肠道病毒71型拮抗I型干扰素反应的机制研究

Type I interferons (IFN-α/β) have been used clinically as antiviral therapy as the first line of defense against virus infections successfully for decades. However, treatment with Type I interferons has not been effective in patients with EV71 infection. In our previous studies, we found that the EV71-infected cells, pre-treated with IFN-β, were not protected from developing a cytopathic effect in which the viral replication was not affected. The mechanism by which EV71 antagonizes interferon signaling, however, has been controversial. Our study indicated that EV71 infection did not inhibit phosphorylation of STAT1/2 induced by IFN-β stimulation, but p-STAT1/2 transport into the nucleus was significantly blocked. We also found that the inhibition rate of ISGs expression by EV71 infection was increased in a time-dependent manner. The translocation of phosphorylated STAT1/2 depends on the interaction with karyopherin-α1(KPNA1), a nuclear import adaptor, and interferon regulatory 9 (IRF9). Also the robust and efficient response of IFNs against virus infection depends on the fuction of U-ISGF3, formed by STAT1, STAT2 and IRF9. . The research aims to study on the mechanism of EV71 blocking the interferon signaling pathway. Specific aims include 1) to understand the mechanism how EV71 is involved in blocking the nuclear importation of STAT1/2, especially the roles of KPNA1 and IRF9 in this process; 2) to figure out whether ISGF3 formation is blocked by EV71 infection and the roles of IRF9 in this process; 3) to figure out whether U-ISGF3 formation is blocked by EV71 infection.

I 型干扰素在临床上被用于EV71感染的治疗，疗效并不显著。其机制亟待进一步研究。我们前期研究发现，EV71感染不影响STAT1/2的蛋白水平和磷酸化水平，但可以抑制p-STAT1/2的核转位。有趣的是，EV71对ISGs的抑制率随着时间的延长而增高。p-STAT1/p-STAT2的核转位与核转运蛋白KPNA1和IRF9密切相关，而IFN诱导抗病毒蛋白的持续高表达依赖于STAT1/STAT2/IRF9组成非磷酸化复合物（U-ISGF3）的形成并入核发挥作用。因此本课题旨在系统研究EV71拮抗干扰素反应的具体机制。将探讨：1）KPNA1/IRF9在EV71抑制p-STAT1/p-STAT2核转位过程中的作用及其机制；2）ISGF3的形成是否受到抑制，以及IRF9在此过程中的作用；3）IFN作用后期，U-ISGF3的形成和入核是否受到抑制。

肠道病毒71型（EV71）是单股正链RNA病毒，是引起手足口病的主要病原体，近年来因其常引起中枢神经系统并发症导致无数患儿死亡而备受关注。至今为止仍没有有效的抗病毒药物。I型干扰素（IFN）作为传统的广谱抗病毒治疗药物，已经成功的应用于HIV，HCV等多种病毒的治疗，但IFN治疗EV71病毒感染效果并不显著。本课题旨在研究EV71与干扰素系统的相互作用机制，从而为EV71的治疗提供新的思路。.本项目研究发现了EV71抑制IFN作用的新机制，即在IFN作用的早期阶段，EV71通过激活caspase-3诱导KPNA1蛋白的降解，从而抑制STAT1的核转位，抑制少量ISGs的产生；在IFN作用的后期阶段，EV71通过下调IFN诱导的STAT2和IRF9的表达，从而阻断U-ISGF3的形成，抑制大量ISGs的持续高表达。.EV71感染能够促进KPNA1的降解，阻断KPNA1与p-STAT1的相互作用，从而抑制p-STAT1/p-STAT2/IRF9三聚体复合物的入核启动一系列抗病毒基因的转录。并且我们发现EV71对KPNA1蛋白的降解作用是依赖于caspase-3的活性，而与细胞内降解蛋白的蛋白酶体途径和自噬溶酶体途径没有关系。本研究结果显示IFN-β明显诱导了STAT1的表达，但是因为STAT2和IRF9的表达被EV71明显抑制了，所以病毒蛋白的复制并没有被抑制，说明在EV71感染的HeLa细胞中STAT1对IFN的抗病毒作用并不是必须的。值得注意的是，在IFN诱导的高表达的STAT1存在的前提下，同时过表达IRF9和STAT2能够显著的逆转EV71对ISGs的产生的抑制作用，说明EV71通过抑制IRF9和STAT2的表达阻断IFN诱导ISGs的产生。.本研究阐明了EV71拮抗IFN作用的新机制，这一发现能够帮助我们进一步认识EV71的致病机理以及其逃逸天然免疫的具体过程，为将来EV71的治疗奠定了理论基础。