前沿速递】王健伟教授团队在EV71病毒研究领域连发两篇JV！厉害了Word 哥！

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编者按
中国医学科学院&北京协和医院病原生物学研究所王健伟教授团队与美国伊利诺伊大学芝加哥分校何斌教授团队合作研究，在EV71病毒感染与免疫领域取得成果，病毒学领域的知名期刊Journal of Virology近日连续刊发其两篇论文。分别为“Enterovirus 71 inhibits pyroptosis through cleavage of GSDMD”和“Enterovirus 3A facilitates viral replication by promoting PI4KB-ACBD3 interaction”，分别于7月5日，12日接收，值得祝贺，详文如下。







本期仅将第2篇进行简单翻译，限于小编水平，还望各位大咖不要拍砖哦！


题目：Enterovirus 3A facilitates viral replication by promoting PI4KB-ACBD3 interaction
​作者：Xia Xiao, Xiaobo Lei, Zhenzhen Zhang, Yijie Ma, Jianli Qi, Chao Wu, Yan Xiao, Li Li, Bin He* and Jianwei Wang*

像其他肠道病毒一样，EV71依赖于磷脂酰肌醇-4-激酶IIIβ（PI4KB）进行基因组RNA复制。然而，如何将PI4KB引入EV71的基因组复制位点仍然难以捉摸。最近，研究者们报道通过与病毒3A蛋白相互作用，EV71复制需要宿主因子ACBD3。在这里，研究者们的研究表明了ACBD3是招募PI4KB到RNA复制位点所必需的。病毒3A或EV71感染的过表达刺激PI4KB和ACBD3的相互作用。同样的，EV71感染诱导也会产生磷脂酰肌醇-4-磷酸（PI4P）。此外，PI4KB，ACBD3和3A均位于病毒RNA复制位点。因此，由siRNA引起的PI4KB或ACBD3消耗导致EV71感染后PI4P产生减少。3A中的I44A或H54Y替代中断PI4KB和ACBD3的刺激。进一步的分析表明，ACBD3-PI4KB相互作用的刺激对于肠道病毒68的复制也是重要的，但对人类鼻病毒16是不利的。这些结果揭示了肠病毒复制的机制，其涉及将p4KB募集到RNA复制位点的选择性策略。
重要性肠道病毒71与其他人肠道病毒一样，在宿主细胞内复制其基因组，其中病毒蛋白质有效利用细胞机制。虽然涉及多个因素，但病毒复制如何受到控制却是不清楚的。研究者们显示肠道病毒71的3A蛋白通过与ACBD3相互作用而招募了磷脂酰肌醇-4-激酶IIIβ，其通过产生脂质PI4P改变细胞膜。因此，病毒和宿主蛋白形成复制位点上RNA合成所必需的大复合物。值得注意的是，PI4KB-ACBD3相互作用也差异地介导肠病毒68和鼻病毒16的复制。

PI4KB阻碍抑制EV71病毒复制





探讨PI4KB表型，研究者们确定的存在或缺失的情况，确认了PI4KB抑制病毒RNA的复制，其中包括pik93恩韦肟和GW5074。EV71病毒RNA的复制增加感染的进展在控制细胞。治疗pik93，恩韦肟或GW5074明显减少病毒RNA的复制。在这些条件中，PI4KB抑制剂没有毒性作用。具体实验评估PI4KB，主要体现在进行siRNA敲除实验。如图1C所示，添加sirna-pi4kb导致减少EV71病毒RNA作为对照，说明要求在EV71复制PI4KB。Western blot分析表明，PI4KB表达的siRNA有效降低但不抢PI4KBsiRNA。这些表型是由于毒性作用。与此一致的是，小干扰RNA抑制病毒PI4KB降低生产效率。此外，作为如图所示，1G，siRNA敲除PI4KB表达也抑制RNA的复制其他EV71分离株。这些结果表明，PI4KB严格要求的EV71复制。

讨论：

最近，研究者们已经报道，EV71 3A蛋白介导的病毒复制的宿主因子ACBD3相互作用。在这里，研究者们表明，相互作用betweenev71 3A和ACBD3方便招募PI4KB和病毒RNA复制的3D聚合酶。此外，ev68，但不是HRV16，利用基因组复制ACBD3。这些结果支持这一概念，pi4kb-acbd3差异有助于pi4kb-acbd3是病毒复制的关键，但与病毒的关系是一个谜。
ACBD3不涉及病毒和柯萨奇病毒复制。另一方面，ACBD3抑制病毒复制的脊髓灰质炎病毒感染后。以往的研究表明，肠道病毒71型复制依赖PI4KB但潜在的机制是未知的。在与PI4KB抑制剂EV71病毒复制处理的细胞减少。
研究者们发现，特定的消耗PI4KB也大幅受损的病毒RNA的复制和EV71后续生产。的确，EV71 3A和ACBD3互动的关键在于PI4KB招募。当感染EV71，PI4KB被招募到离散区域的病毒RNA和病毒3a，ACBD3 PI4KB。这表明，EV71 3A，ACBD3 PI4KB形成功能复合物介导病毒RNA的复制。在这个模型中，EV71 3A、3D、ACBD3 PI4KB被检测到在一个大的复杂的。
研究者们推测一个高阶蛋白复合物很可能代表功能复制机制。这种复杂的基于EV71 3A和ACBD3互动因为耗尽ACBD3防止PI4KB招募和3a与感染细胞PI4KB不再相互作用。研究者们的数据表明，肠道病毒71型刺激在ACBD3依赖PI4P的合成。这是由于EV71和ACBD3的相互作用。研究者们注意到，针对EV71 3a PI4KB在感染细胞的离散区域。耗尽ACBD3 PI4P生产受损，这与病毒的复制缺陷。虽然ACBD3绑定到未受感染的细胞之间的相互作用很弱，PI4KB。EV71感染3a或表达强烈增加它们之间的相互作用。相反，在ev713a颠覆性的突变表型的逆转。一个可能的解释是，EV71 3a可以稳定ACBD3 PI4KB互动。或者，它可以作为改造膜所需的伴侣。
因为通过ACBD3 PI4KB膜招募的增加酶的活性，研究者们相信，通过结合ACBD3病毒3a可以改变其构象，导致PI4KB活化和PI4P生产。除了EV71，ev68调节ACBD3其RNA复制。耗尽ACBD3严重降低复制的ev68，进一步突出了ACBD3至关重要的作用。另一方面，ACBD3是HRV16复制可有可无，这是类似于柯萨奇病毒和鼻病毒3a。这些表型与3A与ACBD3能力。研究者们赞成的论点3A介导的acbd3-pi4kb复合稳定负责。在与ACBD3击倒或敲除HRV16 RNA复制的增加表明ACBD3可能存在不利于HRV16。这似乎与脊髓灰质炎病毒和病毒复制的观察一致ACBD3敲除细胞，在病毒复制的刺激。在HRV16感染，PI4KB可能招募到RNA的复制网站其他宿主因素但不是ACBD3和GBF1。
总的来说，这些结果表明，在招募和病毒复制PI4KB ACBD3的作用可能依赖于病毒的种类和品系。是否与肠道病毒3a结构的差异有关，值得进一步研究。总之，肠道病毒的细胞膜结构改造为专门whereviral复制复合物组装的协调。这项研究显示，EV71和ev68复制ACBD3不可或缺的作用。然而，ACBD3可有可无在细胞与鼻病毒16。这可能是相关的不同的生物可能是ACBD3通路选择性地利用额外的RNA病毒。


