ERK通路调控肠道病毒2A蛋白酶对干扰素抗病毒的拮抗作用及其可能的临床应用

Interferons (IFNs) are a group of signaling proteins made and released by host cells in response to the presence of viruses etc., which have been clinically applied as the only kind of broad-spectrum antiviral drug for the treatments of a variety of important viral diseases. However, clinical application of IFN shows limitations because of its potential toxicity and poor curative effects for some viruses. As we known, HFMD caused by Enterovirus A 71(EV-A71) has a great public health especially in the Asia-Pacific region because of death and severe neurologic complications in infants and children. EV-A71 belongs to Enteroviruses (EV), which develop a generally antagonistic effect on IFN. It has been reported that EV 2Apro would cleave or down-regulate receptors of IFN, which could explain why IFN has a less therapeutic effect on Enteroviruses. Our previous study has shown that blockade of ERK pathway could effectively recover anti-enteroviral effect of IFN to EV-A71 and inhibits cleavage activity of 2Apro, which implicates that the positive regulation of ERK pathway to cleavage activity of 2Apro would be important for EVs to antagonize IFN. Thus by adopting EV-A71 and CVB3 as the model virus, this project intends to validate the importance of ERK pathway in EVs-associated IFN antagonistic effect, further verify the hypothesis that blocking ERK pathway could promote the antiviral activity of IFN both in vivo and in vitro, and in addition, to explore the possible mechanism(s). The output of this study would provide a new strategy for the clinical application of IFN on the treatment of EVs infection, for instance, severe hand, foot and mouth disease (HFMD) and moreover shed lights on the development of practical therapeutic strategies, which may in the future be applied to the infections of viruses outside the genus Enterovirus.

干扰素(IFN)是目前临床唯一应用的广谱抗病毒药物，可治疗多种重要病毒性疾病。已知肠道病毒（EV）普遍存在对IFN拮抗，即临床IFN治疗效果不佳，这可能与肠道病毒蛋白酶（如2A）切割或下调IFN受体及其后续信号通路活性作用有关。我们前期研究发现，特异性阻断ERK通路在有效抑制EV-A71 2A酶切活性同时，也伴随显著恢复外源IFN对EV-A71抗病毒作用；提示ERK通路通过对2A酶切活性的正向调控，在肠道病毒对IFN拮抗效应中发挥重要作用。本项目拟用EV-A71和柯萨奇病毒B3为模式病毒，在体内、外模型中验证阻断ERK通路可促进干扰素抗病毒作用，并通过“联合用药”为临床IFN抗病毒疗效不佳提供可能的解决方案，并进一步阐述ERK通路对肠道病毒2A酶活性调控的作用机制。本项目通过IFN对临床病毒性疾病治疗中存在的重要问题进行基础研究，故具有重要的理论意义和良好的临床应用前景。

肠道病毒是引起人类高发和新发传染病的重要病原，种类繁多，通过消化道、呼吸道等途径传播，临床表现复杂多样，部分可致严重症状甚至死亡，是全球5岁以下儿童致死的重要病因之一，因此亟待特异性药物和疫苗进行治疗和预防。.干扰素-α (IFN-α) 作为一种广谱抗病毒制剂，用于临床抗病毒治疗已数十年。研究发现，EV-A71具有拮抗 IFN-α 的抗病毒作用，主要机制与病毒 2Apro 介导的干扰素-α受体 1 (IFNAR1)表达下调有关。.本团队前期研究发现，细胞ERK通路能正向调控2Apro活性的作用。本研究中，我们以肠道病毒2Apro为研究重点，发现：①肠道病毒2A蛋白S/T125存在单一位点磷酸化现象；该修饰作用受ERK激酶调控，并在有效保障肠道病毒2A蛋白酶功能和病毒复制方面均发挥了重要作用。②肠道病毒2Apro通过激活细胞半胱氨酸酶caspase3，在DIID42基序位点切割胞内IFNAR1蛋白，推测可导致该基序远端的信号肽缺失，故胞内表达的IFNAR1蛋白无法转移到细胞膜上，最终表现为膜IFNAR1表达下调，并影响到其下游信号通路活性，以及抗病毒蛋白的表达和发挥抗病毒功能。.基于上述两个重要原创机制，我们采用联合用药策略，①IFN-α联合抑制2Apro磷酸化的ERK抑制剂（U0126，或soraphenic-索拉菲尼），②IFN-α联合caspase3抑制剂（泛半胱天冬酶抑制剂VAD），发现低浓度且对细胞无毒性和无抗病毒作用的两种联合用药策略，在蛋白水平能有效恢复膜IFNAR1表达水平，在细胞水平能有效发挥广谱抗肠道病毒效果。联合用药结果支持上述原创机制。.我们的研究证明，肠道病毒2Apro 的S/T125磷酸化和激活caspase3介导的干扰素拮抗作用是病毒重要的宿主适应性机制，对保障肠道病毒复制和致病具有重要作用。本研究对后续抗肠道病毒联合用药以及疫苗研究提供了新思路。