马立克氏病病毒ICP0蛋白调节宿主I型干扰素产生的分子机制研究

Marek’s disease virus (MDV) is the pathogen caused to tumor with the characteristic of proliferative lymphocyte in chicken. The mediate-early ICP0 protein of MDV is an important factor in the process of infection. Type I interferon (IFN-I) plays a vital role during MDV latent infection. However, it is unclear so far how ICP0 effects on chicken IFN-I production and ability against MDV infection of host cell. On this basis, we will firstly construct the MDV over-expressed ICP0 as well MDV deleted ICP0 by bacterial artificial chromosome (BAC) manipulation. The dynamic production of IFN-I and viral replication will been monitored in chicken embryo fibroblast infected with recombined MDV over-expressing ICP0 or ICP0 deletion. The ICP0 targeting protein will be pulled down and identified. The interaction of ICP0 with targeting protein will be further analyzed. Then, the effect of targeting protein on viral proliferation and the signal pathway of IFN-I will be investigated as well. Finally, the replication and oncogenicity of MDV regulated by ICP0 protein will be evaluated while activities of IFN-1 were inhibited or activated. The aims of this study is to illuminate the molecular mechanism of MDV Immediate-Early ICP0 protein regulating production of IFN-I in chicken, which will provide evidence for mechanism of immunity and infection of MDV.

马立克氏病病毒（Marek’s disease virus, MDV）是引起鸡淋巴细胞增生肿瘤病的病原体。ICP0蛋白是MDV的早期转录因子，是MDV感染的重要调控因子。I型干扰素（type I interferon，IFN-I）在宿主抗MDV感染中发挥重要作用。然而，ICP0如何影响宿主细胞产生IFN-I抗MDV功能尚不清楚。基于此，本课题拟利用细菌人工染色体操作构建ICP0过表达及缺失的重组MDV；在它们分别感染宿主细胞后，检测细胞内ICP0的表达，监测细胞IFN-I表达水平及病毒增殖动态；筛选及鉴定ICP0相互作用的宿主细胞靶蛋白，进一步分析ICP0对细胞靶蛋白功能、以及细胞靶蛋白对IFN-I信号通路及MDV增殖影响；最终评估在IFN-I抑制及活化状态下ICP0介导的MDV复制及致瘤情况。旨在阐明MDV ICP0调节宿主IFN-I产生的分子机制，为揭示MDV感染与免疫机制提供依据。

疱疹病毒的ICP0蛋白在病毒感染诱导的先天性免疫反应中发挥重要作用，研究发现，马立克氏病病毒(MDV) ICP0蛋白在感染鸡胚成纤维细胞后显著表达，然而，马立克氏病病毒的ICP0蛋白是否与哺乳动物疱疹病毒具有相似的功能尚不清楚。在本研究中，我们发现MDV ICP0蛋白在病毒感染后主要定位于宿主细胞的细胞核中，与宿主细胞DDX5蛋白相互作用，MDV ICP0蛋白增强了宿主细胞DDX5的表达及细胞核内的定位。进一步的研究表明，DDX5通过调控宿主细胞细胞核内的转录因子IRF1进一步调节抗病毒TLR3/MDA5等重要信号通路先天性免疫抗病毒基因的表达，抑制了β干扰素表达，增强了马立克氏病病毒逃避先天性免疫反应。为确证DDX5蛋白对MDV病毒复制的影响，将DDX5从成纤维细胞中敲降表达后，对MDV复制情况进行检测，结果表明，DDX5显著降低了MDV的复制效率，表明DDX5有利于病毒的复制，与此同时，对MDV诱导的IFN-I的表达进行了检测，研究发现，DDX5表达的降低增强了MDV诱导的IFN-I的产生。以上结果表明，MDV 通过其ICP0蛋白通过调节宿主DDX5蛋白抑制先天性免疫增强病毒复制。