单纯疱疹病毒性角膜炎（HSK）是致盲率最高的角膜病变，抗宿主细胞凋亡是单纯疱疹病毒-1（HSV-1）在角膜组织复制传播，病情进展的重要机制。我们的前期研究发现，在HSV-1感染的角膜上皮细胞中Ret受体表达下调，进一步敲除/过表达Ret，细胞凋亡相应减少/增加，提示Ret具有促凋亡作用；此外，我们发现HSK中泛素化Ret水平增高。据此，我们推测Ret泛素化降解增加是HSV-1抑制角膜上皮细胞凋亡的重要机制。但Ret泛素化降解的特异性泛素连接酶（E3）及作用机制尚不清除，本课题拟开展如下研究：（1）文库筛选：使用CRISPR文库筛选技术明确Ret降解的特异性E3酶；（2）功能验证：过表达或者敲除筛选出的E3基因，验证其对HSK中角膜上皮细胞凋亡的作用；（3）靶向干预：在HSV-1体内外模型中观察E3抑制剂对细胞凋亡和病毒复制的干预作用。本课题的研究将为靶向抑制病毒复制和治疗HSK提供新策略。

Herpes simplex keratitis (HSK) is the leading cause of blindness in corneal infective disease. The antiapoptotic characterisics of herpes simplex virus-1 (HSV-1) in infected host cells is the vital mechanism of virus replication and propagation, and the deterioration of corneal injury. We found previously that Ret expression was reduced in human corneal epithelial cells (HCE) infected with HSV-1. Furthermore, cell apoptosis was down-regulated in Ret knock-down HCEs, while cell apoptosis was up-regulated in Ret overexpressed HCEs, which indicating that Ret promotes the apoptosis of HCE infected with HSV-1. Besides, ubiquitinated Ret was found increased upon infection. Therefore, we hypothesize that the enhancement of Ret ubiquitination is a vital mechanism of the antiapoptotic characterisics of HSV-1 in HCE. However, the specific ubiquitin-protein ligase (E3) of Ret and the regulatory mechanism on HCE apoptosis still remain unknown. Therefore, we aim to conduct the below studies: (1) screening the specific ubiquitin ligase (E3) of Ret using the customized CRISPR Knock -Out pooled sgRNA library of E3. (2) investigating the regulatory role of specific E3 of Ret on apoptosis of HCE in HSK. (3) assessing the effects of specific E3 inhibitors on the apoptosis of HCE and the replication of virus in vivo and in vitro experiments. This project would provide new insights into the mechanisms of the antiapoptotic characterisics of specific E3 of Ret in HSK. In addition, the outcome of this study may lead to a new therapeutic strategy using E3 inhibitors to treat HSK.