P53介导的鳜传染性脾肾坏死病毒免疫逃逸分子机制研究

Immune escape is a key strategy for virus to avoiding the host immune clearance and establishing infection. It has been confirmed that tumor suppressor p53 plays the most important role in the immune escape of virus through inducing host cell apoptosis and enforcing the type I IFN response.But how does the p53 mediate immune escape of ISKNV and what is the molecular mechanisms of the immune escape? Interestingly the infection of ISKNV could regulate cell apoptosis and type I IFN pathway based on preliminary experiment results as follows: at the beginning of ISKNV infection the expression of apoptosis factors, antiviral factors and p53 were all down-regulated, but at the late stage of ISKNV infection the expression of apoptosis factors and p53 were up-regulated and that of antiviral factors was still down-regulated, which was accordant with p53-mediated immune escape. Based on the above, we hypothesis that ISKNV can escape from mandarin fish immune recognition and clearance to establish infection through inhibiting p53-dependent apoptosis and the type I IFN response at the beginning of ISKNV infection, then release viruses to infect other cells by inducing apoptosis at the late stage of infection. Now we have approved that all p53 pathway functions are perfect in a mandarin fish cell line (CPB). And based on this CPB model, we will illuminate the scientific question of the molecular mechanisms of p53-mediated immune escape of ISKNV, including that (1) the kenetic change pattern of P53 protein will be investigated in the ISKNV infected cells, (2) ISKNV proteins interacting with the p53 will be identifed and viral proteins how to regulate P53 activity will be explored, (3) biological effects will be determined after the interaction of viral protein with p53. This is the first study to explore the molecular mechanisms of immune escape mediated by p53 in aquatic animal virus. The results of this study will provide the novel ideas for screening effective antiviral target protein in ISKNV and can be used for reference to immune escape study in other aquatic viruses.

免疫逃逸是病毒逃避宿主免疫清除的重要策略。研究发现肿瘤抑制因子p53可参与介导病毒免疫逃逸。但p53如何介导ISKNV免疫逃逸？其分子机制是怎样的呢？预实验结果显示ISKNV感染CPB细胞后p53与凋亡相关蛋白表达均呈现“先抑后扬”趋势、而干扰素相关蛋白下调。据此假设病毒蛋白与宿主p53互作先抑制宿主干扰素及凋亡途径，利于病毒增殖；后诱导凋亡，便于病毒释放与下轮侵染，从而实现病毒免疫逃逸。本项目在前期已建立具有完整p53信号通路细胞模型基础上，研究ISKNV复制与p53活性变化关系，确定与p53互作病毒蛋白，采用双荧光素酶检测系统、流式细胞技术等开展病毒蛋白对p53活性调控及其生物学效应，最终解析p53介导ISKNV免疫逃逸分子机制。本项目率先在水生动物病毒中开展p53介导病毒免疫逃逸分子机制研究，为找寻ISKNV防控靶点提供新的思路，同时可为其它水生动物病毒免疫逃逸机制研究提供借鉴。

鳜鱼是我国主要的高值经济养殖鱼类之一，随着养殖密度的提高，病害越发严重，其中鳜传染性脾肾坏死病毒病（ISKNVD）和鳜弹状病毒病（SCRVD）是阻碍鳜鱼养殖业健康发展的最主要病毒病，因此对其开展防治研究非常必要。近年来，p53蛋白作为新发现参与细胞先天免疫反应的抗病毒因子，已成为病毒防治领域的新热点。本课题以鳜鱼为研究模型，克隆、表达了鳜p53基因，制备了其多克隆抗体，对ISKNV和SCRV感染后的响应模式、抗病毒功能、以及其介导谷氨酰胺代谢重编程的免疫逃逸机制进行了研究。取得的主要结果如下：.①通过RACE克隆鳜p53（Sc-p53）全长，采用Gene walking 获得其基因组；重组表达P53并制备了多克隆抗体；qPCR及WB确定ISKNV或SCRV感染鱼体或细胞后Sc-p53的表达模式，ISKNV可诱导p53表达双向上升变化，而SCRV为单向变化。.②通过RACE克隆获得Sc-p53可变剪接异构体Sc-p53I6，发现其对ISKNV及SCRV的感染应答模式与Sc-p53相同。.③确定p53具有抑制ISKNV及SCRV复制增殖的抗病毒功能。通过抑制或促进p53表达，采用qPCR、WB、TCID50等实验方法，检测p53的表达变化及病毒增殖量，发现促进p53表达，ISKNV及SCRV增殖量显著降低，而抑制p53表达，ISKNV及SCRV增殖量显著升高。.④确定ISKNV及SCRV病毒通过p53介导代谢重编程进行免疫逃逸的新机制。通过饥饿实验，发现ISKNV、SCRV增殖依赖于谷氨酰胺；通过抑制或促进p53表达，采用qPCR及WB实验检测谷氨酰胺代谢途径相关酶的表达变化及病毒增殖量，发现ISKNV、SCRV通过抑制p53表达促进谷氨酰胺酵解途上调满足病毒的增殖复制，从而逃逸p53的抗病毒功能。.