伪狂犬病毒UL13蛋白激酶调控宿主细胞DNA损伤应答通路的分子机制

DNA损伤应答（DDR）是细胞感知并修复受损DNA以维持基因组稳态的级联应激反应，在病毒增殖和致病力方面发挥重要作用。伪狂犬病毒（PRV）是危害养猪业的重要病原之一。申请者前期研究发现，PRV感染激活DDR。同时，过表达UL13也可激活DDR的标记分子γH2AX，且依赖于UL13的激酶活性。为探究UL13对DDR的调控机理，阐明该过程在PRV增殖和致病力中的作用。本项目拟开展以下研究：利用Western-blot和流式细胞术，探究UL13对DDR的调控机理；利用抑制剂和RNA沉默技术，探究UL13介导的DDR对PRV增殖的作用；利用CRISPR/Cas9和同源重组技术，构建UL13激酶活性缺陷的点突变毒株，从病毒感染水平评价UL13对DDR、PRV增殖和致病力的影响。本项目试图从DDR的角度揭示UL13在PRV增殖和致病力中的作用，为PRV基因缺失疫苗和抗PRV药物的研制提供参考靶点。

DNA damage response (DDR), a cascade stress signaling pathway to sustain the cellular genome integrity through detecting and repairing the damaged DNA, plays an important role in viral replication and pathogenesis. Pseudorabies virus (PRV) is one of the most important pathogens that threat the swine industry. Our previous study showed that PRV infection could activate DNA damage response. Moreover, we also found that overexpression of UL13 protein could activate the DNA damage response marker γH2AX in its kinase activity-dependent manner. To explore the mechanisms of how UL13 regulates DNA damage response, and the role of this regulation in PRV replication and pathogenesis, the following experiments will be performed. Firstly, explore the regulation mechanisms of UL13 to DDR signaling pathway by using Western-blot and Flow cytometry. Secondly, explore the role of DNA damage response mediated by UL13 to the replication of PRV by using DNA damage inhibitors and siRNA technology. Finally, explore the influence of UL13 to DDR, PRV replication and pathogenesis under infection with PRV UL13-kinase dead point mutation strain which is constructed by using CRISPR/Cas9 and homologous recombination technology. Through this study, the role of UL13 in PRV replication and pathogenesis from the aspect of DNA damage response will be figured out. This study will provide potential target for the design of PRV gene-deleted vaccine and anti-PRV compound.