原文摘要：
Like other enteroviruses, EV71 relies on phosphatidylinositol-4-kinase IIIβ (PI4KB) for genome RNA replication. However, how PI4KB is recruited to the genome replication sites of EV71 remains elusive. Recently, we reported that a host factor ACBD3 is needed for EV71 replication by interacting with viral 3A protein. Here, we show that ACBD3 is required for the recruitment of PI4KB to RNA replication sites. Overexpression of viral 3A or EV71 infection stimulates the interaction of PI4KB and ACBD3. Consistently, EV71 infection induces the production of phosphatidylinositol-4-phosphate (PI4P). Furthermore, PI4KB, ACBD3 and 3A are all localized to the viral RNA replication sites. Accordingly, PI4KB or ACBD3 depletion by siRNA leads to a reduction in the PI4P production after EV71 infection. I44A or H54Y substitution in 3A interrupts the stimulation of PI4KB and ACBD3. Further analysis suggests that stimulation of ACBD3-PI4KB interaction is also important for the replication of enterovirus 68 but disadvantageous to human rhinovirus 16. These results reveal a mechanism of enterovirus replication that involves a selective strategy for recruitment of PI4KB to the RNA replication sites.

IMPORTANCE Enterovirus 71, like other human enteroviruses, replicates its genome within host cells, where viral proteins efficiently utilize cellular machineries. While multiple factors are involved, it is largely unclear how viral replication is controlled. We show that the 3A protein of enterovirus 71 recruits an enzyme phosphatidylinositol-4-kinase IIIβ by interacting with ACBD3, which alter cellular membranes through the production of a lipid PI4P. Consequently, the viral and host proteins form a large complex that is necessary for RNA synthesis at replication sites. Notably, PI4KB-ACBD3 interaction also differentially mediates the replication of enterovirus 68 and rhinovirus 16. These results provide a new insight into the molecular network of enterovirus replication.


原文链接：
http://jvi.asm.org/content/early ... 17.abstract?papetoc


附第1篇论文信息：
Title：Enterovirus 71 inhibits pyroptosis through cleavage of GSDMD
Author：Xiaobo Lei, Zhenzhen Zhang, Xia Xiao, Jianli Qi, Bin He* and Jianwei Wang*
Abstract：Enterovirus 71 (EV71) can cause hand, foot, and mouth disease (HFMD) in young children. Severe infection with EV71 can lead to neurological complications and even death. However, the molecular basis of viral pathogenesis remain poorly understand. Here, we report that EV71 induces degradation of GSDMD, an essential component of pyroptosis. Remarkably, the viral protease 3C directly targets GSDMD and induces its cleavage, which is dependent on the protease activity. Further analyses show that the Q193-G194 pair within GSDMD is the cleavage site of 3C. This cleavage produces a shorter N-terminal fragment spanning amino acids 1-193. However, unlike the N-terminal fragment produced by casaspe-1 cleavage, this fragment fails to trigger cell death or inhibits EV71 replication. Importantly, T239D or F240D substitution abrogates the activity of GSDMD composed of amino acids 1-275. This is correlated with the lack of pyroptosis or inhibition of viral replication. These results reveal a previously unrecognized strategy for EV71 to evade the antiviral response.
Importance：Recently, it has been reported that GSDMD plays a critical role in regulating lipopolysaccharide and NLRP3-mediated IL-1β secretion. In this process, the N-terminal domain p30 released from GSDMD acts as an effector in cell pyroptosis. We show that EV71 infection down-regulates GSDMD. EV71 3C cleaves GSDMD at the Q193-G194 pair, resulting in a truncated N—terminal fragment disrupted for inducing cell pyroptosis. Notably, the 1-275aa fragment (p30) inhibits EV71 replication whereas the 1-193aa fragment does not. These results reveal a new strategy for EV71 to evade the antiviral response.
DOI：10.1128/JVI.01069-17
原文链接：http://jvi.asm.org/content/early/2017/06/29/JVI.01069-17.abstract