3C蛋白酶介导的EV71逃避ZAP抗病毒作用机制研究

Large outbreaks of foot and mouth disease infected by EV71 have occurred in many places in China during the past several years, and the proportion of severe infections has also raised. Intensive study on the immune escape of EV71 is crucial for the understanding of EV71 pathogenesis. Zinc antiviral protein (ZAP) is a recently identified antiviral protein which severely inhibiting the replication of many viruses through degradation of viral mRNA. Presently, reports on the interaction between EV71 and ZAP are not available. Preliminary work indicates that ZAP mRNA was significantly up-regulated during EV71 infection. However, replication of EV71 was not significantly impaired in the presence of overexpressed ZAP. Further work showed that ZAP was cleaved during EV71 infection or in the presence of EV71 3C proteinase. Studies have proved that 3C could modulate host immune responses by cleaving host factors. So, we hypothesize that EV71 may escape from antiviral function of ZAP by 3C. In this project, we will firstly check the time phase, cleavage and localization of ZAP during EV71 infection . Then we will elucidate the mechanism employed by 3C for the cleavage of ZAP. Finally, we will construct ZAP mutants resistant to 3C cleavage through site directed mutagenesis and further evaluate the potential inhibition activity of these mutants on EV71 replication. This project will help to elucidate the mechanism employed by EV71 to escape form ZAP’s antiviral function, and provide better understanding of immune escape of EV71. This project might also help to develop new insight for prevention and therapy of EV71 infection.

近年来我国多地爆发EV71感染引起的手足口病，且重症患者比例呈上升趋势。研究EV71免疫逃逸机制对了解病毒致病机理至关重要。ZAP是一种抗病毒蛋白，通过降解病毒mRNA对多种病毒复制发挥抑制作用，有关ZAP与EV71的相互作用未见报道。前期研究结果表明EV71感染使ZAP mRNA显著上调，过量表达ZAP时EV71复制却未见显著抑制。此外EV71感染或过量表达EV71 3C蛋白均可引起ZAP切割。多项研究表明3C蛋白可以切割宿主蛋白调控免疫反应，我们推测3C蛋白可能介导EV71逃避ZAP的抗病毒作用。本课题将首先检测EV71感染条件下ZAP的表达时相、切割和定位，然后探索3C切割ZAP的具体机制，最后构建逃避3C切割的ZAP突变体验证其对EV71复制的影响。本课题的开展有利于阐明EV71逃避ZAP抗病毒作用的机制，为深入了解EV71免疫逃逸机制提供依据，也为EV71预防和治疗提供新的思路。

EV-71 免疫逃逸机制的深入研究对于了解 EV-71 发病机制至关重要。宿主锌指抗病毒蛋白（ZAP）是一种 IFN 刺激的产物，主要通过降解病毒 mRNA 的方式特异性抑制多种病毒的复制。我们发现 EV-71 感染后上调293T 、RD 和 HeLa 细胞中的 ZAP mRNA的转录。过表达ZAP使293T 细胞对 EV71 感染具有抗性，并且内源性 ZAP 敲降会增强 EV-71 感染。 EV-71 感染刺激 ZAP 两种剪切体的特异性位点蛋白水解，导致40kDa 的 ZAP N 末端片段在病毒感染的细胞中积累。我们的研究揭示了 EV-71 通过其3C 蛋白酶（3Cpro）切割ZAP 切割，并找打Gln-369 是 ZAP 中的切割位点。尽管 ZAP 过量表达抑制了 EV-71 的复制，但切割后的片段没有显示出同样效果。值得注意的是，我们的研究进一步发现 ZAP 直接特异结合 EV-71 病毒RNA 的 3'UTR 和 5'UTR。除了 EV-71 之外，包括脊髓灰质炎病毒（PV）和柯萨奇 A 病毒 16（CVA16）在内的其他人类肠病毒中也发现 3Cpro 切割 ZAP 的现象。我们的研究结果表明 ZAP 和肠病毒 3Cpro 之间存在一种动态平衡控制着病毒感染。综上所述，本研究揭示了病毒 3Cpro 介导的 ZAP 切割可能是一种逃避宿主抗病毒反应的机制，为深入了解 EV-71 免疫逃逸机制提供依据，并为EV-71 预防和治疗提供新的思路。