV71复制的限制性因子。PML通过抑制感染细胞中的自噬来抑制EV71的复制。EV71感染诱导PML的降解，不依赖于蛋白酶体途径，而是病毒3C蛋白酶介导PML降解。该研究由国家传染病重大专项，国家重点研发计划资助。This study was supported by grants from the National Health and Family Planning Commission of China (2018ZX10301406-001 to ZW), the Ministry of Science and Technology of China (2016YFC1201000 to ZW), and Nature Science Foundation of Jiangsu Province (BY2015069-02 to ZW). 博士研究生陈德燕为第一作者，吴稚伟教授为通讯作者。

　　Abstract

　　The promyelocytic leukemia (PML) protein, also known as TRIM19, functions as a major organizer of PML nuclear bodies (NBs) in most mammalian cells and plays important roles in antiviral activities against both DNA and RNA viruses. In this study, we found that the downregulation of PML rendered HeLa cells more susceptible to infection by enterovirus 71 (EV71), and the overexpression of the PMLIII or PMLIV isoforms inhibited viral protein expression and resulted in viral titers that were 2–3 log units lower than those in the control. Using short interfering RNAs, the downregulation of either the PMLIII or PMLIV isoform increased both viral protein VP1 expression and viral production. The PML repression of EV71 replication was partially mediated by the inhibition of autophagy, and PML deficiency triggered autophagy. Furthermore, the EV71 infection resulted in a reduction in PML independent of the proteasome pathway. Instead, PML degradation was mediated by virus protease 3Cpro. In conclusion, PML contributes to a cellular antiviral effect by inhibiting autophagy, which is countered by a disruption of promyelocytic leukemia protein-nuclear bodies mediated by viral protease 3Cpro.

　　Auhor Summary

　　In this article, we describe the roles of PML as a cellular restriction factor in EV71 infection. PML inhibited EV71 replication by inhibiting autophagy in the infected cells, and PML deficiency triggered autophagy, which facilitated viral replication. In addition, the IFN-β-mediated inhibition of EV71 replication required the presence of PML, suggesting that the ISGs likely require the presence of PML for the establishment of an antiviral status. Moreover, the EV71 infection induced PML degradation, which was mediated by virus protease 3Cpro independent of the proteasome pathway. These findings demonstrate the important roles of PML in innate immune defense against EV71 and describe a novel mechanism by which the virus evades innate immunity.

　　DOI：10.3389/fimmu.2018.01268

　　本期编辑：hantavirus